U.S. patent application number 17/173897 was filed with the patent office on 2021-09-02 for methods for treating pten deficient epithelial cancers using a combination of anti-pi3kbeta and anti-immune checkpoint agents.
The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Johann Bergholz, Gordon J. Freeman, Thomas M. Roberts, Jean Zhao.
Application Number | 20210267991 17/173897 |
Document ID | / |
Family ID | 1000005579655 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210267991 |
Kind Code |
A1 |
Zhao; Jean ; et al. |
September 2, 2021 |
METHODS FOR TREATING PTEN DEFICIENT EPITHELIAL CANCERS USING A
COMBINATION OF ANTI-PI3KBETA AND ANTI-IMMUNE CHECKPOINT AGENTS
Abstract
The present invention relates to methods for treating PTEN
deficient epithelial cancers using a combination of anti-PI3Kbeta
and anti-immune checkpoint agents.
Inventors: |
Zhao; Jean; (Brookline,
MA) ; Bergholz; Johann; (Cambridge, MA) ;
Freeman; Gordon J.; (Brookline, MA) ; Roberts; Thomas
M.; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
1000005579655 |
Appl. No.: |
17/173897 |
Filed: |
February 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16315298 |
Jan 4, 2019 |
10960008 |
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PCT/US2017/041490 |
Jul 11, 2017 |
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17173897 |
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62360596 |
Jul 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 35/04 20180101; A61K 47/6803 20170801; A01K 2267/0331
20130101; C12Q 1/6827 20130101; A01K 2227/105 20130101; C07K 16/32
20130101; A01K 67/0278 20130101; A61K 31/713 20130101; A61K 31/5377
20130101; A61K 31/7105 20130101; C12Q 1/6886 20130101 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; A61K 47/68 20060101 A61K047/68; A61P 35/04 20060101
A61P035/04; A01K 67/027 20060101 A01K067/027; A61K 31/7105 20060101
A61K031/7105; A61K 31/713 20060101 A61K031/713; C07K 16/32 20060101
C07K016/32; C12Q 1/6827 20060101 C12Q001/6827; C12Q 1/6886 20060101
C12Q001/6886 |
Goverment Interests
STATEMENT OF RIGHTS
[0002] This invention was made with government support under grant
numbers R01 CA 172461, RO1 CA 187918, T32 CA009361-34, and POI
AI056299 awarded by The National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of treating a subject afflicted with a cancer that is
deficient in phosphatase and tensin homolog (PTEN) comprising
administering to the subject a therapeutically effective amount of
at least one agent that inhibits or blocks both phosphoinositide
3-kinase isoform beta (PI3Kbeta) and an immune checkpoint, wherein
the cancer is an epithelial cancer.
2. The method of claim 1, wherein the at least one agent is a
single agent that inhibits or blocks both PI3Kbeta and the immune
checkpoint.
3. The method of claim 1, wherein the at least one agent comprises
a first agent that selectively inhibits or blocks PI3Kbeta and a
second agent that selectively inhibits or blocks the immune
checkpoint, optionally wherein the first agent and the second agent
comprise a small molecule that inhibits or blocks PI3Kbeta and/or
the immune checkpoint.
4. (canceled)
5. The method of claim 1, wherein the at least one agent comprises
an RNA interfering agent which inhibits expression of PI3Kbeta
and/or the immune checkpoint, optionally wherein the RNA
interfering agent is a small interfering RNA (siRNA), small hairpin
RNA (shRNA), or a microRNA (miRNA).
6. (canceled)
7. The method of claim 1, wherein the at least one agent comprises
a) an antisense oligonucleotide complementary to PI3Kbeta and/or
the immune checkpoint, b) a peptide or peptidomimetic that inhibits
or blocks PI3Kbeta and/or the immune checkpoint; and/or c) an
aptamer that inhibits or blocks PI3Kbeta and/or the immune
checkpoint.
8-9. (canceled)
10. The method of claim 1, wherein the at least one agent is an
antibody and/or an intrabody, or an antigen binding fragment
thereof, which specifically binds to PI3Kbeta protein and/or the
immune checkpoint protein, optionally wherein the antibody and/or
intrabody, or antigen binding fragment thereof, is murine,
chimeric, humanized, composite, or human.
11. (canceled)
12. The method of claim 10, wherein the antibody and/or intrabody,
or antigen binding fragment thereof, is detectably labeled,
comprises an effector domain, comprises an Fc domain, and/or is
selected from the group consisting of Fv, Fav, F(ab')2), Fab',
dsFv, scFv, sc(Fv)2, and diabodies fragments.
13. The method of claim 10, wherein the antibody and/or intrabody,
or antigen binding fragment thereof, is conjugated to a cytotoxic
agent, optionally wherein the cytotoxic agent is selected from the
group consisting of a chemotherapeutic agent, a biologic agent, a
toxin, and a radioactive isotope.
14. (canceled)
15. The method of claim 1, wherein the immune checkpoint is
selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2,
B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B,
PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA,
SIRPalpha, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4,
TIGIT, IDO, CD39, arginase, CD73, and A2aR, optionally wherein a)
the immune checkpoint is selected from the group consisting of
CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, and LAG-3 and/or b) wherein the
immune checkpoint is PD-1.
16-17. (canceled)
18. The method of claim 1, wherein the at least one agent comprises
a selective PI3Kbeta inhibitor, optionally wherein the selective
PI3Kbeta inhibitor has at least 2-fold more selectivity for the
PI3Kbeta isoform as compared to a non-PI3Kbeta isoform selected
from the group consisting of PI3Kalpha, PI3Kdelta, and
PI3Kgamma.
19. (canceled)
20. The method of claim 1, wherein the at least one agent comprises
5-(2,6-dimorpholin-4-ylpyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine
(BKM120) and/or
(-)-2-[[(1R)-1-[7-Methyl-2-(3-morpholinyl)-4-ox-4H-pyrido[1,2-a]-pyrimidi-
n-9-yl]ethyl]amino]benzoic acid (KIN193).
21. The method of claim 1, wherein the at least one agent a)
reduces the number of proliferating cells in the cancer and/or
reduces the volume or size of a tumor of the cancer, b) increases
the number of viable CD8+ T cells within a tumor of the cancer,
and/or c) is administered in a pharmaceutically acceptable
formulation.
22-23. (canceled)
24. The method of claim 1, further comprising administering to the
subject a therapeutic agent or regimen for treating the cancer,
optionally wherein the therapeutic agent is hormone therapy.
25. The method of claim 1, wherein the PTEN deficiency comprises a
mutation to a genomic nucleic acid sequence encoding PTEN,
optionally wherein the mutation is selected from the group
consisting of a missense mutation, a nonsense mutation, a
frameshift mutation, an insertion mutation, a deletion mutation,
and a rearrangement mutation.
26. (canceled)
27. The method of claim 1, wherein the PTEN deficiency is
determined to be very low or null, as assessed by
immunohistochemistry, or is a mutation to a genomic nucleic acid
sequence encoding PTEN protein and the mutation is a missense
mutation, a nonsense mutation, a frameshift mutation, an insertion
mutation, a deletion mutation, or a rearrangement mutation of a
PTEN codon C71, R130, R233, D268, T319 or X70, or phosphatase or C2
domains, optionally wherein the PTEN deficiency is a) a null
mutation and/or 2) a germline or somatic null mutation.
28-29. (canceled)
30. The method of claim 1, wherein the cancer has a p53
deficiency.
31. The method of claim 30, wherein the p53 deficiency is a
mutation to a genomic nucleic acid sequence encoding p53 protein
and the mutation is a missense mutation, a nonsense mutation, a
frameshift mutation, an insertion mutation, a deletion mutation, or
a rearrangement mutation of a p53 codon L45, Y126, P151, S166,
R175, C176, H179, G187, H193, L194, R196, R213, Y220, C242, G245,
R248, R249, R273, R280, D281, R282, E286, E294, or transactivation,
DNA-binding or oligomerization domains, optionally wherein the p53
deficiency is a) a null mutation and/or 2) a germline or somatic
null mutation.
32. The method of claim 31, wherein the p53 deficiency is a
germline or somatic p53 null mutation.
33. The method of claim 32, wherein the epithelial cancer is a
breast cancer, an ovarian cancer, or a prostate cancer, optionally
wherein a) the breast cancer is triple negative breast cancer
(TNBC) and/or metastatic TNBC, or b) wherein the ovarian cancer is
serous ovarian cancer.
34-36. (canceled)
37. The method of claim 1, wherein the subject is a) an animal
model of the epithelial cancer, optionally wherein the animal model
is an orthotopic xenograft animal model of a human-derived
epithelial cancer and/or a mouse model; b) a mammal, optionally
wherein the mammal is a mouse or a human.
38-42. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/360,596, filed on 11 Jul. 2016, the entire
contents of said application are incorporated herein in their
entirety by this reference.
BACKGROUND OF THE INVENTION
[0003] Breast cancer is one of the most common types of cancer,
with over 230,000 new cases diagnosed annually and accounting for
more than 40,000 deaths every year in the United States alone. For
example, triple-negative breast cancers (TNBCs), as defined by the
absence of estrogen receptor (ER), progesterone receptor (PR), and
human epidermal growth factor receptor 2 (HER2) expression, account
for 15 to 20% of breast cancer cases, are often clinically
aggressive, and typically exhibit high rates of recurrence and
mortality. In addition, they do not respond to current targeted
therapies used in the clinic that are highly effective in the
treatment of luminal or HER2-positive breast cancer. While standard
chemotherapy can be effective for early stage TNBC, patients with
advanced TNBC typically respond poorly and may progress to
metastatic disease, in which cancer cells invade into other
tissues. Of note, metastasis accounts for over 90% of breast
cancer-related deaths and currently there are no effective
treatments against metastatic TNBC. Despite the lack of a common
genetic determinant, loss of phosphatase and tensin homolog (PTEN)
tumor suppressor function is observed in 25 to 30% of TNBCs (Cancer
Genome Atlas Network (2012) Nature 490:61-70). Since PTEN is the
major negative regulator of PI3K activity in cells, PTEN-deficient
cancers often exhibit oncogenic addiction to PI3K signaling. Thus,
the PI3K pathway and, in particular, class I PI3K isoforms
(including p110.alpha., p110.beta., p110.gamma. and p110.delta.)
have emerged as attractive therapeutic targets (Thorpe et al.
(2015) Nat. Rev. Cancer 15:7-24). Yet, clinical trials with
pan-PI3K inhibitors have showed only limited efficacy and reduced
therapeutic windows as monotherapies, largely due to off-target and
on-target effects arising from inhibiting all PI3K isoforms (Fruman
and Rommel (2014) Nat. Rev. Drug Disc. 13:140-156 and Brachmann et
al. (2012) Mol. Cancer Ther. I1:1747-1757).
[0004] Pre-clinical studies and emerging clinical trial data
suggest that selective targeting of individual isoforms may achieve
greater efficacy with fewer side effects than using pan-PI3K
inhibitors, and thus isoform-specific PI3K inhibitors are currently
undergoing clinical trials as both mono- and combination therapies
in multiple cancer types, including breast cancer (Fruman and
Rommel (2014) Nat. Rev. Drug Disc. 13:140-156; Thorpe et al. (2015)
Nat. Rev. Cancer 15:7-24). Compounds targeting PI3Kalpha (also
known as p110.alpha.) have garnered much of the efforts from both
academic and pharmaceutical research groups. This is largely
because mutations in PIK3CA, the gene encoding PI3Kalpha, are found
in a significant number of cancers (Cancer Genome Atlas Network
(2012) Nature 490:61-70). Also, tumors driven by receptor tyrosine
kinases, such as Her2+ breast cancers, or Ras hyperactivation,
mainly require signaling via PI3Kalpha for survival (Schmit et al.
(2014) Proc. Natl. Acad. Sci. USA 111:6395-400).
[0005] However, PTEN-deficient cancers often depend on the beta
isoform of PI3 Kinase (PI3Kbeta; p110.beta.) encoded by PIK3CB for
survival, and pharmacological targeting of p110.beta. can
effectively decrease the growth of PTEN-deficient tumors in mouse
models (Jia et al. (2008) Nature 454:776-779; Ni et al. (2012)
Cancer Discov. 2:425-433; Peng et al. (2016) Cancer Disc.
6:202-216). In TNBC, activating mutations in PIK3CA encoding
PI3Kalpha were found by The Cancer Genome Atlas (TCGA) in only 7%
of cases, but genetic PTEN loss occurred in over 35% of cases
(Cancer Genome Atlas Network (2012) Nature 490:61-70).
[0006] Aside from targeted therapies, additional modalities for
treating cancer have been explored. For example, immune checkpoint
blockade (ICB), whereby anti-tumor T-cell response is enhanced by
blocking immuno-inhibitory signals. The two major targets of ICB
agents developed to date are CTLA-4 (Cytotoxic T
lymphocyte-associated protein 4) and PD-1 (Programmed death-1)
receptors, which are expressed on the surface of activated T-cells
and are responsible for dampening immune response. CTLA-4 blockade
is thought to occur in secondary lymphoid organs, thereby affecting
systemic immune function. By contrast, PD-1 ligands are often
overexpressed by cancer cells. Therefore, PD-1 blockade takes place
primarily within the tumor microenvironment and is associated with
fewer side effects (Topalian et al. (2015) Cancer Cell 27:450-461).
Although immune checkpoint blockade (ICB) immunotherapy has
demonstrated promising results, ICB agents used as monotherapies
have yielded therapeutic efficacy in only a limited percentage of
patients. Accumulating evidence indicates that targeted therapies
can synergize with immunotherapy in multiple cancer types, although
which targeted therapies in combination with which immunotherapies
are efficacious for treating which cancers is currently unknown and
difficult to predict (Vannemann and Dranoff (2012) Nat. Rev. Cancer
12:237-251; Sagiv-Barfi et al. (2015) Proc. Natl. Acad. Sci. USA
112:e966-e972).
[0007] Accordingly, a great need exists in the art to identify
particular therapeutic interventions to effectively treat cancers,
such as epithelial cancers like TNBCs. Similarly, serous ovarian
cancer (SOC), which has also historically been difficult to treat,
has many underlying genetic similarities to TNBCs and which are
quite different from those of other cancers, like melanoma. For
example, both types of cancers are associated with high histologic
grades and poor prognosis. At the genetic level, the two cancers
contain similar types and frequencies of genomic mutations, e.g.
high frequency of TP53 (encoding the p53 tumor suppressor in
humans) mutations, inactivation of BRCA1, genetic and epigenetic
loss of PTEN, and amplification and high expression of the cMYC
oncogene. Because of the similarities between TNBC and SOC,
researchers have proposed that the two cancers could possibly be
targeted by similar therapies (Wang et al. (2012) Clin. Cancer Res.
18:5806-5815).
SUMMARY OF THE INVENTION
[0008] The present invention is based, at least in part, on the
discovery that inhibiting or blocking both PI3Kbeta and an immune
checkpoint overcomes traditional barriers to therapeutically
treating cancers having PTEN deficiency, with or without additional
p53 deficiency. Such results are unexpected given the previously
unappreciated role of PI3Kbeta modulation in treating cancer due to
the focus on other isoforms of PI3K, such as PI3Kalpha.
[0009] In one aspect, a method of treating a subject afflicted with
a cancer that is deficient in phosphatase and tensin homolog (PTEN)
comprising administering to the subject a therapeutically effective
amount of at least one agent that inhibits or blocks both
phosphoinositide 3-kinase isoform beta (PI3Kbeta) and an immune
checkpoint, wherein the cancer is an epithelial cancer (e.g., a
breast cancer, an ovarian cancer, a prostate cancer, etc.), is
provided.
[0010] Numerous embodiments are further provided that can be
applied to any aspect of the present invention and/or combined with
any other embodiment described herein. For example, in one
embodiment, the at least one agent is a single agent that inhibits
or blocks both PI3Kbeta and the immune checkpoint. In another
embodiment, the at least one agent comprises a first agent that
selectively inhibits or blocks PI3Kbeta and a second agent that
selectively inhibits or blocks the immune checkpoint. In still
another embodiment, the first agent and the second agent comprise a
small molecule that inhibits or blocks PI3Kbeta and/or the immune
checkpoint. In yet another embodiment, the at least one agent
comprises an RNA interfering agent which inhibits expression of
PI3Kbeta and/or the immune checkpoint (e.g., the RNA interfering
agent is a small interfering RNA (siRNA), small hairpin RNA
(shRNA), or a microRNA (miRNA)). In another embodiment, the at
least one agent comprises an antisense oligonucleotide
complementary to PI3Kbeta and/or the immune checkpoint. In still
another embodiment, the at least one agent comprises a peptide or
peptidomimetic that inhibits or blocks PI3Kbeta and/or the immune
checkpoint.
[0011] In yet another embodiment, the at least one agent comprises
an aptamer that inhibits or blocks PI3Kbeta and/or the immune
checkpoint. In another embodiment, the at least one agent is an
antibody and/or an intrabody, or an antigen binding fragment
thereof, which specifically binds to PI3Kbeta protein and/or the
immune checkpoint protein (e.g., the antibody and/or intrabody, or
antigen binding fragment thereof, is murine, chimeric, humanized,
composite, or human). In still another embodiment, the antibody
and/or intrabody, or antigen binding fragment thereof, is
detectably labeled, comprises an effector domain, comprises an Fc
domain, and/or is selected from the group consisting of Fv, Fav,
F(ab')2), Fab', dsFv, scFv, sc(Fv)2, and diabodies fragments. In
yet another embodiment, the antibody and/or intrabody, or antigen
binding fragment thereof, is conjugated to a cytotoxic agent (e.g.,
the cytotoxic agent is selected from the group consisting of a
chemotherapeutic agent, a biologic agent, a toxin, and a
radioactive isotope).
[0012] In another embodiment, the immune checkpoint is selected
from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3,
PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B,
KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha,
CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, IDO,
CD39, arginase, CD73, and A2aR. In still another embodiment, the
immune checkpoint is selected from the group consisting of CTLA-4,
PD-1, PD-L1, PD-L2, TIM-3, and LAG-3. In yet another embodiment,
the immune checkpoint is PD-1. In another embodiment, the at least
one agent comprises a selective PI3Kbeta inhibitor (e.g., the
selective PI3Kbeta inhibitor has at least 2-fold more selectivity
for the PI3Kbeta isoform as compared to a non-PI3Kbeta isoform
selected from the group consisting of PI3Kalpha, PI3Kdelta, and
PI3Kgamma). In still another embodiment, the at least one agent
comprises
5-(2,6-dimorpholin-4-ylpyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine
(BKM120) and/or
(-)-2-[[(1R)-1-[7-Methyl-2-(4-morpholinyl)-4-oxo-4H-pyrido[1,2-a]pyrimidi-
n-9-yl]ethyl]amino]benzoic acid (KIN193). In yet another
embodiment, the at least one agent reduces the number of
proliferating cells in the cancer and/or reduces the volume or size
of a tumor of the cancer. In another embodiment, the at least one
agent increases the number of viable CD8+ T cells within a tumor of
the cancer. In still another embodiment, the at least one agent is
administered in a pharmaceutically acceptable formulation. In yet
another embodiment, the method further comprises administering to
the subject a therapeutic agent or regimen for treating the
cancer.
[0013] In another embodiment, the cancer described herein further
has a p53 deficiency. In still another embodiment, the p53
deficiency and/or PTEN deficiency is selected from the group
consisting of a mutation to a genomic nucleic acid sequence
encoding p53 and/or PTEN protein. In yet another embodiment, the
mutation is selected from the group consisting of a missense
mutation, a nonsense mutation, a frameshift mutation, an insertion
mutation, a deletion mutation, and a rearrangement mutation. In
another embodiment, the PTEN deficiency is determined to be very
low or null, as assessed by immunohistochemistry, or is a mutation
to a genomic nucleic acid sequence encoding PTEN protein and the
mutation is a missense mutation, a nonsense mutation, a frameshift
mutation, an insertion mutation, a deletion mutation, or a
rearrangement mutation of a PTEN codon C71, R130, R233, D268, T319
or X70, or phosphatase or C2 domains. Instill another embodiment,
the PTEN deficiency is a null mutation. In yet another embodiment,
the PTEN deficiency is a germline or somatic PTEN null mutation. In
another embodiment, the p53 deficiency is a mutation to a genomic
nucleic acid sequence encoding p53 protein and the mutation is a
missense mutation, a nonsense mutation, a frameshift mutation, an
insertion mutation, a deletion mutation, or a rearrangement
mutation of a p53 codon L45, Y126, P151, S166, R175, C176, H179,
G187, H193, L194, R196, R213, Y220, C242, G245, R248, R249, R273,
R280, D281, R282, E286, E294, or transactivation, DNA-binding or
oligomerization domains. In still another embodiment, the p53
deficiency is a null mutation. In yet another embodiment, the p53
deficiency is a germline or somatic p53 null mutation. In another
embodiment, the epithelial cancer described herein is a breast
cancer, an ovarian cancer, or a prostate cancer. In still another
embodiment, the breast cancer is a triple-negative breast cancer
(TNBC). In yet another embodiment, the TNBC is metastatic TNBC. In
another embodiment, the ovarian cancer is serous ovarian cancer. In
still another embodiment, the subject is an animal model of TNBC or
ovarian cancer. In another embodiment, the animal model is an
orthotopic xenograft animal model of a human-derived epithelial
cancer. In still another embodiment, the animal model is a mouse
model. In yet another embodiment, the subject is a mammal, such as
a mouse or a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 includes 2 panels, identified as panels A and B,
which show that Pten deletion in mammary cells causes breast tumor
formation (Panel A) that is dependent on Pik3cb, but not on Pik3ca
(Panel B).
[0015] FIG. 2 includes 3 panels, identified as panels A, B, and C,
which show that Pten;Trp53-null (PP, Trp53 encodes p53 in mice)
TNBC primary cells with additional deletion of either Pik3ca (PPA)
or Pik3cb maintain biochemical and phenotypic characteristics of
parental tumors, including expression of diagnostic markers (Panel
A) and sensitivity to isoform-specific PI3K inhibition using a
pan-PI3K inhibitor (BKM120), a p110.alpha. inhibitor (BYL719) and a
p110.beta. inhibitor (KIN-193) (Panels B-C).
[0016] FIG. 3 includes 4 panels, identified as panels A, B, C, and
D, which show that PI3Kbeta signaling regulates a cell invasion and
motility program (Panels A-D).
[0017] FIG. 4 shows a representative image of tumor (T) and
endothelial (E) cells in sections of a distal TNBC metastasis
having PTEN deficiency stained with hematoxylin and eosin (H&E)
or analyzed using immunohistochemistry (IHC) with a PTEN
antibody.
[0018] FIG. 5 includes 3 panels, identified as panels A, B, and C,
which show that PP TNBC tumors grow faster (Panel A) and form
metastatic nodules (Panels B-C) in immunocompromised (nude)
mice.
[0019] FIG. 6 shows that PP and PPA TNBC tumors grow in
immunocompetent mice, while PPB tumors do not form tumors in
immunocompetent mice.
[0020] FIG. 7 includes 3 panels, identified as panels A, B, and C,
which show the results of combined PI3K targeted therapy and ICB
therapy toward inhibiting PP tumor growth (Panel A) and increasing
CD8+ T cell infiltration (Panels B-C).
[0021] FIG. 8 includes 4 panels, identified as panels A, B, C, and
D, which show the results of combined PI3Kbeta targeted therapy and
ICB therapy synergizing to inhibit PP TNBC tumor growth (Panels
A-C) and increasing CD84 T cell infiltration (Panel D) on cohort
1.
[0022] FIG. 9 includes 2 panels, identified as panels A and B,
which show the results of combined PI3Kbeta targeted therapy and
ICB therapy synergizing to inhibit PP TNBC tumor growth on cohort
2.
[0023] FIG. 10 shows that gene signatures involved in anti-tumor
immune response are enriched to a much greater extent (represented
by a greater normalized enrichment score (NES) value) and with a
much greater degree of statistical significance (represented by a
lower q-value) in combined PI3Kbeta targeted therapy and ICB
therapy compared to the vehicle control than either agent alone
compared to the vehicle control.
[0024] FIG. 11 shows that CD8+ T cell infiltration negatively
correlates with tumor size.
[0025] FIG. 12 shows that adding a PI3Kbeta targeted inhibitor to a
treatment regimen of lapatinib (Lap; an EGFR and Her2/Neu dual
inhibitor) leads to reduced tumor growth in a model of
Her2-positive breast cancer.
[0026] FIG. 13 includes 3 panels, identified as panels A, B, and C,
which show that PDX models faithfully recapitulate patient-derived
samples as shown by IHC analyses (Panel A), whole-exome sequencing
(Panel B), and live imaging of metastatic lesions (Panel C).
[0027] FIG. 14 shows that combined PI3Kbeta targeted therapy and
ICB therapy synergize to inhibit PTEN/p53-deficient ovarian cancer
tumor growth.
[0028] FIG. 15 includes 2 panels, identified as panels A, and B,
which show the unique role of p110.beta. in PTEN-null tumors (Panel
A) and the cycle of Rac/p110.beta. activation.
[0029] FIG. 16 includes 4 panels, identified as panels A, B, C, and
D, which show the efficacy of Rac inhibitors in
p110.beta.-dependent tumors arising from PTEN-null hematopoietic
stem cells.
[0030] FIG. 17 includes 3 panels, identified as panels A, B, and C,
which show that p110.beta.-dependent PTEN-null tumor cell lines are
sensitive to statins but can be rescued by activated alleles of
p110.alpha. PC3 cells. Cells with wild-type Pten and PTEN-null
cells that have had PTEN expression restored are not sensitive.
[0031] FIG. 18 shows that PP TNBC tumors with additional deletion
of p110.beta. (PPB TNBC) failed to grow in immunocompetent mice,
but did form tumors in immunodeficient mice.
[0032] Note that for every figure containing a histogram, the bars
from left to right for each discrete measurement correspond to the
figure boxes from top to bottom in the figure legend as
indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0033] It has been determined herein based on genetic,
pharmacologic, and molecular analyses of PTEN-deficient TNBC
models, with or without additional p53 deficiency, that inhibiting
or blocking PI3Kbeta in combination with inhibiting or blocking an
immune checkpoint (ICB) overcomes traditional barriers to
therapeutically treating cancers having PTEN deficiency. For
example, KIN-193 (i.e., a PI3Kbeta-selective inhibitor), but not
BYL719 (i.e., a PI3Kalpha-selective inhibitor), synergizes with ICB
to inhibit PTEN/p53-null TNBC tumor growth.
[0034] The great majority of samples analyzed from a panel of TNBC
patient-derived samples showed the absence of PTEN expression (see
Table 1 below, at least three retained a wild type PTEN gene),
suggesting that the percentage of TNBC with deficient PTEN
expression may be higher than what has been determined based on
genetic deletion.
TABLE-US-00001 TABLE 1 TNBC patient-derive sample data PDX # PTEN
(IHC) P-AKT PTEX gene Scoring scale: Orthotopic PDX models of TNBC
brain metastases DF-BM362 0 na na 0 = Null DF-BM456 0 na na 1 = Low
DF-BM640 0 na na 2 = Medium DF-BM656 0 na na 3 = High
Patient-derived xenograft (PDX) models of TNBC (non-CNS) DFBC-1501
0-1 1 nd 0 = Null DFBC-1504 0 3 nd 1 = Low DFBC-1507 0 nd WT 2 =
Medium DFBC-1510 0 2 WT 3 = High DFBC-l520 0 2-3 WT DFBC-1524 nd nd
nd DFBC-1611 nd nd nd DFBC-1613 nd nd nd
[0035] The importance of PI3Kbeta for overcoming anti-tumor immune
response and supporting growth of PTEN-deficient tumors is further
demonstrated by the inability of PTEN/p53/PI3Kbeta triple-null
tumor cells to form tumors in immunocompetent mice, while
PTEN/p53/PI3Kalpha triple-null tumor cells grow aggressive tumors
in immunocompetent mice. On the other hand, PTEN/p53 double-null,
PTEN/p53/PI3Kbeta triple-null and PTEN/p53/PI3Kalpha triple-null
tumor cells all form tumors in immunodeficient mice lacking T
cells. Notably, combined ICB and PI3Kbeta-selective inhibition
strongly increases CD8+ T cell infiltration into the tumor,
indicating an important role for this combination treatment in
immunomodulation of tumor growth. In addition, the results show
that PI3Kbeta, but not PI3Kalpha, is required for inducing cell
motility in primary breast cancer cells with PTEN/p53 co-loss. In
line with these finding, PTEN is lost in all samples of distal
metastases analyzed from patients with TNBC. Thus, it is believed
that PI3Kbeta inhibitors are effective against advanced and
metastatic TNBC.
[0036] In addition, PI3Kbeta inhibition combined with immune
checkpoint blockade (ICB) also inhibits growth of a Her2-positive
model of breast cancer driven by Her2/Neu over-expression and
harboring wild type p53 (see at least FIG. 12). Moreover, combining
a PI3Kbeta inhibitor and ICB led to stronger tumor growth
inhibition than either agent alone in a model of serous ovarian
cancer (SOC) with PTEN/p53 loss and Myc over-expression (see at
least FIG. 14).
[0037] These results are surprising for at least several reasons.
First, anti-cancer agents are generally focused on inhibiting or
blocking PI3Kalpha or PI3K isoforms other than PI3Kbeta. Yet, a
remarkable benefit of specifically targeting PI3Kbeta rather than
all PI3K proteins or PI3Kalpha alone is that PI3Kbeta inhibition is
less toxic than pan-PI3K or PI3Kalpha inhibition because PI3Kalpha
is the major effector of insulin function in normal cells, and
therefore inhibiting PI3Kalpha can lead to significant side
effects. Second, TNBC and SOC are believed to be especially
well-suited to the combination of PI3Kbeta inhibition/block and ICB
because of their particular genotypic background regarding PTEN
deficiencies, as distinguished from other cancer types. For
example, TNBC and SOC are characterized by significant levels of
PTEN deficiency and a high degree of PTEN/p53 co-deficiency. Also,
unlike melanoma and lung cancer, for which ICB therapies are
currently approved by the Food and Drug Administration (FDA), TNBC
and SOC do not tend to have a high mutational load, and are
therefore predicted to not respond well to IBC therapy alone
Finally, PI3Kbeta inhibition is known to generate resistance in
some cancers, such as in PTEN-deficient prostate cancers (Schwartz
et al. (2015) Cancer Cell 27:109-22), highlighting the need to find
novel therapeutic targets to combine with PI3Kbeta inhibitors.
[0038] Accordingly, the present invention relates, in part, to
methods for treating PTEN-deficient epithelial cancers (e.g.,
breast cancer and SOC) with a combination of PI3Kbeta and immune
checkpoint inhibitors. In another aspect, the present invention
provides diagnostic, prognostic, and prophylactic methods of
stratifying patients and predicting responses of cancers to
treatment with a combination of PI3Kbeta and immune checkpoint
inhibitors based upon a determination and analysis of biomarkers
described herein.
I. Definitions
[0039] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0040] The term "administering" is intended to include routes of
administration which allow an agent to perform its intended
function. Examples of routes of administration for treatment of a
body which can be used include injection (subcutaneous,
intravenous, parenterally, intraperitoneally, intrathecal, etc.),
oral, inhalation, and transdermal routes. The injection can be
bolus injections or can be continuous infusion. Depending on the
route of administration, the agent can be coated with or disposed
in a selected material to protect it from natural conditions which
may detrimentally affect its ability to perform its intended
function. The agent may be administered alone, or in conjunction
with a pharmaceutically acceptable carrier. The agent also may be
administered as a prodrug, which is converted to its active form in
vivo.
[0041] The term "altered amount" or "altered level" refers to
increased or decreased copy number (e.g., germline and/or somatic)
of a biomarker nucleic acid, e.g., increased or decreased
expression level in a cancer sample, as compared to the expression
level or copy number of the biomarker nucleic acid in a control
sample. The term "altered amount" of a biomarker also includes an
increased or decreased protein level of a biomarker protein in a
sample, e.g., a cancer sample, as compared to the corresponding
protein level in a normal, control sample. Furthermore, an altered
amount of a biomarker protein may be determined by detecting
posttranslational modification such as methylation status of the
marker, which may affect the expression or activity of the
biomarker protein.
[0042] The amount of a biomarker in a subject is "significantly"
higher or lower than the normal amount of the biomarker, if the
amount of the biomarker is greater or less, respectively, than the
normal level by an amount greater than the standard error of the
assay employed to assess amount, and preferably at least 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%,
500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
Alternately, the amount of the biomarker in the subject can be
considered "significantly" higher or lower than the normal amount
if the amount is at least about two, and preferably at least about
three, four, or five times, higher or lower, respectively, than the
normal amount of the biomarker. Such "significance" can also be
applied to any other measured parameter described herein, such as
for expression, inhibition, cytotoxicity, cell growth, and the
like.
[0043] The term "altered level of expression" of a biomarker refers
to an expression level or copy number of the biomarker in a test
sample, e.g., a sample derived from a patient suffering from
cancer, that is greater or less than the standard error of the
assay employed to assess expression or copy number, and is
preferably at least twice, and more preferably three, four, five or
ten or more times the expression level or copy number of the
biomarker in a control sample (e.g., sample from a healthy subjects
not having the associated disease) and preferably, the average
expression level or copy number of the biomarker in several control
samples. The altered level of expression is greater or less than
the standard error of the assay employed to assess expression or
copy number, and is preferably at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%,
700%, 800%, 900%, 1000% or more times the expression level or copy
number of the biomarker in a control sample (e.g., sample from a
healthy subjects not having the associated disease) and preferably,
the average expression level or copy number of the biomarker in
several control samples. In some embodiments, the level of the
biomarker refers to the level of the biomarker itself, the level of
a modified biomarker (e.g., phosphorylated biomarker), or to the
level of a biomarker relative to another measured variable, such as
a control (e.g., phosphorylated biomarker relative to an
unphosphorylated biomarker).
[0044] The term "altered activity" of a biomarker refers to an
activity of the biomarker which is increased or decreased in a
disease state, e.g., in a cancer sample, as compared to the
activity of the biomarker in a normal, control sample. Altered
activity of the biomarker may be the result of, for example,
altered expression of the biomarker, altered protein level of the
biomarker, altered structure of the biomarker, or, e.g., an altered
interaction with other proteins involved in the same or different
pathway as the biomarker or altered interaction with
transcriptional activators or inhibitors.
[0045] The term "altered structure" of a biomarker refers to the
presence of mutations or allelic variants within a biomarker
nucleic acid or protein, e.g., mutations which affect expression or
activity of the biomarker nucleic acid or protein, as compared to
the normal or wild-type gene or protein. For example, mutations
include, but are not limited to substitutions, deletions, or
addition mutations. Mutations may be present in the coding or
non-coding region of the biomarker nucleic acid.
[0046] Unless otherwise specified here within, the terms "antibody"
and "antibodies" broadly encompass naturally-occurring forms of
antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies, as well as fragments and derivatives
of all of the foregoing, which fragments and derivatives have at
least an antigenic binding site. Antibody derivatives may comprise
a protein or chemical moiety conjugated to an antibody.
[0047] The term "antibody" as used herein also includes an
"antigen-binding portion" of an antibody (or simply "antibody
portion"). The term "antigen-binding portion", as used herein,
refers to one or more fragments of an antibody that retain the
ability to specifically bind to an antigen (e.g., a biomarker
polypeptide or fragment thereof). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VII regions pair to form monovalent polypeptides (known as
single chain Fv (scFv); see e.g., Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:
778). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Any VH and VL sequences of specific scFv can be linked to
human immunoglobulin constant region cDNA or genomic sequences, in
order to generate expression vectors encoding complete IgG
polypeptides or other isotypes. VH and VL can also be used in the
generation of Fab, Fv or other fragments of immunoglobulins using
either protein chemistry or recombinant DNA technology. Other forms
of single chain antibodies, such as diabodies are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure
2:1121-1123).
[0048] Still further, an antibody or antigen-binding portion
thereof may be part of larger immunoadhesion polypeptides, formed
by covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion polypeptides include use of the streptavidin
core region to make a tetrameric scFv polypeptide (Kipriyanov, S.
M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use
of a cysteine residue, biomarker peptide and a C-terminal
polyhistidine tag to make bivalent and biotinylated scFv
polypeptides (Kipriyanov, S. M., et al. (1994) Mol. Immunol.
31:1047-1058). Antibody portions, such as Fab and F(ab').sub.2
fragments, can be prepared from whole antibodies using conventional
techniques, such as papain or pepsin digestion, respectively, of
whole antibodies. Moreover, antibodies, antibody portions and
immunoadhesion polypeptides can be obtained using standard
recombinant DNA techniques, as described herein.
[0049] By contrast, antigen-binding portions can be adapted to be
expressed within cells as "intracellular antibodies." (Chen et al.
(1994) Human Gene Ther. 5:595-601). Methods are well-known in the
art for adapting antibodies to target (e.g., inhibit) intracellular
moieties, such as the use of single-chain antibodies (scFvs),
modification of immunoglobulin VL domains for hyperstability,
modification of antibodies to resist the reducing intracellular
environment, generating fusion proteins that increase intracellular
stability and/or modulate intracellular localization, and the like.
Intracellular antibodies can also be introduced and expressed in
one or more cells, tissues or organs of a multicellular organism,
for example for prophylactic and/or therapeutic purposes (e.g., as
a gene therapy) (see, at least PCT Publs. WO 08/020079, WO
94/02610, WO 95/22618, and WO 03/014960; U.S. Pat. No. 7,004,940,
Cattaneo and Biocca (1997) Intracellular Antibodies: Development
and Applicalions (Landes and Springer-Verlag pubis.); Kontermann
(2004) Methods 34:163-170; Cohen et al. (1998) Oncogene
17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412;
Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303.19-39).
[0050] Antibodies may be polyclonal or monoclonal; xenogeneic,
allogeneic, or syngeneic; or modified forms thereof (e.g.
humanized, chimeric, etc.). Antibodies may also be fully human.
Preferably, antibodies of the present invention bind specifically
or substantially specifically to a biomarker polypeptide or
fragment thereof. The terms "monoclonal antibodies" and "monoclonal
antibody composition", as used herein, refer to a population of
antibody polypeptides that contain only one species of an antigen
binding site capable of immunoreacting with a particular epitope of
an antigen, whereas the term "polyclonal antibodies" and
"polyclonal antibody composition" refer to a population of antibody
polypeptides that contain multiple species of antigen binding sites
capable of interacting with a particular antigen. A monoclonal
antibody composition typically displays a single binding affinity
for a particular antigen with which it immunoreacts.
[0051] Antibodies may also be "humanized", which is intended to
include antibodies made by a non-human cell having variable and
constant regions which have been altered to more closely resemble
antibodies that would be made by a human cell. For example, by
altering the non-human antibody amino acid sequence to incorporate
amino acids found in human germline immunoglobulin sequences. The
humanized antibodies of the present invention may include amino
acid residues not encoded by human germline immunoglobulin
sequences (e.g., mutations introduced by random or site-specific
mutagenesis in vitro or by somatic mutation in vivo), for example
in the CDRs. The term "humanized antibody", as used herein, also
includes antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0052] The term "assigned score" refers to the numerical value
designated for each of the biomarkers after being measured in a
patient sample. The assigned score correlates to the absence,
presence or inferred amount of the biomarker in the sample. The
assigned score can be generated manually (e.g., by visual
inspection) or with the aid of instrumentation for image
acquisition and analysis In certain embodiments, the assigned score
is determined by a qualitative assessment, for example, detection
of a fluorescent readout on a graded scale, or quantitative
assessment. In one embodiment, an "aggregate score," which refers
to the combination of assigned scores from a plurality of measured
biomarkers, is determined.
[0053] In one embodiment the aggregate score is a summation of
assigned scores. In another embodiment, combination of assigned
scores involves performing mathematical operations on the assigned
scores before combining them into an aggregate score. In certain,
embodiments, the aggregate score is also referred to herein as the
"predictive score."
[0054] The term "biomarker" refers to a measurable entity of the
present invention that has been determined to be predictive of PI3K
and mTOR combinatorial inhibitor therapy effects on a cancer.
Biomarkers can include, without limitation, nucleic acids and
proteins, including those shown in the Tables, the Examples, the
Figures, and otherwise described herein. As described herein, any
relevant characteristic of a biomarker can be used, such as the
copy number, amount, activity, location, modification (e.g.,
phosphorylation), and the like.
[0055] A "blocking" antibody or an antibody "antagonist" is one
which inhibits or reduces at least one biological activity of the
antigen(s) it binds. In certain embodiments, the blocking
antibodies or antagonist antibodies or fragments thereof described
herein substantially or completely inhibit a given biological
activity of the antigen(s).
[0056] The term "body fluid" refers to fluids that are excreted or
secreted from the body as well as fluids that are normally not
(e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma,
cerebrospinal fluid, cerumen and earwax, cowper's fluid or
pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate,
interstitial fluid, intracellular fluid, lymph, menses, breast
milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum,
sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous
humor, vomit).
[0057] The terms "cancer" or "tumor" or "hyperproliferative" refer
to the presence of cells possessing characteristics typical of
cancer-causing cells, such as uncontrolled proliferation,
immortality, metastatic potential, rapid growth and proliferation
rate, and certain characteristic morphological features. Unless
otherwise stated, the terms include metaplasias. In some
embodiments, such cells exhibit such characteristics in part or in
full due to the reduced expression, activity, and/or loss of PTEN.
In other embodiments, such cells exhibit such characteristics in
part or in full due to the reduced expression, activity, and/or
loss of PTEN and p53 Cancer cells are often in the form of a tumor,
but such cells may exist alone within an animal, or may be a
non-tumorigenic cancer cell, such as a leukemia cell. As used
herein, the term "cancer" includes premalignant as well as
malignant cancers. Cancers include, but are not limited to, B cell
cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia,
the heavy chain diseases, such as, for example, alpha chain
disease, gamma chain disease, and mu chain disease, benign
monoclonal gammopathy, and immunocytic amyloidosis, melanomas,
breast cancer, lung cancer, bronchus cancer, colorectal cancer,
prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer,
urinary bladder cancer, brain or central nervous system cancer,
peripheral nervous system cancer, esophageal cancer, cervical
cancer, uterine or endometrial cancer, cancer of the oral cavity or
pharynx, liver cancer, kidney cancer, testicular cancer, biliary
tract cancer, small bowel or appendix cancer, salivary gland
cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma,
chondrosarcoma, cancer of hematologic tissues, and the like. Other
non-limiting examples of types of cancers applicable to the methods
encompassed by the present invention include human sarcomas and
carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor,
testicular cancer, lung carcinoma, small cell lung carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute
lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,
promyelocytic, myelomonocytic, monocytic and erythroleukemia);
chronic leukemia (chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia); and polycythemia vera, lymphoma
(Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, and heavy chain disease. In some
embodiments, cancers are epithelial in nature and include but are
not limited to, bladder cancer, breast cancer, cervical cancer,
colon cancer, gynecologic cancers, renal cancer, laryngeal cancer,
lung cancer, oral cancer, head and neck cancer, ovarian cancer,
pancreatic cancer, prostate cancer, or skin cancer. In other
embodiments, the cancer is breast cancer, prostate cancer, lung
cancer, or colon cancer. In still other embodiments, the epithelial
cancer is non-small-cell lung cancer, nonpapillary renal cell
carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous
ovarian carcinoma), or breast carcinoma. The epithelial cancers may
be characterized in various other ways including, but not limited
to, serous, endometrioid, mucinous, clear cell, Brenner, or
undifferentiated.
[0058] In some embodiments, the cancer is "an epithelial cancer,"
which refers to cancers developed from an epithelial cell/tissue
(e.g., epithelium) origin. Epithelial tissues line the cavities and
surfaces of blood vessels and organs throughout the body. There are
three principal shapes of epithelial cell: squamous, columnar, and
cuboidal. These can be arranged in a single layer of cells as
simple epithelium, either squamous, columnar, cuboidal,
pseudo-stratified columnar or in layers of two or more cells deep
as stratified (layered), either squamous, columnar or cuboidal. All
glands are made up of epithelial cells. Malfunction of any of these
different types of epithelial cells may result in a carcinoma, or
an epithelial cancer. Different types of epithelial cancers,
according to their different cell origins, include, for example,
squamous cell carcinoma (starting in squamous cells, which are the
flat, surface covering cells found in areas such as the skin or the
lining of the throat or food pipe (oesophagus)), adenocarcinoma
(starting in glandular cells called adenomatous cells that produce
fluids to keep tissues moist), transitional cell carcinoma
(starting in cells that can stretch as an organ expands and make up
tissues called transitional epithelium. An example is the lining of
the bladder.), basal cell carcinoma (starting from the deepest
layer of skin cells), etc. Among them, basal cell cancer grows
slowly and can damage the tissue around it but is unlikely to
spread to distant areas or result in death (Cakir et al. (2012)
Facialplasic surgery clinics of North America. 20:419-422). It
often appears as a painless raised area of skin, that may be shiny
with small blood vessel running over it or may present as a raised
area with an ulcer. Squamous-cell skin cancer is more likely to
spread (Cakir et al. (2012), supra). It usually presents as a hard
lump with a scaly top but may also form an ulcer Exemplary
epithelial cancers include, at least, lung cancers (e.g.,
non-small-cell lung cancer), nonpapillary renal cell carcinoma,
cervical carcinoma, ovarian carcinoma (e.g., serous ovarian
carcinoma), breast carcinoma, bladder cancers, colonrectal cancers,
prostate cancers, etc. The epithelial cancers may be characterized
in various other ways including, but not limited to, serous,
endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
Epithelial cancers may be caused by exposure to ultraviolet
radiation from the Sun. Decreasing exposure to ultraviolet
radiation and the use of sunscreen (usually containing zinc oxide
and/or titanium oxide) are appear to be effective methods of
preventing squamous-cell skin cancer. The effect to basal cell
cancer is unknown. Other treatments generally include surgical
removal and, less commonly, radiation therapy or topical
medications such as fluorouracil. Topical chemotherapy might be
indicated for large superficial basal-cell carcinoma and other
epithelial cancers. For low-risk diseases, radiation therapy
(external beam radiotherapy or brachytherapy), topical chemotherapy
(e.g., imiquimod or 5-fluorouracil) and cryotherapy (e.g., freezing
the cancer off) can provide adequate control. However, they may
have lower overall cure rates than certain type of surgery. Other
modalities of treatment such as photodynamic therapy, topical
chemotherapy, electrodessication and curettage can be found for
basal-cell carcinomas and squamous-cell carcinomas. Mohs'
micrographic surgery (Mohs surgery) is a technique used to remove
the cancer with the least amount of surrounding tissue and the
edges are checked immediately to see if tumor is found. In the case
of disease that has spread (metastasized), further surgical
procedures or chemotherapy may be required. Chimeric antigen
receptor (CAR) or T cell receptor (TCR) gene therapies have been
found promising for epithelial cancers, performed by first
harvesting mononuclear cells from the peripheral blood of the
patient through a leukapheresis procedure. T cells from the
leukapheresis product are then transduced with a viral vector other
gene transfer platform encoding a CAR to TCR. The genetically
engineered T cells are expanded in the laboratory and administered
to the patient intravenously. Lymphocyte-depleting chemotherapy may
be given prior to cell infusion to enhance engraftment and function
of the engineered T cells. T cell infusion may be followed by
systemic administration of cytokines, such as interleukin-2, to
support the T cells. CARs are synthetic molecules composed of
antibody single-chain variable fragments (ScFv) that bind to a
target tumor antigen, and domains from CD3 signaling chains and T
cell costimulatory receptor molecules that provide intracellular
signaling. For a review of CARs and TCRs, see Hinrichs (2016) Clin
Cancer Res. 22:1559-1564. Many types of epithelial derived tumors
have an elevated level of Notch, which is also associated with poor
outcomes. A new group of therapies target stem-like cells in
epithelial cancers, identifying multiple druggable targets and
pathways. For a review of exemplary treatments and clinical trials,
see Ahmed et al. (2017) Stem Cells 35:839-850. For example, Wnt
inhibitors affecting many cancer sten cekk (CSC) regulators (such
as Hedgehog, Notch, PI3K/Akt/mTOR pathway, etc.), such as Curcumin,
analog GO-Y030, and diflurinated curcumin (CDF), have been tested
in Phase 2 for colon CSC, pancreatic CSC, and breast CSC (Naujokat
and Laufer (2013) J. Cancer Res. Updates 2:36-67; Ramasamy et al.
(2015) Cancer Cell Int. 15. doi: 10.1186/s12935-015-0241-x).
Multiple compounds treating breast cancers, lung cancers,
colonrectal cancers, and pancreatic cancers are also summarized in
Ahmed et al. (2017), supra.
[0059] In some embodiments, the cancer is "triple negative breast
cancer" or "TNBC," which refers to breast cancers that are estrogen
receptor (ER) negative, progesterone receptor (PR) negative, and
human epidermal growth factor receptor 2 (HER-2) negative (Pegram
et al. (1998) J. Clin. Oncol. 16:2659-2671; Wiggans et al. (1979)
Cancer Chemother. Pharmacol. 3:45-48; Carey et al. (2007) Clin.
Cancer Res. 13:2329-2334). Several subtypes of TNBC are known,
including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a
mesenchymal (M), a mesenchymal stem-like (MSL), and a luminal
androgen receptor (LAR) subtype (see, for example, U.S. Pat. Publ.
2014/0303133). Determination of TNBC or a subtype thereof can be pe
accomplished using well-known and standard techniques, such as
immunohistochemical and/or nucleic acid analysis of ER, PR, and
HER-2 receptor status (Chebil et al. (2003) Acta Oncol. 42:43-47;
Chebil et al. (2003) Acta Oncol. 42:719-725; Yamashita et al.
(2006) Rinsho Byori 54:27-30; Schaller et al. (2001) Ann. Oncol.
12:S97-S100; and Kallioniemi et al. (1992) Proc. Natl. Acad. Sci.
U.S.A. 89:5321-5325).
[0060] TNBCs constitute 10-20% of all breast cancers, more
frequently affect younger patients, and are more prevalent in
African-American women (Morris et al. (2007) Cancer 110:876-884).
TNBC tumors are generally larger in size, are of higher grade, have
lymph node involvement at diagnosis, and are biologically more
aggressive (Haffty et al. (2006) J. Clin. Oncol. 24:5652-5657).
Despite having higher rates of clinical response to presurgical
(neoadjuvant) chemotherapy, TNBC patients have a higher rate of
distant recurrence and a poorer prognosis than women with other
breast cancer subtypes (Haffly et al. (2006) J. Clin. Oncol.
24:5652-5657; Dent et al. (2007) Clin. Cancer Res. 13:4429-4434).
Less than 30% of women with metastatic TNBC survive 5 years, and
almost all die of their disease despite adjuvant chemotherapy,
which is the mainstay of treatment (Dent et al. (2007) Clin. Cancer
Res. 13:4429-4434).
[0061] Two of the three major modalities for treating hormone
receptor-positive breast cancers (i.e., endocrine and biologic) do
not apply to TNBC due to the lack of hormone receptor expression.
Thus, well-established therapies, such as selective ER modulation
using tamoxifen or anti-estrogens, aromatase inhibitors,
nonsteroidal drugs (e.g., letrozol, anastrozol, and vostrozol),
steroidal drugs (e.g., exemestane), ovarian ablation surgery,
ovarian ablation radiotherapy, LHRH analog therapy, anti-HER-2
antibodies, anti-ER antibodies, anti-PR antibodies, and the like,
are generally ineffective against TNBC. The general standard of
care for treating TNBC is therefore chemotherapy. Frequently
applied chemotherapeutic drugs in breast cancer are drugs from the
anthracycline class, the taxane class and to a lower extent
antimetabolites (e.g., capecitabine, gemcitabine, alkylating
agents, vinca alkaloids, and the like). These drugs are used in two
basic applications schemes. The drugs can be applied as single
agents in a sequential fashion or they can be used in a combination
regime. The two treatment sub-modalities can be combined to some
extent. The anthracyclines, and especially doxorubicin and
epirubicin, have been shown to be active agents in the treatment of
breast cancer and anthracycline-containing combination regimes are
common first line treatments in patients who have not received
anthracyclines in an adjuvant setting. Common combination treatment
consists for example of doxorubicin/epirubicin plus
cyclophosphamide, doxorubicin/epirubicin plus cyclophosphamide and
5-fluorouracil, or combinations of anthracyclines and capecitabine
or gemcitabine. With the common use of anthracyclines in early
stages of breast cancer treatment, the likelihood of anthracycline
resistant forms of breast cancer, however, increases (Bernard-Marty
et al. (2004) Oncologist 9:617-632).
[0062] In certain embodiments, the cancer is "ovarian cancer." The
ovarian cancer can be a type of ovarian cancer, such as epithelia
ovarian cancer, or a sub-type thereof, such as serous ovarian
cancer. Ovarian cancer ranks as the fifth most common cancer in
women and has the highest mortality rate among gynecologic
malignancies (U.S. Pat. Publ. 2016/0097102Suh et al. (2010) Exp.
Rev. Mol. Diagn. 10:1069-1083; Landen et al. (2008) J. Clin. Oncol.
26:995-1005). Although the 5-year survival rate of ovarian cancer
is around 90% when detected in early stages (I/II), nearly 80% of
the new cases are diagnosed in advanced stages (III/IV) because of
the asymptomatic nature of the disease at stage I and early stage
II. Unfortunately, the 5-year survival rate of advanced ovarian
cancer is as low as 11% (Altekruse et al., SEER Cancer Statistics
Review, 1975-2007, National Cancer Institute. Bethesda, Md.).
[0063] Generally, ovarian cancers are grouped into 3 major
categories: (1) epithelial tumors (tumors arising from cells that
line or cover the ovaries); (2) germ cell tumors (tumors that
originate from cells that are destined to form eggs within the
ovaries; and (3) sex cord-stromal cell tumors (tumors that begin in
connective cells that hold the ovaries together and produce female
hormones). The most common ovarian cancers are epithelial tumors,
which account for about 90% of all ovarian cancers. Ovarian
epithelial tumors are divided into subtypes which include serous,
papillary serous, endometrioid, mucinous and clear cell tumors.
[0064] The serous subtype of ovarian carcinoma accounts for
approximately 60-80% of ovarian cancer cases and exhibits the most
aggressive histology (Levanon et al. (2008) J. Clin. Oncol.
26:5284-5293). Fewer than 25% of serous ovarian cancer cases are
detected at an early stage (stages I and II), which reflects grimly
on survival figures (Seidman et al. (2004) Int. J. Gynecol. Pathol.
23:41-44). High-grade serous carcinoma involves the surface of the
ovary, often bilaterally, and the peritoneal membranes, with rapid
onset of carcinomatosis, a fact that restricts the surgical options
to debulking only (Levanon et al. (2008) J. Clin. Oncol.
26:5284-5293). Despite the introduction of taxanes to therapeutic
protocols and the prolonged survival with intraperitoneal
chemotherapy administration, there has been little progress in
improving cure rates, a parameter that is still solely dependent on
the disease stage at the time of presentation (Levanon et al.
(2008) J. Clin. Oncol. 26:5284-5293).
[0065] Papillary serous carcinoma of the ovary is one of the most
common and lethal malignant tumors (Tong et al. (2007) Modern
Pathol. 20:856-863).
[0066] Papillary serous histology accounts for 75% of ovarian
cancers and its histological pattern simulates the lining of the
fallopian tube (Jelovac and Armstrong (2011) CA 61:183-203). Most
cases of papillary serous ovarian cancer are diagnosed at advanced
stages, when the tumors have already metastasized (Kim et al.
(2012) Proc. Natl. Acad. Sci. USA 109:3921-3926). Despite the
steady improvement of surgery and chemotherapy, greater than 90% of
women with advanced ovarian cancers die after relapse (Bukowski et
al. (2007) Semin. Oncol. 34:S1-S15). Early detection of these
high-grade serous carcinomas is thus key to reducing ovarian cancer
deaths (Bast et al. (2009) Nat. Rev. Cancer 9:415-428).
[0067] Ovarian endometrioid carcinomas account for only 10% of
ovarian carcinomas (McConechy et al. (2007) Modern Pathol.
27:128-134). The majority of ovarian endometrioid carcinomas are
low-grade carcinomas with good prognosis (Chen et al. (2005) Modern
Pathol. 18:903-911)
[0068] The mucinous cell type accounts for approximately 10% of all
primary epithelial ovarian carcinomas (Chan et al. (2008) Gynecol.
Oncol. 109:370-376). Most mucinous epithelial ovarian carcinomas
are diagnosed early (International Federation of Gynecology and
Obstetrics (FIGO) stages I-IIA) and confined to one ovary. In stage
1, mucinous epithelial ovarian carcinomas, the 5-year disease-free
survival rate is about 90%, which is slightly better than the 76%
observed for patients with serous epithelial ovarian carcinomas
(Vergote et al. (2001) Lancet 357:176-182). Less frequently,
primary mucinous epithelial ovarian carcinoma is associated with
peritoneal carcinomatosis and/or extraperitoneal metastases (FIGO
stages IIB-IV). Unlike FIGO stage I tumors, advanced mucinous
epithelial ovarian carcinomas reportedly have poorer prognoses than
serous epithelial ovarian carcinomas (Omura et al. (1991) J. Clin.
Oncol. 9:1138-1150; Teramukai et al. (2007) J. Clin. Oncol.
25:3302-3306).
[0069] Ovarian clear cell adenocarcinomas account for <5% of all
ovarian malignancies and 3.7-12.1% of all epithelial ovarian
carcinomas (Tan and Kaye (2007) J. Clin. Pathol. 60:355-360).
Compared to other epithelial ovarian cancer (EOC) subtypes, when at
an advanced stage, they are associated with a poorer prognosis and
are relatively resistant to conventional platinum-based
chemotherapy (Sugiyama et al. (2000) Cancer 88:2584-2589). By
contrast, early-stage clear cell ovarian cancer carries a
relatively good prognosis (Tan and Kaye (2007) J. Clin. Pathol.
60:355-360). Hence, early detection is the key to improve prognosis
and reduce deaths associated with this type of ovarian cancer.
[0070] The process used to determine whether ovarian cancer has
spread within the ovaries or to other parts of the body (i.e.,
metastasized) is called staging. It is important to determine the
stage of ovarian cancer because the stage will determine the type
of treatment plan selected to combat the disease. The results of
tests used to diagnose ovarian cancer are often also used to stage
the disease. Such tests include ultrasound, computerized tomography
(CT) scan, positron emission tomography (PET) scan, magnetic
resonance imaging (MRI), X-ray and biopsy. Ovarian cancer staging
guidelines have been developed by the International Federation of
Gynecologists and Obstetricians (FIGO). The FIGO staging system for
ovarian cancer is well-known in the art.
[0071] In addition to staging, an ovarian tumor can also be
described by grade (G) GX, GB, and G1-G4. Grading determines how
similar ovarian cancer tissue is to normal tissue. Tumor grade is
determined by microscopic examination of cancer tissue; with
healthy cells appearing as well-differentiated That is, the more
differentiated the ovarian tumor, the better the prognosis. The
ovarian cancer grading system is well-known in the art.
[0072] Serous ovarian cancer is not graded in this way and only
considers a low-grade and a high-grade classification. Low-grade
serous carcinomas exhibit low-grade nuclei with infrequent mitotic
figures. They evolve from adenofibromas or borderline tumors, have
frequent mutations of the KRAS, BRAF, or ERBB2 genes, and can lack
TP53 mutations (Type I pathway). Low-grade tumors are indolent and
have better outcome than high-grade tumors. In contrast, high-grade
serous carcinomas have high-grade nuclei and numerous mitotic
figures (Vang et al. (2009) Adv. Anat. Pathol. 16:267-282).
[0073] A number of well-known and standard-of-care therapies are
available to treat ovarian cancer. Due to the lack of effective
screening programs, ovarian cancer is diagnosed at an early stage
only in about 25% of cases (Kim et al. (2012) J. Exp. Clin. Cancer
Res. 31:14). In most of these cases, surgery is able to cure the
disease, and the five-year survival rate for early-stage (stage I
or II) ovarian cancer is around 90% (Hennessy et al. (2009) Lancet
374:1371-1382). Adjuvant chemotherapy for early stage ovarian
cancer is still controversial, but some studies have shown its
benefit under confined conditions. According to these studies,
patients with IA or IB FIGO stage, non-clear-cell histology,
well-differentiated (G1) tumors, and an "optimal" surgery (i.e.,
performed according to international guidelines, with pelvic and
retroperitoneal assessment), appear not to benefit from
chemotherapy (Trimbos et al. (2003) J. Natl. Cancer Inst.
95:105-112). Thus, it is commonly believed that, at least in these
cases, chemotherapy can probably be avoided and patients can be
advised to undergo clinical and instrumental follow-up. In all the
other (early stage) patients, (adjuvant) chemotherapy is indicated
(Hennessy et al. (2009) Lancet 374:1371-1382).
[0074] By contrast, the standard treatment for patients with
advanced ovarian cancer is maximal surgical cytoreduction (i.e.,
total abdominal hysterectomy, bilateral salpingo-oophorectomy,
pelvic and para-aortic lymphadenectomy and omentectomy) followed by
systemic platinum-based chemotherapy (e.g., cisplatin followed by
carboplatin-based combinations, cisplatin with paclitaxel,
cisplatin with cyclophosphamide, cisplatin with doxorubicin, etc.).
The expected 5-year survival for these patients is 10-30% (Hennessy
el al. (2009) Lancet 374:1371-1382). The concept of primary
debulking surgery is to diminish the residual tumor burden to a
point at which adjuvant therapy will be optimally effective. The
percentage of patients with advanced ovarian cancer who can
optimally undergo cytoreductive surgery seems to range from 17%-87%
(Ramirez et al. (2011) Cancer Control 18:22-30). This percentage
can largely depend on the experience of the surgeon.
[0075] In addition, a deeper knowledge of ovarian cancer biology
has led to the identification of multiple molecular targets, such
as growth factor receptors, signal transduction pathways, cell
cycle regulators, and angiogenic mechanisms (Kim et al. (2012) J.
Exp. Clin. Cancer Res. 31:14). For example, VEGF expression is
higher in ovarian cancer tumors than in normal ovarian tissue or
benign ovarian tumors, and increasing VEGF expression in either
cytosolic fractions derived from ovarian cancer tumors or serum
VEGF levels in preoperative serum is considered to be associated
with advanced stage and poor prognosis (Kim et al. (2012) J. Exp.
Clin. Cancer Res. 31:14). In order to inhibit the VEGF pathway,
there are two primary strategies: (1) inhibition of the VEGF ligand
with antibodies or soluble receptors and (2) inhibition of the VEGF
receptor (VEGFR) with tyrosine kinase inhibitors (TKI5), or
receptor antibodies. Of the VEGF targeting therapies, the one most
employed has been inhibition of the VEGF ligand with bevacizumab.
However, oral inhibitors of the VEGF receptor (VEGFR) tyrosine
kinase have been shown to have activity in patients with recurrent
ovarian cancer, resulting in tumor responses and stabilization of
disease, delaying tumor progression (Friedlander et al. (2010)
Gynecol. Oncol. 119:32-37; Ledermann et al. (2011) J. Clin. Oncol.
29:3798-3804; Matulonis et al. (2009) J. Clin. Oncol. 27:5601-5606:
Biagi et al. (2011) Ann. Oncol. 22:335-340; Matei et al. (2011) J.
Clin. Oncol. 29:69-75).
[0076] Other targets to inhibit include the epidermal growth factor
receptor (EGFR) overexpressed in up to 70% of ovarian cancer
patients (Kohler et al. (1992) Eur. J. Cancer 28a:1432-1437) such
as by erlotinib treatment; insulin growth factor 1 (IGF 1) is
involved in the inhibition of apoptosis, tumor progression and
metastase, such as by aMG479 monoclonal antibody or OSI-906
treatment; poly (ADP-ribose) polymerase (PARP) (Rouleau et al.
(2010) Nat. Rev. Cancer 10:293-301), such as by olaparib treatment
(Fong et al. (2010) J. Clin. Oncol. 28:2512-2519).
[0077] In some embodiments, the cancer is "prostate cancer," which
refers to cancers that occur in male's prostate. Prostate cancer is
one of the most common types of cancer in men. Prostate cancer
usually grows slowly and initially remains confined to the prostate
gland, where it may not cause serious harm. While some types of
prostate cancer grow slowly and may need minimal or no treatment,
other types are aggressive and can spread quickly. Prostate cancer
that is detected early--when ifs still confined to the prostate
gland--has a better chance of successful treatment. In the vast
majority of cases, the prostate cancer starts in the gland
cells--this is called adenocarcinoma.
[0078] The stage of prostate cancers, or how far they have spread
helps to define the prognosis and treatment. The most common system
for determining cancer stages is the TNM (Tumor/Nodes/Metastases).
This involves defining the size of the tumor, how many lymph nodes
are involved, and whether there are any other metastases. When
defining with the TNM system, it is crucial to distinguish between
cancers that are still restricted just to the prostate, and those
that have spread elsewhere. Clinical T1 and T2 cancers are found
only in the prostate, and nowhere else, while T3 and T4 have spread
outside the prostate. There are many ways to find out whether the
cancer has spread. Computer tomography will check for spread inside
the pelvis, bone scans will decide whether the cancer has spread to
the bones, and endorectal coil magnetic resonance imaging will
evaluate the prostatic capsule and the seminal vesicles.
[0079] Prostate cancer may cause no signs or symptoms in its early
stages. Prostate cancer that is more advanced may cause signs and
symptoms such as: trouble urinating (little or no urine output,
difficulty starting (straining) or stopping the urine stream,
decreased force in the stream of urine, increased frequency for
urine), blood in urine or semen and/or deep back, discomfort in the
hip, pelvic, or abdominal area, bone pain, erectile dysfunction,
urinary problems, frequent urination, dribbling, pain or burning
during urination), erectile dysfunction or painful ejaculation.
Other symptoms may include weight loss, bone pain (often in the
spine (vertebrae), pelvis, or ribs), leg weakness (if cancer has
spread to the spine and compressed the spinal cord), urinary
incontinence (if cancer has spread to the spine and compressed the
spinal cord), fecal incontinence (if cancer has spread to the spine
and compressed the spinal cord) and lower extremity swelling. In
addition to tumor translocation, prostate cancer is related to, at
least, BPH (benign prostatic hyperplasia), acute and chronic
bacterial prostatitis and chronic prostatitis (non-bacterial).
[0080] The specific causes for prostate cancers are not well know,
although both genetic and environmental factors may be relevant.
There are so many possible factors, including age, race, lifestyle,
medications, and genetics, to name a few. Age is considered as the
primary risk factor The older a man is, the higher is his risk.
Prostate cancer is rare among men under the age of 45, but much
more common after the age of 50. Among genetic factors, BRCA1 and
BRCA2 have been reported as relevant genes for prostate cancers
(Castro el al (2013)J. Clin. Oncol. 31:1748-1757). Many other loci
on chromosomes are also related to prostate cancers, including
genes such as MSMB, TBP2, JAZF1, PNE3, IL16, LATK2, KLK2, KLK3, and
CDH13 (Thomas et al. (2008) Nat Genet. 40:310-315; Eeles et al.
(2008) Nat Genet. 40:316-321). PRSS3/mesotrypsin is also a
therapeutic target for metastatic prostate cancer (Hockla et al.
(2008) Mol Cancer Res. 10:1555-1566). Men with chronic inflammation
in non-cancerous prostate tissue were found to have nearly double
the risk of developing prostate cancer, representing a clear
association between prostate inflammation and prostate cancer
(Gurel et al. (2014) Cancer Epidemiol Biomarkers Prev. 23:847-856).
Diet, obesity, sexually transmitted diseases (STDs, such as
gonorrhea) are also risk factors.
[0081] Prostate screening tests might include, at least, digital
rectal exam (DRE) and prostate-specific antigen (PSA) test. Further
diagnostic methods include ultrasound detection and/or prostate
biopsy. A Gleason score is used to represent the aggressiveness of
the cancer, which combines two numbers and usually ranges from 2
(nonaggressive cancer) to 10 (very aggressive cancer). Further
scans (such as bone scan, ultrasound, computerized tomography (CT)
scan, magnetic resonance imaging (MRI), positron emission
tomography (PET) scan, etc.) may be used to determine the degree of
the prostate cancer spreading to other tissues and/or organs.
[0082] Treatment for the early stage of prostate cancers (e.g.,
when the cancer tissues are small and contained/localized) may
include, at least: 1) watchful waiting--not immediate treatment is
carried out. PSA blood levels are regularly monitored; 2) radical
prostatectomy--the prostate is surgically removed; 3)
brachytherapy--radioactive seeds are implanted into the prostate;
4) conformal radiotherapy--the radiation beams are shaped so that
the region where they overlap is as close to the same shape as the
organ or region that requires treatment, thus minimizing healthy
tissue exposure to radiation; and 5) intensity modulated
radiotherapy--beams with variable intensity are used. An advanced
form of conformal radiotherapy usually delivered by a
computer-controlled linear accelerator.
[0083] Treatment recommendations really depend on individual cases.
In general, if there is a good prognosis and the cancer is in its
early stages, all options can be considered. However, they all have
their advantages and disadvantages. The patient should discuss
available options thoroughly with his doctor. More aggressive, or
advanced, prostate cancer may require a combination of radiotherapy
and hormone therapy (e.g., preventing testosterone production).
Other surgery methods include, at least, salvage radical
prostatectomy, image-guided, intensity-modulated radiation therapy
(TG-IMRT), stereotactic high-precision radiosurgery (similar to
CyberKnife), stereotactic hypofractionated accelerated radiation
therapy (SHARP), and low-dose-rate permanent seed implants and
high-dose-rate temporary seed implants (both forms of
brachytherapy). For men with small, localized prostate tumors,
focal therapy, or partial gland ablation, can be used.
Chemotherapies and immunotherapies are also important treatment
methods. For example, Sipuleucel-T (APC8015, trade name
Provenge.RTM.) is a cell-based cancer immunotherapy for prostate
cancer. Other immunotherapies for prostate cancers include, at
least, PROSTVAC (similar to Provenge.RTM. in that it uses
re-engineered cells to attack prostate cancer cells. This vaccine
is targeted to the PSA antigen and has been tested for treating
prostate cancers alone or in combination with ipilimumab
(Yervoy.RTM.)), GVAX (alone or in combination with ipilimumab
(Yervoy.RTM.)), etc.
[0084] In certain embodiments, the cancer is a "PI3Kbeta-dependent
cancer," which can refer to a cancer that is functionally dependent
on PI3Kbeta. For instance, even if the expression level of PI3Kbeta
(e.g., PI3Kbeta mRNA, PI3Kbeta protein, newly synthesized PI3Kbeta
protein, etc.) in a tumor tissue is comparable to its expression
level in normal tissue, a cancer is PI3Kbeta-dependent if
inhibition of the PI3Kbeta mRNA and/or protein, directly or
indirectly such as by using RNAi or any other means, or deletion of
the PI3Kbeta gene (e.g., by knock-out or clustered regularly
interspaced short palindromic repeats (CRISPR) technology) leads to
inhibition of oncogenesis, tumor cell proliferation, tumor
metastasis or induces tumor cell differentiation. The term
"PI3Kbeta-dependent cancer" also refers to a cancer in which
PI3Kbeta is expressed (e.g., PI3Kbeta mRNA, PI3Kbeta protein, newly
synthesized PI3Kbeta protein, etc.) at a significantly higher level
than the normal amount of PI3Kbeta expressed in a non-cancerous
cell of the same cell type as the PI3Kbeta-dependent cancer. A
significantly modulated amount of PI3Kbeta relative to the normal
amount of PI3Kbeta is an amount less than or greater than,
respectively, the standard error of the assay employed to assess
amount, and preferably at least 5%, 10%, 15% 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%,
300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more than
the normal (control) amount. Alternately, the amount of the
biomarker in the subject can be considered "significantly"
modulated relative to the normal (control) amount if the amount is
at least about 20%, 25%, 30%, 35%, 40% 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%,
160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 5000, 600%,
700%, 800%, 900%, 1000% or more, higher or lower, respectively,
than the normal (control) amount of PI3Kbeta.
[0085] The term "micrometastasis" as used herein is preferably
defined as a group of confluent cancer cells measuring from greater
than 0.2 mm and/or having greater than 200 cells to 2 mm in maximum
width. More preferably "micrometastasis" is defined as a group of
confluent cancer cells from 0.2 mm to 2 mm in maximum width (see
Edge et al. (2010) AJCC Cancer StagingAamal and Handbook (7th
ed.)). An alternative preferred definition of"micrometastasis" is a
confluent group of at least 1000 cancer cells and at least 0.1 mm
in widest dimension up to 1 mm in widest dimension. Micrometastasis
is generally not visible in standard contrast MRI imaging or other
clinical imaging techniques. However, in certain cancers,
radioactive antibodies directed to tumor selective antigens (e.g.,
Her2 for breast cancer metastasis) allows for visualization of
micrometastasis. Other indirect detection methods include contrast
media leakage at brain micrometastasis sites due to VEGF induced
vascular leakage (Yano et al. (2000) Cancer Res. 60:4959-49067;
U.S. Pat. Publ. 2015/0352113). More sensitive imaging techniques
may also be applied to detect micrometastases. For example, blood
volume may be imaged by MRI using the alternative contrast agent,
USPIO (Molday Iron, Biopal, Worcester, Mass.) to detect
micrometastasis (Yin et al. (2009) Clin. Exp. Metastasis.
26:403-414).
[0086] The term "coding region" refers to regions of a nucleotide
sequence comprising codons which are translated into amino acid
residues, whereas the term "noncoding region" refers to regions of
a nucleotide sequence that are not translated into amino acids
(e.g., 5' and 3' untranslated regions).
[0087] The term "complementary" refers to the broad concept of
sequence complementarity between regions of two nucleic acid
strands or between two regions of the same nucleic acid strand. It
is known that an adenine residue of a first nucleic acid region is
capable of forming specific hydrogen bonds ("base pairing") with a
residue of a second nucleic acid region which is antiparallel to
the first region if the residue is thymine or uracil. Similarly, it
is known that a cytosine residue of a first nucleic acid strand is
capable of base pairing with a residue of a second nucleic acid
strand which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion.
[0088] The term "control" refers to any reference standard suitable
to provide a comparison to the expression products in the test
sample. In one embodiment, the control comprises obtaining a
"control sample" from which expression product levels are detected
and compared to the expression product levels from the test sample.
Such a control sample may comprise any suitable sample, including
but not limited to a sample from a control cancer patient (can be
stored sample or previous sample measurement) with a known outcome;
normal tissue or cells isolated from a subject, such as a normal
patient or the cancer patient, cultured primary cells/tissues
isolated from a subject such as a normal subject or the cancer
patient, adjacent normal cells/tissues obtained from the same organ
or body location of the cancer patient, a tissue or cell sample
isolated from a normal subject, or a primary cells/tissues obtained
from a depository. In another preferred embodiment, the control may
comprise a reference standard expression product level from any
suitable source, including but not limited to housekeeping genes,
an expression product level range from normal tissue (or other
previously analyzed control sample), a previously determined
expression product level range within a test sample from a group of
patients, or a set of patients with a certain outcome (for example,
survival for one, two, three, four years, etc.) or receiving a
certain treatment (for example, standard of care cancer therapy).
It will be understood by those of skill in the art that such
control samples and reference standard expression product levels
can be used in combination as controls in the methods of the
present invention. In one embodiment, the control may comprise
normal or non-cancerous cell/tissue sample. In another preferred
embodiment, the control may comprise an expression level for a set
of patients, such as a set of cancer patients, or for a set of
cancer patients receiving a certain treatment, or for a set of
patients with one outcome versus another outcome. In the former
case, the specific expression product level of each patient can be
assigned to a percentile level of expression, or expressed as
either higher or lower than the mean or average of the reference
standard expression level. In another preferred embodiment, the
control may comprise normal cells, cells from patients treated with
combination chemotherapy, and cells from patients having benign
cancer. In another embodiment, the control may also comprise a
measured value for example, average level of expression of a
particular gene in a population compared to the level of expression
of a housekeeping gene in the same population. Such a population
may comprise normal subjects, cancer patients who have not
undergone any treatment (i.e., treatment naive), cancer patients
undergoing standard of care therapy, or patients having benign
cancer. In another preferred embodiment, the control comprises a
ratio transformation of expression product levels, including but
not limited to determining a ratio of expression product levels of
two genes in the test sample and comparing it to any suitable ratio
of the same two genes in a reference standard; determining
expression product levels of the two or more genes in the test
sample and determining a difference in expression product levels in
any suitable control; and determining expression product levels of
the two or more genes in the test sample, normalizing their
expression to expression of housekeeping genes in the test sample,
and comparing to any suitable control. In particularly preferred
embodiments, the control comprises a control sample which is of the
same lineage and/or type as the test sample. In another embodiment,
the control may comprise expression product levels grouped as
percentiles within or based on a set of patient samples, such as
all patients with cancer. In one embodiment a control expression
product level is established wherein higher or lower levels of
expression product relative to, for instance, a particular
percentile, are used as the basis for predicting outcome. In
another preferred embodiment, a control expression product level is
established using expression product levels from cancer control
patients with a known outcome, and the expression product levels
from the test sample are compared to the control expression product
level as the basis for predicting outcome. As demonstrated by the
data below, the methods of the present invention are not limited to
use of a specific cut-point in comparing the level of expression
product in the test sample to the control.
[0089] The "copy number" of a biomarker nucleic acid refers to the
number of DNA sequences in a cell (e.g., germline and/or somatic)
encoding a particular gene product. Generally, for a given gene, a
mammal has two copies of each gene. The copy number can be
increased, however, by gene amplification or duplication, or
reduced by deletion. For example, germline copy number changes
include changes at one or more genomic loci, wherein said one or
more genomic loci are not accounted for by the number of copies in
the normal complement of germline copies in a control (e.g., the
normal copy number in germline DNA for the same species as that
from which the specific germline DNA and corresponding copy number
were determined). Somatic copy number changes include changes at
one or more genomic loci, wherein said one or more genomic loci are
not accounted for by the number of copies in germline DNA of a
control (e.g., copy number in germline DNA for the same subject as
that from which the somatic DNA and corresponding copy number were
determined).
[0090] The "normal" copy number (e.g., germline and/or somatic) of
a biomarker nucleic acid or "normal" level of expression of a
biomarker nucleic acid or protein is the activity/level of
expression or copy number in a biological sample, e.g., a sample
containing tissue, whole blood, serum, plasma, buccal scrape,
saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a
subject, e.g., a human, not afflicted with cancer, or from a
corresponding non-cancerous tissue in the same subject who has
cancer.
[0091] As used herein, the term "costimulate" with reference to
activated immune cells includes the ability of a costimulatory
molecule to provide a second, non-activating receptor mediated
signal (a "costimulatory signal") that induces proliferation or
effector function. For example, a costimulatory signal can result
in cytokine secretion, e.g., in a T cell that has received a T
cell-receptor-mediated signal. Immune cells that have received a
cell-receptor mediated signal, e.g., via an activating receptor are
referred to herein as "activated immune cells."
[0092] The term "determining a suitable treatment regimen for the
subject" is taken to mean the determination of a treatment regimen
(i.e., a single therapy or a combination of different therapies
that are used for the prevention and/or treatment of the cancer in
the subject) for a subject that is started, modified and/or ended
based or essentially based or at least partially based on the
results of the analysis according to the present invention. One
example is starting an adjuvant therapy after surgery whose purpose
is to decrease the risk of recurrence, another would be to modify
the dosage of a particular chemotherapy. The determination can, in
addition to the results of the analysis according to the present
invention, be based on personal characteristics of the subject to
be treated. In most cases, the actual determination of the suitable
treatment regimen for the subject will be performed by the
attending physician or doctor.
[0093] The term "diagnosing cancer" includes the use of the
methods, systems, and code of the present invention to determine
the presence or absence of a cancer or subtype thereof in an
individual. The term also includes methods, systems, and code for
assessing the level of disease activity in an individual.
[0094] A molecule is "fixed" or "affixed" to a substrate if it is
covalently or non-covalently associated with the substrate such
that the substrate can be rinsed with a fluid (e.g. standard saline
citrate, pH 7.4) without a substantial fraction of the molecule
dissociating from the substrate.
[0095] The term "expression signature" or "signature" refers to a
group of one or more coordinately expressed biomarkers related to a
measured phenotype. For example, the genes, proteins, metabolites,
and the like making up this signature may be expressed in a
specific cell lineage, stage of differentiation, or during a
particular biological response. The biomarkers can reflect
biological aspects of the tumors in which they are expressed, such
as the cell of origin of the cancer, the nature of the
non-malignant cells in the biopsy, and the oncogenic mechanisms
responsible for the cancer. Expression data and gene expression
levels can be stored on computer readable media, e.g., the computer
readable medium used in conjunction with a microarray or chip
reading device. Such expression data can be manipulated to generate
expression signatures.
[0096] "Homologous" as used herein, refers to nucleotide sequence
similarity between two regions of the same nucleic acid strand or
between regions of two different nucleic acid strands. When a
nucleotide residue position in both regions is occupied by the same
nucleotide residue, then the regions are homologous at that
position. A first region is homologous to a second region if at
least one nucleotide residue position of each region is occupied by
the same residue. Homology between two regions is expressed in
terms of the proportion of nucleotide residue positions of the two
regions that are occupied by the same nucleotide residue. By way of
example, a region having the nucleotide sequence 5'-ATTGCC-3' and a
region having the nucleotide sequence 5'-TATGGC-3' share 50%
homology. Preferably, the first region comprises a first portion
and the second region comprises a second portion, whereby, at least
about 50%, and preferably at least about 75%, at least about 90%,
or at least about 95% of the nucleotide residue positions of each
of the portions are occupied by the same nucleotide residue. More
preferably, all nucleotide residue positions of each of the
portions are occupied by the same nucleotide residue.
[0097] The term "immune cell" refers to cells that play a role in
the immune response. Immune cells are of hematopoietic origin, and
include lymphocytes, such as B cells and T cells; natural killer
cells; myeloid cells, such as monocytes, macrophages, eosinophils,
mast cells, basophils, and granulocytes.
[0098] The term "immune checkpoint" refers to a group of molecules
on the cell surface of CD4+ and/or CD8+ T cells that fine-tune
immune responses by down-modulating or inhibiting an anti-tumor
immune response. Immune checkpoint proteins are well-known in the
art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2,
B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B,
PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA,
SIRPalpha, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4,
T1GIT, IDO, CD39, arginase, CD73, and A2aR (see, for example, WO
2012/177624). The term further encompasses biologically active
protein fragment, as well as nucleic acids encoding full-length
immune checkpoint proteins and biologically active protein
fragments thereof. In some embodiment, the term further encompasses
any fragment according to homology descriptions provided
herein.
[0099] Immune checkpoints and their sequences are well-known in the
art and representative embodiments are described below. For
example, the term "PD-1" refers to a member of the immunoglobulin
gene superfamily that functions as a coinhibitory receptor having
PD-L1 and PD-L2 as known ligands. PD-1 was previously identified
using a subtraction cloning based approach to select for genes
upregulated during TCR-induced activated T cell death. PD-1 is a
member of the CD28/CTLA-4 family of molecules based on its ability
to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the
surface of T-cells in response to anti-CD3 (Agata et al. 25 (1996)
Int. Immunol. 8:765). In contrast to CTLA-4, however, PD-1 is also
induced on the surface of B-cells (in response to anti-IgM).
[0100] PD-1 is also expressed on a subset of thymocytes and myeloid
cells (Agata et al. (1996) supra; Nishimura et al. (1996) Int.
Immunol. 8:773).
[0101] The nucleic acid and amino acid sequences of a
representative human PD-1 biomarker is available to the public at
the GenBank database under NM_005018.2 and NP_005009.2 and is shown
in Table 3 (see also Ishida et al. (1992) 20 EMBO J 11:3887;
Shinohara et al. (1994) Genomics 23:704; U.S. Pat. No. 5,698,520).
PD-1 has an extracellular region containing immunoglobulin
superfamily domain, a transmembrane domain, and an intracellular
region including an immunoreceptor tyrosine-based inhibitory motif
(ITIM) (Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al.
(1994) Genomics 23:704; and U.S. Pat. No. 5,698,520) and an
immunoreceptor tyrosine-based switch motif (ITSM). These features
also define a larger family of polypeptides, called the
immunoinhibitory receptors, which also includes gp49B, PIR-B, and
the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997)
Immunol. Today 18:286). It is often assumed that the tyrosyl
phosphorylated ITIM and/or ITSM motif of these receptors interacts
with SH2-domain containing phosphatases, which leads to inhibitory
signals. A subset of these immunoinhibitory receptors bind to MHC
polypeptides, for example the KIRs, and CTLA4 binds to B7-1 and
B7-2. It has been proposed that there is a phylogenetic
relationship between the MHC and B7 genes (Henry et al. (1999)
Immunol. Today 20(6):285-8). Nucleic acid and polypeptide sequences
of PD-1 orthologs in organisms other than humans are well known and
include, for example, mouse PD-1 (NM 008798.2 and NP 032824.1), rat
PD-1 (NM 001106927.1 and NP 001100397.1), dog PD-1 (XM_543338.3 and
XP_543338.3), cow PD-1 (NM_001083506.1 and NP_001076975.1), and
chicken PD-1 (XM_422723.3 and XP_422723.2).
[0102] PD-1 polypeptides are inhibitory receptors capable of
transmitting an inhibitory signal to an immune cell to thereby
inhibit immune cell effector function, or are capable of promoting
costimulation (e.g., by competitive inhibition) of immune cells,
e.g., when present in soluble, monomeric form. Preferred PD-1
family members share sequence identity with PD-1 and bind to one or
more B7 family members, e.g., B7-1, B7-2, PD-1 ligand, and/or other
polypeptides on antigen presenting cells.
[0103] The term "PD-1 activity," includes the ability of a PD-1
polypeptide to modulate an inhibitory signal in an activated immune
cell, e.g., by engaging a natural PD-1 ligand on an antigen
presenting cell, tumor cell or other cell in the tumor
microenvironment. Modulation of an inhibitory signal in an immune
cell results in modulation of proliferation of, and/or cytokine
secretion by, an immune cell. Thus, the term "PD-1 activity"
includes the ability of a PD-1 polypeptide to bind its natural
ligand(s), the ability to modulate immune cell costimulatory or
inhibitory signals, and the ability to modulate the immune
response.
[0104] The term "PD-1 ligand" refers to binding partners of the
PD-1 receptor and includes both PD-L1 (Freeman et al. (2000) J.
Exp. Med. 192:1027) and PD-L2 (Latchman et al. (2001) Nat. Immunol.
2:261). At least two types of human PD-1 ligand polypeptides exist.
PD-1 ligand proteins comprise a signal sequence, and an IgV domain,
an IgC domain, a transmembrane domain, and a short cytoplasmic
tail. Both PD-L1 (See Freeman et al. (2000) J. Exp. Med. 192:1027
for sequence data) and PD-L2 (See Latchman et al. (2001) Nat.
Immunol. 2:261 for sequence data) are members of the B7 family of
polypeptides. Both PD-L1 and PD-T2 are expressed in placenta,
spleen, lymph nodes, thymus, and heart Only PD-L2 is expressed in
pancreas, lung and liver, while only PD-L1 is expressed in fetal
liver. Both PD-1 ligands are upregulated on activated monocytes and
dendritic cells, although PD-L1 expression is broader. For example,
PD-L1 is known to be constitutively expressed and upregulated to
higher levels on murine hematopoietic cells (e.g., T cells, B
cells, macrophages, dendritic cells (DCs), and bone marrow-derived
mast cells) and non-hematopoietic cells (e.g., endothelial,
epithelial, and muscle cells), whereas PD-L2 is inducibly expressed
on DCs, macrophages, and bone marrow-derived mast cells (see Butte
et al. (2007) Immunity 27:111).
[0105] PD-1 ligands comprise a family of polypeptides having
certain conserved structural and functional features. The term
"family" when used to refer to proteins or nucleic acid molecules,
is intended to mean two or more proteins or nucleic acid molecules
having a common structural domain or motif and having sufficient
amino acid or nucleotide sequence homology, as defined herein. Such
family members can be naturally or non-naturally occurring and can
be from either the same or different species. For example, a family
can contain a first protein of human origin, as well as other,
distinct proteins of human origin or alternatively, can contain
homologues of non-human origin. Members of a family may also have
common functional characteristics. PD-1 ligands are members of the
B7 family of polypeptides. The term "B7 family" or "B7
polypeptides" as used herein includes costimulatory polypeptides
that share sequence homology with B7 polypeptides, e.g., with B7-1,
B7-2, B7h (Swallow et al. (1999) Immunity 11:423), and/or PD-1
ligands (e.g., PD-L1 or PD-L2). For example, human B7-1 and B7-2
share approximately 26% amino acid sequence identity when compared
using the BLAST program at NCBI with the default parameters
(Blosum62 matrix with gap penalties set at existence 11 and
extension 1 (See the NCBI website). The term B7 family also
includes variants of these polypeptides which are capable of
modulating immune cell function. The B7 family of molecules share a
number of conserved regions, including signal domains, IgV domains
and the IgC domains. IgV domains and the IgC domains are
art-recognized Ig superfamily member domains. These domains
correspond to structural units that have distinct folding patterns
called Ig folds. Ig folds are comprised of a sandwich of two 0
sheets, each consisting of anti-parallel p strands of 5-10 amino
acids with a conserved disulfide bond between the two sheets in
most, but not all, IgC domains of Ig, TCR, and MHC molecules share
the same types of sequence patterns and are called the C1-set
within the Ig superfamily. Other IgC domains fall within other
sets. IgV domains also share sequence patterns and are called V set
domains. IgV domains are longer than IgC domains and contain an
additional pair of P strands.
[0106] Preferred B7 polypeptides are capable of providing
costimulatory or inhibitory signals to immune cells to thereby
promote or inhibit immune cell responses. For example, B7 family
members that bind to costimulatory receptors increase T cell
activation and proliferation, while B7 family members that bind to
inhibitory receptors reduce costimulation. Moreover, the same B7
family member may increase or decrease T cell costimulation. For
example, when bound to a costimulatory receptor, PD-1 ligand can
induce costimulation of immune cells or can inhibit immune cell
costimulation, e.g., when present in soluble form. When bound to an
inhibitory receptor, PD-1 ligand polypeptides can transmit an
inhibitory signal to an immune cell. Preferred B7 family members
include B7-1, B7-2, B7h, PD-L1 or PD-L2 and soluble fragments or
derivatives thereof. In one embodiment, B7 family members bind to
one or more receptors on an immune cell, e.g., CTLA4, CD28, ICOS,
PD-1 and/or other receptors, and, depending on the receptor, have
the ability to transmit an inhibitory signal or a costimulatory
signal to an immune cell, preferably a T cell.
[0107] Modulation of a costimulatory signal results in modulation
of effector function of an immune cell. Thus, the term "PD-1 ligand
activity" includes the ability of a PD-1 ligand polypeptide to bind
its natural receptor(s) (e.g. PD-1 or B7-1), the ability to
modulate immune cell costimulatory or inhibitory signals, and the
ability to modulate the immune response.
[0108] The term "PD-L1" refers to a specific PD-1 ligand. Two forms
of human PD-L1 molecules have been identified. One form is a
naturally occurring PD-L1 soluble polypeptide, i.e., having a short
hydrophilic domain and no transmembrane domain, and is referred to
herein as PD-LIS (shown in Table 3). The second form is a
cell-associated polypeptide, i.e., having a transmembrane and
cytoplasmic domain, referred to herein as PD-L1M (shown in SEQ ID
NO: 6). The nucleic acid and amino acid sequences of representative
human PD-L1 biomarkers regarding PD-LIM are also available to the
public at the GenBank database under NM_014143.3 and NP_054862.1.
PD-L1 proteins comprise a signal sequence, and an IgV domain and an
IgC domain. The signal sequence of SEQ ID NO: 4 is shown from about
amino acid 1 to about amino acid 18. The signal sequence of SEQ ID
NO: 6 is shown: from about amino acid 1 to about amino acid 18. The
IgV domain of SEQ ID NO: 4 is shown from about amino acid 19 to
about amino acid 134 and the IgV domain of SEQ ID NO: 6 is shown
from about amino acid 19 to about amino acid 134. The IgC domain of
SEQ ID NO: 4 is shown from about amino acid 135 to about amino acid
227 and the IgC domain of SEQ ID NO: 6 is shown from about amino
acid 135 to about amino acid 227. The hydrophilic tail of the PD-L1
exemplified in SEQ ID NO: 4 comprises a hydrophilic tail shown from
about amino acid 228 to about amino acid 245. The PD-L1 polypeptide
exemplified in SEQ ID NO: 6 comprises a transmembrane domain shown
from about amino acids 239 to about amino acid 259 of SEQ ID NO: 6
and a cytoplasmic domain shown from about 30 amino acid 260 to
about amino acid 290 of SEQ ID NO: 6. In addition, nucleic acid and
polypeptide sequences of PD-L1 orthologs in organisms other than
humans are well known and include, for example, mouse PD-L1
(NM_021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and
NP_001178883.1), dog PD-L1 (XM_541302.3 and XP_541302.3), cow PD-L1
(NM_001163412.1 and NP_001156884.1), and chicken PD-L1 (XM_424811.3
and XP_424811.3).
[0109] The term "PD-L2" refers to another specific PD-1 ligand.
PD-L2 is a B7 family member expressed on various APCs, including
dendritic cells, macrophages and bone-marrow derived mast cells
(Zhong et al. (2007) Eur. J. Immunol. 37:2405). APC-expressed PD-L2
is able to both inhibit T cell activation through ligation of PD-1
and costimulate T cell activation, through a PD-1 independent
mechanism (Shin et al. (2005) J. Exp. Med. 201:1531). In addition,
ligation of dendritic cell-expressed PD-L2 results in enhanced
dendritic cell cytokine expression and survival (Radhakrishnan et
al. (2003) J. Immunol. 37:1827; Nguyen et al. (2002) J. Exp. Med.
196:1393). The nucleic acid and amino acid sequences of
representative human PD-L2 biomarkers (e.g., SEQ ID NOs: 7 and 8)
are well known in the art and are also available to the public at
the GenBank database under NM_025239.3 and NP_079515.2. PD-L2
proteins are characterized by common structural elements. In some
embodiments, PD-L2 proteins include at least one or more of the
following domains: a signal peptide domain, a transmembrane domain,
an IgV domain, an IgC domain, an extracellular domain, a
transmembrane domain, and a cytoplasmic domain. For example, amino
acids 1-19 of SEQ ID NO: 8 comprises a signal sequence. As used
herein, a "signal sequence" or "signal peptide" serves to direct a
polypeptide containing such a sequence to a lipid bilayer, and is
cleaved in secreted and membrane bound polypeptides and includes a
peptide containing about 15 or more amino acids which occurs at the
N-terminus of secretory and membrane bound polypeptides and which
contains a large number of hydrophobic amino acid residues For
example, a signal sequence contains at least about 10-30 amino acid
residues, preferably about 15-25 amino acid residues, more
preferably about 18-20 amino acid residues, and even more
preferably about 19 amino acid residues, and has at least about
35-65%, preferably about 38-50%, and more preferably about 40-45%
hydrophobic amino acid residues (e.g., valine, leucine, isoleucine
or phenylalanine). In another embodiment, amino acid residues
220-243 of the native human PD-L2 polypeptide and amino acid
residues 201-243 of the mature polypeptide comprise a transmembrane
domain. As used herein, the term "transmembrane domain" includes an
amino acid sequence of about 15 amino acid residues in length which
spans the plasma membrane. More preferably, a transmembrane domain
includes about at least 20, 25, 30, 35, 40, or 45 amino acid
residues and spans the plasma membrane. Transmembrane domains are
rich in hydrophobic residues, and typically have an alpha-helical
structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%,
90%, 95% or more of the amino acids of a transmembrane domain are
hydrophobic, e.g., leucines, isoleucines, tyrosines, or
tryptophans. Transmembrane domains are described in, for example,
Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19: 235-263. In
still another embodiment, amino acid residues 20-120 of the native
human PD-L2 polypeptide and amino acid residues 1-101 of the mature
polypeptide comprise an IgV domain. Amino acid residues 121-219 of
the native human PD-L2 polypeptide and amino acid residues 102-200
of the mature polypeptide comprise an IgC domain. As used herein,
IgV and IgC domains are recognized in the art as Ig superfamily
member domains. These domains correspond to structural units that
have distinct folding patterns called Ig folds. Ig folds are
comprised of a sandwich of two B sheets, each consisting of
antiparallel (3 strands of 5-10 amino acids with a conserved
disulfide bond between the two sheets in most, but not all,
domains. IgC domains of Ig, TCR, and MHC molecules share the same
types of sequence patterns and are called the Cl set within the Ig
superfamily. Other IgC domains fall within other sets. IgV domains
also share sequence patterns and are called V set domains. IgV
domains are longer than C-domains and form an additional pair of
strands. In yet another embodiment, amino acid residues 1-219 of
the native human PD-L2 polypeptide and amino acid residues 1-200 of
the mature polypeptide comprise an extracellular domain. As used
herein, the term "extracellular domain" represents the N-terminal
amino acids which extend as a tail from the surface of a cell. An
extracellular domain of the present invention includes an IgV
domain and an IgC domain, and may include a signal peptide domain.
In still another embodiment, amino acid residues 244-273 of the
native human PD-L2 polypeptide and amino acid residues 225-273 of
the mature polypeptide comprise a cytoplasmic domain. As used
herein, the term "cytoplasmic domain" represents the C-terminal
amino acids which extend as a tail into the cytoplasm of a cell. In
addition, nucleic acid and polypeptide sequences of PD-L2 orthologs
in organisms other than humans are well known and include, for
example, mouse PD-L2 (NM_021396.2 and NP_067371.1), rat PD-L2
(NM_001107582.2 and NP_001101052.2), dog PD-L2 (XM_847012.2 and
XP_852105.2), cow PD-L2 (XM_586846.5 and XP_586846.3), and
chimpanzee PD-L2 (XM_001140776.2 and XP_001140776.1).
[0110] The term "PD-L2 activity," "biological activity of PD-L2,"
or "functional activity of PD-L2," refers to an activity exerted by
a PD-L2 protein, polypeptide or nucleic acid molecule on a
PD-L2-responsive cell or tissue, or on a PD-L2 polypeptide binding
partner, as determined in vivo, or in vitro, according to standard
techniques. In one embodiment, a PD-L2 activity is a direct
activity, such as an association with a PD-L2 binding partner. As
used herein, a "target molecule" or "binding partner" is a molecule
with which a PD-L2 polypeptide binds or interacts in nature, such
that PD-L2-mediated function is achieved. In an exemplary
embodiment, a PD-L2 target molecule is the receptor RGMb.
Alternatively, a PD-L2 activity is an indirect activity, such as a
cellular signaling activity mediated by interaction of the PD-L2
polypeptide with its natural binding partner (i.e., physiologically
relevant interacting macromolecule involved in an immune function
or other biologically relevant function), e.g., RGMb. The
biological activities of PD-L2 are described herein. For example,
the PD-L2 polypeptides of the present invention can have one or
more of the following activities: 1) bind to and/or modulate the
activity of the receptor RGMb. PD-1, or other PD-L2 natural binding
partners, 2) modulate intra- or intercellular signaling, 3)
modulate activation of immune cells, e.g., T lymphocytes, and 4)
modulate the immune response of an organism, e.g., a mouse or human
organism.
[0111] The term "TIM-3" refers to a type I cell-surface
glycoprotein that comprises an N-terminal immunoglobulin (Ig)-like
domain, a mucin domain with O-linked glycosylations and with
N-linked glycosylations close to the membrane, a single
transmembrane domain, and a cytoplasmic region with tyrosine
phosphorylation motif(s) (see, for example, U.S. Pat. Publ.
2013/0156774). TIM-3 is a member of the T cell/transmembrane,
immunoglobulin, and mucin (TIM) gene family. Nucleic acid and
polypeptide sequences of human TIM-3 are well known in the art and
are publicly available, for example, as described in NM_032782.4
and NP_116171.3. The term, as described above for useful markers
such as PD-L1 and PD-1, encompasses any naturally occurring
allelic, splice variants, and processed forms thereof. Typically,
TIM-3 refers to human TIM-3 and can include truncated forms or
fragments of the TIM-3 polypeptide. In addition, nucleic acid and
polypeptide sequences of TIM-3 orthologs in organisms other than
humans are well known and include, for example, mouse TIM-3
(NM_134250.2 and NP_599011.2), chimpanzee TIM-3 (XM_518059.4 and
XP_518059.3), dog TIM-3 (NM_001254715.1 and NP_001241644.1), cow
TIM-3 (NM_001077105.2 and NP_001070573.), and rat TIM-3
(NM_001100762.1 and NP_001094232.1). In addition, neutralizing
anti-TIM-3 antibodies are well known in the art (see, at least U.S.
Pat. Publ. 2013/0183688, Ngiow et al. (2011) Cancer Res.
71:3540-3551; and antibody 344823 from R&D Biosystems, as well
as clones 2C23, 5D12, 2E2, 4A4, and IG5, which are all published
and thus publicly available).
[0112] TIM-3 was originally identified as a mouse Th1-specific cell
surface protein that was expressed after several rounds of in vitro
Th1 differentiation, and was later shown to also be expressed on
Th17 cells. In humans, TIM-3 is expressed on a subset of activated
CD4+ T cells, on differentiated Th1 cells, on some CD8+ T cells,
and at lower levels on Th17 cells (Hastings et al. (2009) Eur. J.
Immunol. 39:2492-2501). TIM-3 is also expressed on cells of the
innate immune system including mouse mast cells, subpopulations of
macrophages and dendritic cells (DCs), NK and NKT cells, human
monocytes, human dendritic cells, and on murine primary bronchial
epithelial cell lines. TIM-3 expression is regulated by the
transcription factor T-bet. TIM-3 can generate an inhibitory signal
resulting in apoptosis of Th1 and Tc1 cells, and can mediate
phagocytosis of apoptotic cells and cross-presentation of antigen.
Polymorphisms in TIM-1 and TIM-3 can reciprocally regulate the
direction of T-cell responses (Freeman et al. (2010) Immunol. Rev.
235:172-89).
[0113] TIM-3 has several known ligands, including galectin-9,
phosphatidylserine, and HMGB1. For example, galectin-9 is an S-type
lectin with two distinct carbohydrate recognition domains joined by
a long flexible linker, and has an enhanced affinity for larger
poly-N-acetyllactosamine-containing structures. Galectin-9 does not
have a signal sequence and is localized in the cytoplasm However,
it can be secreted and exerts its function by binding to
glycoproteins on the target cell surface via their carbohydrate
chains (Freeman et al. (2010) Immunol. Rev. 235:172-89). Engagement
of TIM-3 by galectin-9 leads to Th1 cell death and a consequent
decline in IFN-gamma production. When given in vivo, galectin-9 had
beneficial effects in several murine disease models, including an
EAE model, a mouse model of arthritis, in cardiac and skin
allograft transplant models, and contact hypersensitivity and
psoriatic models (Freeman et al. (2010) Immunol. Rev. 235:172-89).
Residues important for TIM-3 binding to galectin-9 include
TIM-3(44), TIM-3(74), and TIM-3(100), which undergo N- and/or
O-glycosylation. It is also known that TIM-3 mediates T-cell
dysfunction associated with chronic viral infections (Golden-Mason
et al. (2009). J. Virol. 83:9122-9130; Jones et al. (2008) J. Exp.
Med. 205:2763-2779) and increases HIV-1-specific T cell responses
when blocked ex vivo (Golden-Mason et al. (2009) J. Virol.
83:9122-9130). In addition, in chronic HCV infection, TIM-3
expression was increased on CD4+ and CD8+ T cells, specifically
HCV-specific CD8+ cytotoxic T cells (CTLs) in chronic HCV infection
and treatment with a blocking monoclonal antibody to TIM-3 reversed
HCV-specific T cell exhaustion (Jones et al. (2008) J. Exp. Med.
205:2763-2779).
[0114] The term "LAG-3," also known as CD223, refers to a member of
the immunoglobulin supergene family and is structurally and
genetically related to CD4 (see, U.S. Pat. Publ. 2011/0150892).
LAG-3 is generally known as a membrane protein encoded by a gene
located on the distal part of the short arm of chromosome 12, near
the CD4 gene, suggesting that the LAG-3 gene may have evolved
through gene duplication (Triebel et al. (1990) J. Exp. Med.
171:1393-1405). However, secreted forms of the protein are known
(e.g., for human and mouse TIM-3). Nucleic acid and polypeptide
sequences of human LAG-3 are well known in the art and are publicly
available, for example, as described in NM_002286.5 and
NP_002277.4.
[0115] The term encompasses any naturally occurring allelic, splice
variants, and processed forms thereof. Typically. LAG-3 refers to
human LAG-3 and can include truncated forms or fragments of the
LAG-3 polypeptide. In addition, nucleic acid and polypeptide
sequences of LAG-3 orthologs in organisms other than humans are
well known and include, for example, mouse LAG-3 (NM_008479.2 and
NP_032505.1), chimpanzee LAG-3 (XM_508966.4 and XP_508966.2),
monkey LAG-3 (XM_001108923.2 and XP_001108923.1), cow LAG-3
(NM_00124949.1 and NP_001232878.1), rat LAG-3 (NM_212513.2 and
NP_997678.2), and chicken LAG-3 (XM_416510.3, XP_416510.2,
XM_004938117.1, and XP_004938174.1). In addition, neutralizing
anti-LAG-3 antibodies are well known in the art (see, at least U.S.
Pat. Publs, 2011/0150892 and 2010/0233183; Macon-Lemaitre and
Triebel (2005) Immunology 115:170-178; Drake et al. (2006) J. Clin.
Oncol. 24:2573; Richter et al. (2010) Int. Immunol. 22:13-23).
[0116] LAG-3 is not expressed on resting peripheral blood
lymphocytes but is expressed on activated T cells and NK cells and
has a number of functions (see, U.S. Pat. Publ. 2011/0150892).
Similar to CD4, LAG-3 has been demonstrated to interact with MHC
Class II molecules but, unlike CD4, LAG-3 does not interact with
the human immunodeficiency virus gp120 protein (Baixeras et al.
(1992) J. Exp. Med. 176:327-337). Studies using a soluble LAG-3
immunoglobulin fusion protein (sLAG-3Ig) demonstrated direct and
specific binding of LAG-3 to MHC class 11 on the cell surface
(Huard et al. (1996) Eur. J. Immunol. 26:1180-1186). In in vitro
studies of antigen-specific T cell responses, the addition of
anti-LAG-3 antibodies led to increased T cell proliferation and
higher expression of activation antigens such as CD25, supporting a
role for the LAG-/MHC class II interaction in down-regulating
antigen-dependent stimulation of CD4+T lymphocytes (Huard et al.
(1994) Eur. J. Immunol. 24:3216-3221). The intra-cytoplasmic region
of LAG-3 has been demonstrated to interact with a protein termed
LAP, which is thought to be a signal transduction molecule involved
in the downregulation of the CD3/TCR activation pathway (Iouzalen
et al. (2001) Eur. J. Immunol. 31:2885-2891). Furthermore,
CD4+CD25+ regulatory T cells (T.sub.reg) have been shown to express
LAG-3 upon activation and antibodies to LAG-3 inhibit suppression
by induced regulatory T cells, both in vitro and in vivo,
suggesting that LAG-3 contributes to the suppressor activity of
regulatory T cells (Huang et al. (2004) Immunity 21:503-513). Still
further, LAG-3 has been shown to negatively regulate T cell
homeostasis by regulatory T cells in both T cell-dependent and
independent mechanisms (Workman and Vignali (2005) J. Immunol.
174:688-695).
[0117] In certain circumstances, LAG-3 also has been shown to have
immunostimulatory effects. For example, LAG-3 transfected tumor
cells transplanted into syngeneic mice showed marked growth
reduction or complete regression as compared to untransfected tumor
cells, suggesting that LAG-3 expression on the tumor cells
stimulated an anti-tumor response by triggering antigen presenting
cells via MHC class IT molecules (Prigent et al. (1999) Eur. J.
Immunol. 29:3867-3876). Additionally, soluble LAG-3 Ig fusion
protein has been shown to stimulate both humoral and cellular
immune responses when administered to mice together with an
antigen, indicating that soluble LAG-3Ig can function as a vaccine
adjuvant (El Mir and Triebel (2000) J. Immunol. 164:5583-5589).
Furthermore, soluble human LAG-3Ig has been shown to amplify the in
vitro generation of type I tumor-specific immunity (Casati et al.
(2006) Cancer Res. 66:4450-4460). The functional activity of LAG-3
is reviewed further in Triebel (2003) Trends Immunol.
24:619-622.
[0118] "Anti-immune checkpoint" therapy refers to the use of agents
that inhibit immune checkpoint nucleic acids and/or proteins.
Immune checkpoints share the common function of providing
inhibitory signals that suppress immune response and inhibition of
one or more immune checkpoints can block or otherwise neutralize
inhibitory signaling to thereby upregulate an immune response in
order to more efficaciously treat cancer. Exemplary agents useful
for inhibiting immune checkpoints include antibodies, small
molecules, peptides, peptidomimetics, natural ligands, and
derivatives of natural ligands, that can either bind and/or
inactivate or inhibit immune checkpoint proteins, or fragments
thereof; as well as RNA interference, antisense, nucleic acid
aptamers, etc. that can downregulate the expression and/or activity
of immune checkpoint nucleic acids, or fragments thereof. Exemplary
agents for upregulating an immune response include antibodies
against one or more immune checkpoint proteins block the
interaction between the proteins and its natural receptor(s); a
non-activating form of one or more immune checkpoint proteins
(e.g., a dominant negative polypeptide); small molecules or
peptides that block the interaction between one or more immune
checkpoint proteins and its natural receptor(s); fusion proteins
(e.g. the extracellular portion of an immune checkpoint inhibition
protein fused to the Fc portion of an antibody or immunoglobulin)
that bind to its natural receptor(s); nucleic acid molecules that
block immune checkpoint nucleic acid transcription or translation;
and the like. Such agents can directly block the interaction
between the one or more immune checkpoints and its natural
receptor(s) (e.g., antibodies) to prevent inhibitory signaling and
upregulate an immune response. Alternatively, agents can indirectly
block the interaction between one or more immune checkpoint
proteins and its natural receptor(s) to prevent inhibitory
signaling and upregulate an immune response. For example, a soluble
version of an immune checkpoint protein ligand such as a stabilized
extracellular domain can binding to its receptor to indirectly
reduce the effective concentration of the receptor to bind to an
appropriate ligand In one embodiment, anti-PD-1 antibodies,
anti-PD-L1 antibodies, and/or anti-PD-L2 antibodies, either alone
or in combination, are used to inhibit immune checkpoints. These
embodiments are also applicable to specific therapy against
particular immune checkpoints, such as the PD-1 pathway (e.g.,
anti-PD-1 pathway therapy, otherwise known as PD-1 pathway
inhibitor therapy). Numerous immune checkpoint inhibitors are known
and publicly available including, for example, Keytruda.RTM.
(pembrolizumab; anti-PD-1 antibody), Opdivo.RTM. (nivolumab;
anti-PD-1 antibody), Tecentriq.RTM. (atezolizumab; anti-PD-L1
antibody), durvalumab (anti-PD-L1 antibody), and the like.
[0119] The term "immune response" includes T cell mediated and/or B
cell mediated immune responses. Exemplary immune responses include
T cell responses, e.g., cytokine production and cellular
cytotoxicity. In addition, the term immune response includes immune
responses that are indirectly affected by T cell activation, e.g.,
antibody production (humoral responses) and activation of cytokine
responsive cells, e.g., macrophages.
[0120] The term "immunotherapeutic agent" can include any molecule,
peptide, antibody or other agent which can stimulate a host immune
system to generate an immune response to a tumor or cancer in the
subject. Various immunotherapeutic agents are useful in the
compositions and methods described herein.
[0121] The term "inhibit" or "deficient" includes the decrease,
limitation, or blockage, of, for example a particular action,
function, or interaction. In some embodiments, cancer is
"inhibited" if at least one symptom of the cancer is alleviated,
terminated, slowed, or prevented. As used herein, cancer is also
"inhibited" if recurrence or metastasis of the cancer is reduced,
slowed, delayed, or prevented. Similarly, a biological function,
such as the function of a protein, is inhibited if it is decreased
as compared to a reference state, such as a control like a
wild-type state. For example, kinase activity of a mutant PI3
kinase or a PI3 kinase that is contacted with a PI3 kinase
inhibitor is inhibited or deficient if the kinase activity is
decreased due to the mutation and/or contact with the inhibitor, in
comparison to the wild-type PI3 kinase and/or the PI3 kinase not
contacted with the inhibitor. Such inhibition or deficiency can be
induced, such as by application of agent at a particular time
and/or place, or can be constitutive, such as by a heritable
mutation. Such inhibition or deficiency can also be partial or
complete (e.g., essentially no measurable activity in comparison to
a reference state, such as a control like a wild-type state).
Essentially complete inhibition or deficiency is referred to as
blocked.
[0122] The term "interaction", when referring to an interaction
between two molecules, refers to the physical contact (e.g.,
binding) of the molecules with one another. Generally, such an
interaction results in an activity (which produces a biological
effect) of one or both of said molecules.
[0123] An "isolated protein" refers to a protein that is
substantially free of other proteins, cellular material, separation
medium, and culture medium when isolated from cells or produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. An "isolated" or "purified"
protein or biologically active portion thereof is substantially
free of cellular material or other contaminating proteins from the
cell or tissue source from which the antibody, polypeptide, peptide
or fusion protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of a biomarker polypeptide or fragment thereof, in
which the protein is separated from cellular components of the
cells from which it is isolated or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations of a biomarker protein or fragment thereof,
having less than about 30% (by dry weight) of non-biomarker protein
(also referred to herein as a "contaminating protein"), more
preferably less than about 20% of non-biomarker protein, still more
preferably less than about 10% of non-biomarker protein, and most
preferably less than about 5% non-biomarker protein. When antibody,
polypeptide, peptide or fusion protein or fragment thereof, e.g., a
biologically active fragment thereof, is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation.
[0124] A "kit" is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g. a probe or small molecule,
for specifically detecting and/or affecting the expression of a
marker of the present invention. The kit may be promoted,
distributed, or sold as a unit for performing the methods of the
present invention. The kit may comprise one or more reagents
necessary to express a composition useful in the methods of the
present invention. In certain embodiments, the kit may further
comprise a reference standard, e.g., a nucleic acid encoding a
protein that does not affect or regulate signaling pathways
controlling cell growth, division, migration, survival or
apoptosis. One skilled in the art can envision many such control
proteins, including, but not limited to, common molecular tags
(e.g., green fluorescent protein and beta-galactosidase), proteins
not classified in any of pathway encompassing cell growth,
division, migration, survival or apoptosis by GeneOntology
reference, or ubiquitous housekeeping proteins. Reagents in the kit
may be provided in individual containers or as mixtures of two or
more reagents in a single container. In addition, instructional
materials which describe the use of the compositions within the kit
can be included.
[0125] The term "neoadjuvant therapy" refers to a treatment given
before the primary treatment. Examples of neoadjuvant therapy can
include chemotherapy, radiation therapy, and hormone therapy. For
example, in treating breast cancer, neoadjuvant therapy can allows
patients with large breast cancer to undergo breast-conserving
surgery.
[0126] The "normal" level of expression and/or activity of a
biomarker is the level of expression and/or activity of the
biomarker in cells of a subject, e.g., a human patient, not
afflicted with a cancer. An "over-expression" or "significantly
higher level of expression" of a biomarker refers to an expression
level in a test sample that is greater than the standard error of
the assay employed to assess expression, and is preferably at least
10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 times or more higher than the expression
activity or level of the biomarker in a control sample (e.g.,
sample from a healthy subject not having the biomarker associated
disease) and preferably, the average expression level of the
biomarker in several control samples. A "significantly lower level
of expression" of a biomarker refers to an expression level in a
test sample that is at least 10%, and more preferably 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,
9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or
more lower than the expression level of the biomarker in a control
sample (e.g., sample from a healthy subject not having the
biomarker associated disease) and preferably, the average
expression level of the biomarker in several control samples. The
same determination can be made to determine overactivity or
underactivity.
[0127] The term "PI3K signaling pathway" refers to one of the
intracellular signaling pathways activated by the binding of growth
factors to receptor tyrosine kinases. Alterations in the PI3K
signaling pathway involved in the development and recurrence of
cancer commonly occur due to mutations in the catalytic or
regulatory PI3K subunits, activation or amplification of receptor
tyrosine kinases, or loss or inactivation of PTEN.
[0128] In general, upon activation, PI3K phosphorylates
phosphatidylinositol-4,5-bisphosphate (PIP2) to
phsophatidylinositol-3,4,5-trisphosphate (PIP3), a process that is
reversed by PTEN. PIP3 signals activate the kinase PDK1, which in
turn activates the kinase AKT. The AKT protein family, which
members are also called protein kinases B (PKB) plays an important
role in mammalian cellular signaling. Akt kinase is a
serine/threonine kinase which is a downstream effector molecule of
phosphoinositide 3-kinase and is involved in protecting a cell from
apoptosis. Akt kinase is thought to be involved in the progression
of cancer because it stimulates cell proliferation and suppresses
apoptosis. Akt1 is involved in cellular survival pathways, by
inhibiting apoptotic processes. Akt1 is also able to induce protein
synthesis pathways, and is therefore a key signaling protein in the
cellular pathways that lead to skeletal muscle hypertrophy, and
general tissue growth. Since it can block apoptosis, and thereby
promote cell survival, Akt1 has been implicated as a major factor
in many types of cancer. Akt is known to play a role in the cell
cycle. Under various circumstances, activation of Akt was shown to
overcome cell cycle arrest in G1 and G2 phases. Moreover, activated
Akt may enable proliferation and survival of cells that have
sustained a potentially mutagenic impact and, therefore, may
contribute to acquisition of mutations in other genes. AKT
(activation, amplification) and PTEN (mutation, deletion,
epigenetic inactivation) are deregulated in many human cancers
(Altomare et al. (2003). J. Cell Biochem. 88:470-476; Ruggeri et
al. (1998) Mol. Carcin. 21:81-86; Cheng et al. (1996) Proc. Natl.
Acad. Sci. U.S.A. 93:3636-3641; Staal et al. (1987) Proc. Natl.
Acad. Sci. U.S.A. 84:5034-5037; Li et al. (2005) World 0.1.
Gastroenterol. 11:285-288: Li et al. (1997) Science 275:1943-1947;
Goel et al. (2004) Cancer Res. 64:3014-3021). PI3K pathway
activation can be assessed by immunohistochemical analysis of PTEN
or phosphorylated AKT levels in clinical samples (Slipicevic et al.
(2005) Am. J. Clin. Pathol. 124:528-536).
[0129] Molecular targets of such inhibitors include, but are not
limited to, PI3K, AKT, S6K1, mTORC1, PDK1, MYC, cMET, FGFR2, growth
factors (EGF, b-FGF, IGF1, Insulin, or Heregulin) and the like. For
example, mTOR exists in at least 2 distinct multiprotein complexes
described as raptor-mTOR complex (mTORC1) and rictor-mTOR complex
(mTORC2) in mammalian cells (sometimes referred to as just TORC1
and TORC2) (Dowling et al. (2010) Biochim. Biophys. Acta
1804:433-439; Dunlop et al. (2009) (el. Signal. 21:827-8735;
Hoeffer et al. (2010) Trends Neurosci. 33:67-75; Laplante et al.
(2012) Cell 149:274-293; Laplante et al. (2013) J. Cell. Sci.
126:1713-1719; Neufeld (2010) Curr. Opin. Cel Rio. 22:157-168;
Zoncu et al. (2011) Nat. Rev. Mol. Cell Biol. 12:21-35). mTORC1 is
composed of mTOR, G.beta.L and raptor proteins and it binds to
FKBP12-rapamycin. mTORC1 is a rapamycin-sensitive complex as its
kinase activity is inhibited by FKB12-rapamycin in vitro and the
mTORC1 complex positively regulates cell growth. The raptor branch
of the mTOR pathway modulates number of processes, including mRNA
translation, ribosome biogenesis, nutrient metabolism and
autophagy. The two mammalian proteins, S6 Kinase 1 (S6K1) and
4E-BP1, which are linked to protein synthesis, are downstream
targets of mTORC1. S6K1 also phosphorylates S6RP, which is the S6
component of the 40S ribosomal subunit involved in regulating
translation, cell size, cell proliferation, and glucose homeostasis
(Magnuson et al. (2012) Biochem. J. 441:1-21). mTORC1 has been
shown to phosphorylate S6K1 at T389 and is inhibited by
FKBP12-rapamycin in vitro and by rapamycin in vivo. mTORC1 can also
phosphorylate 4E-BP1 at T37/46 in vitro and in vivo. Other
molecular targets are well-known in the art and are described, for
example, in U.S. Pat. Publ. 2011/0015869. In some embodiments, the
PI3K signaling pathway is limited to subsets of biomolecules within
the pathway, such as PI3K, PI3K isoforms, mTORC1, S6RP, and 4E-BP1,
or individual biomolecules within the pathway, such as PI3K, PI3k
isoforms, mTORC1, S6RP, or 4E-BP1.
[0130] Inhibitors of PI3K signaling pathway members are also
well-known in the art and include, mTOR inhibitors, such as RAD001
(also known as Everolimus; Novartis), CCI-779 (also known as
Temsirolimus; Pfizer), AP23573 (Ariad Pharmaceuticals), and
KU-0059475 (Kudus Pharmaceuticals; Mita, M. M. et al. (2003) Cancer
Biology & Therapy 2:4:Supp1.1, S169-S177); S6K1 inhibitors,
such as PF-4708671 (Pearce et al., 2010, Biochem. J. 431:245-255)
and DG2
(3-bromo-4-)4-)2-methoxyphenyl)piperazine-1-yl)-1H-pyrazolo[3,4-d]-pyrimi-
dine (Axon Medchem); AKT antibodies (Shin et al., 2005, Cancer Res.
65:2815-2824) (see also Cheng et al., Oncogene, 2005, 24:7482-7492
for review on inhibitors of AKT pathway); PDK1 inhibitors, such as
AR-12, BX-795, staurosporine, OSU-03012, celecoxib, and others
described in U.S. Pat. Nos. 6,124,272; 7,344,870; and 7,041,687);
and IGF1R inhibitors (such as monoclonal antibody MK-0646 U.S. Pat.
No. 7,241,444).
[0131] As used herein, the term "PI3K" refers to a family of
intracellular signal transducer enzymes capable of phosphorylating
the 3 position hydroxyl group of the inositol ring of
phosphatidylinositol (PtdIns). PI3Ks are divided into four
different classes, known as class I, class II, class III, and class
IV, based on the enzyme primary structure, enzymatic regulation,
and lipid substrate specificity (Leevers et al. (1999) Curr. Opin.
Cell Bio. 11:219-225) Class I PI3Ks are heterodimeric molecules
composed of a regulatory and a catalytic subunit, are activated by
G protein-coupled receptors (GPCRs) and tyrosine kinase receptors,
and are responsible for the production of the following
phosphatidylinositols: PI(3)P, PI(3,4)P2, and PI(3,4,5)P.sub.3.
Class II PI3Ks do not contain a regulatory subunit, lack a critical
Asp residue in the C-terminal C2 domain required for coordinate
binding of calcium ions, can comprise one of three catalytic
isoforms (C2alpha, C2beta, or C2gamma), and catalyze the production
of PI(3)P from PI and PI(3,4)P2 from PIP. Class III PI3Ks are
similar to class II PI3Ks in structure, but only produce PI(3)P
from PI. Finally, class TV PI3Ks is a more distantly related set of
enzymes that are protein serine/threonine kinases and include the
members, mTOR, DNA-PK, ATM, and ATR. In humans, the four class I
catalytic PI3Ks are known as PIK3C alpha, PIK3C beta, PIK3C gamma,
and PIK3C delta. The term "pan-PI3K" refers to the group of PIK3C
alpha, PIK3C beta, PIK3C gamma, and PIK3C delta. For example, a
"pan-PI3K inhibitor" inhibits PIK3C alpha, PIK3C beta, PIK3C gamma,
and PIK3C delta.
[0132] Nucleic acid and amino acid sequences for each PI3K,
including catalytic PI3Ks, are known in the art and are publicly
available in the GenBank database maintained by the U.S. National
Center for Biotechnology Information. For example, PIK3C alpha
(PIK3CA) nucleic acid and amino acid sequences are well-known and
include, for example, human PIK3CA (NM_006218.2 and NP_006209.2),
monkey PIK3CA (NM_001260668.1 and NP_001247597.1), mouse PIK3CA
(XM_006535409.2, XP_006535472.1, XM_006535410.2, and
XP_006535473.1), and rat PIK3CA (NM_133399.2 and NP_596890.2).
PIK3C beta (PIK3CB) nucleic acid and amino acid sequences are
well-known and include, for example, human PIK3CB (NM_006219.2,
NP_006210.1, NM_001256045.1, and NP_001242974.1), monkey PIK3CB
(XM_015132082.1 and XP_014987568.1), mouse PIK3CB (NM_029094.3 and
NP_083370.2), and rat P1K3CB (XM_008766567.1, XP_008764789.1,
XM_006243642.2, and XP_006243704.1). PIK3C gamma (PIK3CG) nucleic
acid and amino acid sequences are well-known and include, for
example, human PIK3CG (NM_002649.3, NP_002640.2, NM_001282426.1,
NP_001269355.1, NM_001282427.1, and NP_001269356.1), monkey PIK3CG
(NM_001266758.1 and NP_001253687.1), mouse PIK3CG (NM_020272.2,
NP_064668.2, NM_001146201.1, NP_001139673.1, NM_001146200.1, and
NP_001139672.1), and rat PIK3CG (XM_006240004.2, XP_006240066.1,
XM_006240005.2, XP_006240067.1, XM_006240003.2, and
XP_006240065.1). PIK3C delta (PIK3CD) nucleic acid and amino acid
sequences are well-known and include, for example, human PIK3CD
(NM_005026.3 and NP_005017.3), chimpanzee PIK3CD (XM_009447951.1,
XP_009446226.1, XM_009447957.1, and XP_009446232.1), mouse PIK3CD
(NM_008840.3, NP_032866.2, NM_001164052.1, NP_001157524.1,
NM_001164051.1, NP_001157523.1, NM_001164050.1, NP_001157522.1,
NM_001164049.1, NP_001157521.1, NM_001029837.2, and
NP_001025008.2), and rat PIK3CD (NM_0011089078.1 and
NP_001102448.1). Anti-PI3K agents, including intrabodies, nucleic
acids, and the like are well-known in the art and include, for
example, pan-PI3K inhibitors having broad inhibitory activity
against all catalytic PI3Ks (e.g., pan-Class 1 PI3K inhibitors) are
known and include BKM120
(5-(2,6-dimorpholin-4-ylpyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amin-
e; Maira et al. (2011) Mol. Cancer Ther. 11:317-348), BEZ235 (Maira
et al. (2011) Mol. Cancer Ther. 11:317-348), wortmannin (Wymann el
cl. (1996) Mol. Cell. Biol. 16:1722-1733), LY294002 (Vlahos et al.
(1994) J. Biol. Chem. 269:5241-5248; Wetzker and Rommel (2004)
Curr. Pharm. Des. 10:1915-1922), and BAY 80-80-6946 (copanlisib).
In addition, PI3K isoform-specific small molecule inhibitors are
known. For example, AZD6482 and GSK2636771 selectively inhibit PI3
KB, AS-252424 and AS-604850 selectively inhibit PI3KG, IC87114
selectively inhibits PI3KD, and GDC0941 selectively inhibits PI3KA
and PI3KD (Finan and Thomas (2004) Biochem. Soc. Trans. 32:378-382;
PCT Publ. WO1/81346; PCT Publ. WO01/372557; U.S. Pat. No.
6,403,588; and PCT Publ. WO01/43266). Other inhibitors of PI3Ks
(e.g., other small molecules that are organic chemical molecules
that are not peptides or nucleic acids) are known. In addition,
antibodies that bind PI3Ks, such as, TA802118, TA801482, and
TA303167 (PIK3CA; OriGene Technol., Inc.); TA308795, TA330901, and
TA329903 (PIK3CB; OriGene Technol., Inc.); TA505226, TA505228, and
TA505227 (PIK3CGI OriGene Technol., Inc.); and OTI2H3, TA325015,
and TA307256 (PIK3CD; OriGene Technol., Inc.), and nucleic acids,
such as SR303520, TF310428, SR421939, and TL501641
(PIK3CA-specific, OriGene Technol., Inc.); SR303521, TL310427,
SR421863, TL515159, SR512202, and TL711892 (PIK3CB-specific,
OriGene Technol., Inc.); SR303524, TL310425, SR422070, TL502804,
TR705298 (PIK3CG-specific, OriGene Technol., Inc.); and SR303523,
TL310426, SR421859, TL515984, SR500333, and TL707500
(PIK3CD-specific, OriGene Technol., Inc.), are well-known in the
art. It is to be noted that the term can further be used to refer
to any combination of features described herein regarding PI3Ks.
For example, any combination of class, sequence composition,
percentage identify, sequence length, domain structure, functional
activity, etc. can be used to describe a PI3K of the present
invention.
[0133] As used herein, the term "P53" refers to the well-known
tumor suppressor, p53 (see, for example, Meek (2015) Biochem J.
469:325-346; Ballinger et al. (2015) Curr. Opin. Oncol. 27:332-337;
Amelio and Melino (2015) Trends Biochem. Sci. 40:425-434, Saha et
al. (2014) Prog. Biophys. Mol. Biol. 117:250-263; Tchelebit et al.
(2014) Subcell. Biochem. 85:133-159; Yeudall (2014) Subcell.
Biochem. 85:105-117; Santoro et al. (2014) Subcell. Biochem.
85:91-103; Girardini et al. (2014) Subcell. Biochem. 85.41-70;
Soussi et al. (2014) Hum. Mutat. 35:766-778; Leroy et al. (2014)
Hum. Mutat. 35:756-765, Leory et al. (2014) Hum. Mutat. 35:672-688;
Nguyen et al. (2014) Hum. Mutat. 35:738-755; Bertheau et a. (2013)
Breast 22:S27-S29; Brachova et al. (2013) Int. J. Mol. Sci.
14:19257-19275; Carvajal and Manfredi (2013) EMBO Rep. 14:414-421;
Tornesello et al. (2013) Gynecol. Oncol. 128:442-448; Lehmann and
Pietenpol (2012) J. Clin. Oncol. 30:3648-3650; Bellini et al.
(2012). J. Biomed Biotechnol. 2012:891961; Li et al. (2012)
Biochim. Biophys. Acta. 1819:684-687; and Naccarati et al. (2012)
Mutagenesis 27:211-218). The gene encoding the p53 protein is
highly conserved among vertebrates and is mutated to cause
deficiency of p53 protein function in greater than 50% of human
cancers (Surget et al. (2013) OncoTargets Therapy 7:57-68). In
humans, the p53 gene, which is located at 17p13.1, encodes at least
15 protein isoforms. The protein structure of the p53 protein is
well-known and is characterized by certain domains. For example, in
one embodiment, wild-type functional human p53 comprises:
[0134] 1) an acidic N-terminus transcription-activation domain
(TAD), also known as activation domain 1 (AD1), which activates
transcription factors (e.g., residues 1-42). The N-terminus
contains two complementary transcriptional activation domains, with
a major one at residues 1-42 and a minor one at residues 55-75,
specifically involved in the regulation of several pro-apoptotic
genes (Venot et al. (1998) EMBO J. 17:4668-4679); 2) activation
domain 2 (AD2), which is important for apoptotic activity (e.g.,
residues 43-63);
[0135] 3) proline rich domain, which is important for the apoptotic
activity of p53 by nuclear exportation via MAPK (e.g., residues
64-92);
[0136] 4) central DNA-binding core domain (DBD), which contains one
zinc atom and several arginine amino acids (e.g., residues
102-292). This region is responsible for binding the p53
co-repressor LMO3 (Larsen et al. (2010) Biochem. Biophys. Res.
Commun. 392-252-257;
[0137] 5) nuclear localization signaling domain (e.g., residues
316-325);
[0138] 6) homo-oligomerization domain (OD) (e.g., residues
307-355). Tetramerization is essential for the activity of p53 in
vivo, and
[0139] 7) a C-terminal domain involved in downregulation of DNA
binding of the central domain (e.g., residues 356-393) (Harms et
al. (2005)Mol. Cell. Biol. 25:2014-2030).
[0140] Mutations that make p53 deficient in cancer usually occur in
the DBD. Most of these mutations destroy the ability of the protein
to bind to its target DNA sequences, and thus prevents
transcriptional activation of these genes. As such, mutations in
the DBD are recessive loss-of-function mutations. Molecules of p53
with mutations in the OD dimerize with wild-type p53, and prevent
them from activating transcription. Therefore, OD mutations have a
dominant negative effect on the function of p53. Mutations in p53
nucleic acids that either do not encode functional p53 protein or
p53 protein having reduced function (collectively, p53 deficiency)
are well-known in the art, as described above, and can be generated
by any number of well-known types of mutation including, for
example, a missense mutation (base change that alters the encoded
amino acid), a nonsense mutation (base change that alters the
encoded amino acid to a premature stop codon), a frameshift
mutation (base addition or loss in a manner that is not a multiple
of 3), an insertion mutation (any base addition, large or small in
number, that alters the function of the encoded protein), a
deletion mutation (any base deletion, large or small in number,
that alters the function of the encoded protein), or a
rearrangement mutation (any alteration, large or small, that alters
the function of the encoded protein while retaining the starting
amount of bases). In some embodiments, mutations can be combined,
such as when rearrangements are accompanied by additions and/or
deletions, or multiple missense mutations are combined. In some
embodiments, the mutation is a genetic null (any mutation that
completes ablates the function of the encoded protein) that arises
in the germline, somatically, or both. This description of mutation
types applies to any marker described herein.
[0141] Assays for determining p53 activity, or reduction thereof,
are well-known and commercially available (see, for example, Qiagen
Cignal.RTM. p53 reporter kit, Active Motif.RTM. TransAM.RTM. p53
reporter kit; Cayman Chemical p53 transcription factor assay kit
item number 600020, Genecopoeia.TM. TF-Detect.TM. human p53
activity assay kit, Hiraki et al. (2015) Cell Chem. Biol.
22:1206-1216; Flaman et al. (1995) Proc. Natl. Acad. Sci. USA
92:3963-3967 (1995); and Kovvali et al. (2001) Nucl. Acids Res.
29:e28).
[0142] Nucleic acid and amino acid sequences for p53 nucleic acids
and protein are known in the art and are publicly available in the
GenBank database maintained by the U.S. National Center for
Biotechnology Information. For example, human p53 nucleic acid and
amino acid sequences are well-known and include, for example,
NM_000546.5 (variant 1) and NP_000537.3 (isoform a); NM_001126112.2
(variant 2) and NP_001119584.1 (isoform a); NM_001126114.2 (variant
3) and NP_001119586.1 (isoform b); NM_001126113.2 (variant 4) and
NP_001119585.1 (isoform c); NM_001126115.1 (variant 5) and NP
001119587.1 (isoform d); NM 001126116.1 (variant 6) and NP
001119588.1 (isoform e); NM 001126117.1 (variant 7) and NP
001119589.1 (isoform f); NM_001126118.1 (variant 8) and
NP_001119590.1 (isoform g); NM_001276695.1 (variant 9) and
NP_001263624.1 (isoform h); NM_001276696.1 (variant 10) and
NP_001263625.1 (isoform i), NM_001276697.1 (variant 10) and
NP_001263626.1 (isoform j); NM_001276698.1 (variant 11) and
NP_001263627.1 (isoform k); NM_001276699.1 (variant 12) and
NP_001263628.1 (isoform l); NM_001276760.1 (variant 13) and
NP_001263689.1 (isoform g); and NM_001276761.1 (variant 14) and
NP_001263690.1 (isoform g). Nucleic acid and amino acid sequences
of p53 orthologs in other species are also well-known and include,
for example, mouse p53 (NM_001127233.1, NP_001120705.1,
NM_011640.3, and NP_035770.2), chimpanzee p53 (XM_001172077.4 and
XP_001172077.2), monkey p53 (NM_001047151.2 and NP_001040616.1),
dog p53 (NM_001003210.1 and NP_001003210.1), cow p53 (NM_174201.2
and NP_776626.1), frog p53 (NM_001001903.1 and NP_001001903.1), and
zebrafish p53 (NM_001271820.1, NP_001258749.1, NM_131327.3, and
NP_571402.1). It is to be noted that the term can further be used
to refer to any combination of features described herein regarding
p53. For example, any combination of class, sequence composition,
percentage identify, sequence length, domain structure, functional
activity, etc. can be used to describe p53 as used according to the
present invention.
[0143] The term "pre-determined" biomarker amount and/or activity
measurement(s) may be a biomarker amount and/or activity
measurement(s) used to, by way of example only, evaluate a subject
that may be selected for a particular treatment, evaluate a
response to a treatment such as combination PI3Kbeta and immune
checkpoint inhibitor therapy, and/or evaluate the disease state. A
pre-determined biomarker amount and/or activity measurement(s) may
be determined in populations of patients with or without cancer.
The pre-determined biomarker amount and/or activity measurement(s)
can be a single number, equally applicable to every patient, or the
pre-determined biomarker amount and/or activity measurement(s) can
vary according to specific subpopulations of patients. Age, weight,
height, and other factors of a subject may affect the
pre-determined biomarker amount and/or activity measurement(s) of
the individual. Furthermore, the pre-determined biomarker amount
and/or activity can be determined for each subject individually. In
one embodiment, the amounts determined and/or compared in a method
described herein are based on absolute measurements. In another
embodiment, the amounts determined and/or compared in a method
described herein are based on relative measurements, such as ratios
(e.g., serum biomarker normalized to the expression of housekeeping
or otherwise generally constant biomarker). The pre-determined
biomarker amount and/or activity measurement(s) can be any suitable
standard. For example, the pre-determined biomarker amount and/or
activity measurement(s) can be obtained from the same or a
different human for whom a patient selection is being assessed. In
one embodiment, the pre-determined biomarker amount and/or activity
measurement(s) can be obtained from a previous assessment of the
same patient. In such a manner, the progress of the selection of
the patient can be monitored over time. In addition, the control
can be obtained from an assessment of another human or multiple
humans, e.g., selected groups of humans, if the subject is a human.
In such a manner, the extent of the selection of the human for whom
selection is being assessed can be compared to suitable other
humans, e.g., other humans who are in a similar situation to the
human of interest, such as those suffering from similar or the same
condition(s) and/or of the same ethnic group.
[0144] The term "predictive" includes the use of a biomarker
nucleic acid and/or protein status, e.g., over- or under-activity,
emergence, expression, growth, remission, recurrence or resistance
of tumors before, during or after therapy, for determining the
likelihood of response of a cancer to combination PI3Kbeta and
immune checkpoint inhibitor therapy (e.g., treatment with a
combination of a PI3Kbeta-selective inhibitor, such as KIN193, and
an immune checkpoint inhibitor, such as an anti-PD-1 antibody).
Such predictive use of the biomarker may be confirmed by, e.g., (1)
increased or decreased copy number (e.g., by FISH, FISH plus SKY,
single-molecule sequencing, e.g., as described in the art at least
at J. Biotechnol., 86:289-301, or qPCR), overexpression or
underexpression of a biomarker nucleic acid (e.g., by ISH, Northern
Blot, or qPCR), increased or decreased biomarker protein (e.g., by
IHC), or increased or decreased activity, e.g., in more than about
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human
cancers types or cancer samples; (2) its absolute or relatively
modulated presence or absence in a biological sample, e.g., a
sample containing tissue, whole blood, serum, plasma, buccal
scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow,
from a subject, e.g. a human, afflicted with cancer; (3) its
absolute or relatively modulated presence or absence in clinical
subset of patients with cancer (e.g., those responding to a
particular combination PI3Kbeta and immune checkpoint inhibitor
therapy or those developing resistance thereto).
[0145] The term "pre-malignant lesions" as described herein refers
to a lesion that, while not cancerous, has potential for becoming
cancerous. It also includes the term "pre-malignant disorders" or
"potentially malignant disorders." In particular this refers to a
benign, morphologically and/or histologically altered tissue that
has a greater than normal risk of malignant transformation, and a
disease or a patient's habit that does not necessarily alter the
clinical appearance of local tissue but is associated with a
greater than normal risk of precancerous lesion or cancer
development in that tissue (leukoplakia, erythroplakia,
erytroleukoplakia lichen planus (lichenoid reaction) and any lesion
or an area which histological examination showed atypia of cells or
dysplasia. In one embodiment, a metaplasia is a pre-malignant
lesion.
[0146] The terms "prevent," "preventing," "prevention,"
"prophylactic treatment," and the like refer to reducing the
probability of developing a disease, disorder, or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disease, disorder, or condition.
[0147] The term "probe" refers to any molecule which is capable of
selectively binding to a specifically intended target molecule, for
example, a nucleotide transcript or protein encoded by or
corresponding to a biomarker nucleic acid. Probes can be either
synthesized by one skilled in the art, or derived from appropriate
biological preparations. For purposes of detection of the target
molecule, probes may be specifically designed to be labeled, as
described herein. Examples of molecules that can be utilized as
probes include, but are not limited to, RNA, DNA. proteins,
antibodies, and organic molecules.
[0148] The term "prognosis" includes a prediction of the probable
course and outcome of cancer or the likelihood of recovery from the
disease. In some embodiments, the use of statistical algorithms
provides a prognosis of cancer in an individual. For example, the
prognosis can be surgery, development of a clinical subtype of
cancer (e.g., solid tumors, such as esophageal cancer and gastric
cancer), development of one or more clinical factors, or recovery
from the disease.
[0149] As used herein, the term "PTEN" or "phosphatase and tensin
homolog" refers to the well-known tumor suppressor, pten (see, for
example, Nakanishi et al. (2014) Int. J. Oncol. 44:1813-1819; Xu el
al (2014) Drug Des. Devel. Ther. 8:1745-1751; Shi et al. (2012) J.
Cell Sci. 125:4687-4692; Leslie (2012) Sci. Signal. 5:pe50;
Conde-Perez and Larue (2012) Future Oncol. 8.1109-1120; Zhang et
al. (2012) Biomed. Pharmacother. 66:485-490; Song et al. (2012)
Nat. Rev. Mol. Cell Biol. 13:283-296; Aguissa-Toure and Li (2012)
Cell Mol. Life Sci. 69:1475-1491; Wallace et al. (2011) Cancer Res.
71:1203-1207; Liu et al. (2008) Anticancer Res. 28:3613-3619;
Keniry and Parsons (2008) Oncogene 27:5477-5485; Yin and Shen
(2008) Oncogene 27:5443-5453; Planchon et al. (2008) J. Cell Sci.
121:249-253; Maehama (2007) Biol. Pharm. Bull. 30:1624-1727;
Vazquez and Devreotes (2006) Cell Cycle 5:1523-1527; Steelman et
al. (2004) Exp. Opin. Ther. Targets 8:537-550; Parsons (2004)
Semin. Cell Dev. Biol. 15:171-176; and Maehama et al. (2004)
Biochem. Soc. Trans. 32:343-347). The gene encoding the PTEN
protein, which is a phosphatidylinositol-3,4,5-trisphosphate
3-phosphatase, is highly conserved among vertebrates and is mutated
to cause deficiency of PTEN protein function (e.g., negatively
regulating phosphatidyl-3,4,5-trisphosphate (PtdIns (3,4,5)P3, also
known as PIP3) levels in cells and negatively regulating AKT/PKB
signaling). In particular, PTEN specifically catalyzes the
dephosphorylation of the 3' phosphate of the inositol ring in PIP3
to generate the bisphosphate product, PtdIns(4,5)P2 (also known as
PIP2), and whose dephosphorylation inhibits the AKT/PKB signaling
pathway. In addition, PTEN has phosphatase activity of proteins,
such as IRS1 and disheveled, involved in cell cycle regulation (Shi
et al. (2014) Nat. Struct. Mol. Biol. 21:522-527; Shnitsar et al.
(2015) Nat. Comm. 6:838). In humans, the pten gene, which is
located at 10q23.3, encodes several different isoforms. The protein
structure of the PTEN protein is well-known and is characterized by
certain domains (see, for example, Lee et al. (1999) Cell
99:323-334; Haynie and Xue (2015) Prot. Sci. 24:874-882, Campbell
et al. (2003) J. Biol. Chem. 278:33617-33620; Iijima et al. (2004)
J. Biol. Chem. 279:16606-16613; McConnachie et al. (2003) Biochem.
J. 371:947-955; Rahdar et a. (2009) Proc. Natl. Acad. Sci. USA
106:480-485; Masson et al. (2015) Biochem. J. 473:135-144; Hopkins
et al. (2013) Science 341:399-402; Liang et al. (2014) Cell
Metabol. 19:836-848; and Malaney et al. (2013) Mol. Biosys.
9:877-2888). For example, in one embodiment, wild-type functional
human PTEN comprises a phosphatase domain, which contains the
active enzymatic site, and a C2 domain, which binds the
phospholipid membrane. PTEN binds cellular membranes through both
the phosphatase and C2 domains in order to bring the active site in
proximity to membrane-bound P1P3 for dephosphorylation. In
addition, wild-type functional human PTEN comprises, but need not
comprise for functionality, several additional domains. For
example, a short 10-amino acid N-terminal region (residues 6-15)
called the PIP2 binding domain (PBD), which increases PTEN's
affinity for the cellular membrane. Similarly, a C-terminal domain
spanning residues 353-403 is constitutively phosphorylated and
enhances PTEN's ability to bind lipid membranes. Finally, PTEN can
be expressed as a long version adding an additional .about.173
amino acids to the N-terminus.
[0150] Mutations that make PTEN deficient in cancer cause
inactivation of its enzymatic activity leading to increased
cellular proliferation and decreased cell death. Assays for
determining PTEN activity are well-known and commercially available
(see, for example, Echelon.RTM. PTEN activity ELISA catalog number
K-4700; Shi et al. (2014) Nat. Struct. Mol. Biol. 21:522-527; and
Zhang et al. (2012) Biochem. J. 444:457-464).
[0151] Nucleic acid and amino acid sequences for PTEN nucleic acids
and protein are known in the art and are publicly available in the
GenBank database maintained by the U.S. National Center for
Biotechnology Information. For example, human PTEN nucleic acid and
amino acid sequences are well-known and include, for example,
NM_000314.6 or NM_001304717.2 (variant 1), which encodes multiple
isoforms due to the use of alternative translation initiation
codons. The longest isoform is known as PTEN-L or PTENalpha, is
derived from the use of an upstream non-AUG (CUG) start codon to
initiate with a leucine, is believed to preferentially associated
with the mitochondrial inner membrane, and has an amino acid
sequence that is publicly available as NP_001291646.2. Two shorter
isoforms are derived from downstream AUG start codons. The most
abundant isoform (PTEN), is derived from the use of the 5'-most AUG
start codon and has an amino acid sequence that is publicly
available as NP_000305.3. Similarly, NM_001304718.1 (variant 2)
both contains and lacks alternate exons in its 5' untranslated
region (UTR) as compared to variant 1. Variant 2 represents
translation initiation at a downstream AUG compared to the more
upstream CUG and AUG start codons, as used in variant 1. Use of the
more 5' initiation codons, as used in variant 1, is associated with
a truncated ORF that would render the transcript a candidate for
nonsense-mediated decay (NMD). Leaky scanning may allow translation
initiation at the downstream AUG to encode an isoform, whose amino
acid sequence is publicly available as NP_001291647.1, which has a
shorter N-terminus, compared to isoform PTEN-L and PTEN described
above.
[0152] Nucleic acid and amino acid sequences of PTEN orthologs in
other species are also well-known and include, for example, mouse
PTEN (NM_008960.2 and NP_032986.1), monkey PTEN (NM_001260965.1 and
NP_001247894.1), dog PTEN (NM_001003192.1 and NP_001003192.1), rat
PTEN (NM_031606.1 and NP_113794.1), frog PTEN (NM_001123471.1 and
NP_001116943.1), chicken PTEN (XM_015278701.1 and XP_015134187.1),
and zebrafish PTEN (NM_001001822.2 and NP_001001822.1). It is to be
noted that the term can further be used to refer to any combination
of features described herein regarding PTEN. For example, any
combination of class, sequence composition, percentage identify,
sequence length, domain structure, functional activity, etc. can be
used to describe PTEN as used according to the present
invention.
[0153] The term "response to anti-cancer therapy" or "response to
combination PI3Kbeta and immune checkpoint inhibitor therapy"
relates to any response of the hyperproliferative disorder (e.g.,
cancer) to an anti-cancer agent(s) such as treatment with a
combination of a PI3Kbeta-selective inhibitor, such as KIN193, and
an immune checkpoint inhibitor, such as an anti-PD-1 antibody,
preferably to a change in tumor mass and/or volume after initiation
of neoadjuvant or adjuvant therapy. Hyperproliferative disorder
response may be assessed, for example for efficacy or in a
neoadjuvant or adjuvant situation, where the size of a tumor after
systemic intervention can be compared to the initial size and
dimensions as measured by CT, PET, mammogram, ultrasound or
palpation. Responses may also be assessed by caliper measurement or
pathological examination of the tumor after biopsy or surgical
resection. Response may be recorded in a quantitative fashion like
percentage change in tumor volume or in a qualitative fashion like
"pathological complete response" (pCR), "clinical complete
remission" (cCR), "clinical partial remission" (cPR), "clinical
stable disease" (cSD), "clinical progressive disease" (cPD) or
other qualitative criteria. Assessment of hyperproliferative
disorder response may be done early after the onset of neoadjuvant
or adjuvant therapy, e.g., after a few hours, days, weeks or
preferably after a few months. A typical endpoint for response
assessment is upon termination of neoadjuvant chemotherapy or upon
surgical removal of residual tumor cells and/or the tumor bed This
is typically three months after initiation of neoadjuvant therapy.
In some embodiments, clinical efficacy of the therapeutic
treatments described herein may be determined by measuring the
clinical benefit rate (CBR). The clinical benefit rate is measured
by determining the sum of the percentage of patients who are in
complete remission (CR), the number of patients who are in partial
remission (PR) and the number of patients having stable disease
(SD) at a time point at least 6 months out from the end of therapy.
The shorthand for this formula is CBR=CR+PR+SD over 6 months. In
some embodiments, the CBR for a particular cancer therapeutic
regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the
response to cancer therapies are related to "survival," which
includes all of the following: survival until mortality, also known
as overall survival (wherein said mortality may be either
irrespective of cause or tumor related); "recurrence-free survival"
(wherein the term recurrence shall include both localized and
distant recurrence); metastasis free survival; disease free
survival (wherein the term disease shall include cancer and
diseases associated therewith). The length of said survival may be
calculated by reference to a defined start point (e.g., time of
diagnosis or start of treatment) and end point (e.g., death,
recurrence or metastasis). In addition, criteria for efficacy of
treatment can be expanded to include response to chemotherapy,
probability of survival, probability of metastasis within a given
time period, and probability of tumor recurrence. For example, in
order to determine appropriate threshold values, a particular
cancer therapeutic regimen can be administered to a population of
subjects and the outcome can be correlated to biomarker
measurements that were determined prior to administration of any
cancer therapy. The outcome measurement may be pathologic response
to therapy given in the neoadjuvant setting. Alternatively, outcome
measures, such as overall survival and disease-free survival can be
monitored over a period of time for subjects following cancer
therapy for which biomarker measurement values are known. In
certain embodiments, the doses administered are standard doses
known in the art for cancer therapeutic agents. The period of time
for which subjects are monitored can vary. For example, subjects
may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement
threshold values that correlate to outcome of a cancer therapy can
be determined using well-known methods in the art, such as those
described in the Examples section.
[0154] The term "resistance" refers to an acquired or natural
resistance of a cancer sample or a mammal to a cancer therapy
(i.e., being nonresponsive to or having reduced or limited response
to the therapeutic treatment), such as having a reduced response to
a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%,
60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,
15-fold, 20-fold or more. The reduction in response can be measured
by comparing with the same cancer sample or mammal before the
resistance is acquired, or by comparing with a different cancer
sample or a mammal that is known to have no resistance to the
therapeutic treatment. A typical acquired resistance to
chemotherapy is called "multidrug resistance." The multidrug
resistance can be mediated by P-glycoprotein or can be mediated by
other mechanisms, or it can occur when a mammal is infected with a
multi-drug-resistant microorganism or a combination of
microorganisms. The determination of resistance to a therapeutic
treatment is routine in the art and within the skill of an
ordinarily skilled clinician, for example, can be measured by cell
proliferative assays and cell death assays as described herein as
"sensitizing." In some embodiments, the term "reverses resistance"
means that the use of a second agent in combination with a primary
cancer therapy (e.g., chemotherapeutic or radiation therapy) is
able to produce a significant decrease in tumor volume at a level
of statistical significance (e.g., p<0.05) when compared to
tumor volume of untreated tumor in the circumstance where the
primary cancer therapy (e.g., chemotherapeutic or radiation
therapy) alone is unable to produce a statistically significant
decrease in tumor volume compared to tumor volume of untreated
tumor. This generally applies to tumor volume measurements made at
a time when the untreated tumor is growing log rhythmically.
[0155] The terms "response" or "responsiveness" refers to an
anti-cancer response, such as in the sense of reduction of tumor
size or inhibiting tumor growth. The terms can also refer to an
improved prognosis, for example, as reflected by an increased time
to recurrence, which is the period to first recurrence censoring
for second primary cancer as a first event or death without
evidence of recurrence, or an increased overall survival, which is
the period from treatment to death from any cause. To respond or to
have a response means there is a beneficial endpoint attained when
exposed to a stimulus. Alternatively, a negative or detrimental
symptom is minimized, mitigated or attenuated on exposure to a
stimulus. It will be appreciated that evaluating the likelihood
that a tumor or subject will exhibit a favorable response is
equivalent to evaluating the likelihood that the tumor or subject
will not exhibit favorable response (i.e., will exhibit a lack of
response or be non-responsive).
[0156] An "RNA interfering agent" as used herein, is defined as any
agent which interferes with or inhibits expression of a target
biomarker gene by RNA interference (RNAi). Such RNA interfering
agents include, but are not limited to, nucleic acid molecules
including RNA molecules which are homologous to the target
biomarker gene of the present invention, or a fragment thereof,
short interfering RNA (siRNA), and small molecules which interfere
with or inhibit expression of a target biomarker nucleic acid by
RNA interference (RNAi).
[0157] "RNA interference (RNAi)" is an evolutionally conserved
process whereby the expression or introduction of RNA of a sequence
that is identical or highly similar to a target biomarker nucleic
acid results in the sequence specific degradation or specific
post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA)
transcribed from that targeted gene (see Coburn and Cullen (2002)
J. Virol. 76:9225), thereby inhibiting expression of the target
biomarker nucleic acid. In one embodiment, the RNA is double
stranded RNA (dsRNA). This process has been described in plants,
invertebrates, and mammalian cells. In nature, RNAi is initiated by
the dsRNA-specific endonuclease Dicer, which promotes processive
cleavage of long dsRNA into double-stranded fragments termed
siRNAs. siRNAs are incorporated into a protein complex that
recognizes and cleaves target mRNAs. RNAi can also be initiated by
introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA
interfering agents, to inhibit or silence the expression of target
biomarker nucleic acids. As used herein, "inhibition of target
biomarker nucleic acid expression" or "inhibition of marker gene
expression" includes any decrease in expression or protein activity
or level of the target biomarker nucleic acid or protein encoded by
the target biomarker nucleic acid. The decrease may be of at least
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared
to the expression of a target biomarker nucleic acid or the
activity or level of the protein encoded by a target biomarker
nucleic acid which has not been targeted by an RNA interfering
agent.
[0158] The term "sample" used for detecting or determining the
presence or level of at least one biomarker is typically brain
tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva,
urine, stool (e.g., feces), tears, and any other bodily fluid
(e.g., as described above under the definition of "body fluids"),
or a tissue sample (e.g., biopsy) such as a small intestine, colon
sample, or surgical resection tissue. In certain instances, the
method of the present invention further comprises obtaining the
sample from the individual prior to detecting or determining the
presence or level of at least one marker in the sample.
[0159] The term "selective inhibition" or "selectively inhibit" as
applied to a biologically active agent refers to the agent's
ability to selectively reduce the target signaling activity as
compared to off-target signaling activity, via direct or interact
interaction with the target.
[0160] For example, an agent that selectively inhibits one isoform
of PI3K over another isoform of PI3K has an activity against a
first isoform that is at least 2.times. (times) more than the
compound's activity against the second isoform (e.g., at least
about 3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times., 15.times., 20.times., 25.times., 30.times.,
35.times., 40.times., 45.times., 50.times., 55.times., 60.times.,
65.times., 70.times., 75.times., 80.times., 85.times., 90.times.,
95.times., 100.times., 105.times., 110.times., 120.times.,
125.times., 150.times., 200.times., 250.times., 300.times.,
350.times., 400.times., 450.times., 500.times., 600.times.,
700.times., 800.times., 900.times., 1000.times., or greater, or any
range in between, inclusive) (Frazzetto et al. (2008) Biochem J.
414:383-390). The PI3Kbeta-selective inhibitor, AZD6482, has an
IC50 for PI3Kbeta of 10 nM and is 8.times. (IC50 80 nM), 87.times.
(IC50 870 nM), and 109.times. (IC50 1090 nM) more selective toward
PI3Kbeta than to PI3Kdelta, PI3Kalpha, and PI3Kgamma, respectively,
as determined in cell-free assays (PCT Publ. WO 2009/093972; Ni et
al. (2012) Cancer Discov. 2:425-433; Nylander et al. (2012) J.
Thromb. Haemost. 10:2127-2136). The PI3Kbeta-selective inhibitor,
TGC-221
(7-methyl-2-morpholino-9-(1-(phenylamino)ethyl)-4H-pyrido[1,2-a]pyrimidin-
-4-one) has an IC50 of 5 nM, 0.1 uM, 5 uM, and less than 10 uM for
PI3Kbeta, PI3Kdelta, PI3Kalpha, and PI3Kgamma, respectively
(Jackson et al. (2005) Nat. Med. 11:507-514). GSK2636771
(2-methyl-1-[[2-methyl-3-(trifluoromethyl)phenyl]methyl]-6-(4-morpholinyl-
)-1H-benzimidazole-4-carboxylic acid) is also a PI3Kbeta-selective
inhibitor (Macauley et al. (2012) Drugs Fut. 37:451; Weigelt et al.
(2013) Clin. Cancer Res. 19:3533-3544).
TABLE-US-00002 TABLE 2 IC50 values for PI3K chemical inhibitors
(adapted from the World Wide Web at
selleckchem.com/pathways_PI3K.html) IC50 (nM) Pan- PI3K P13K P13K
PI3K PI3K alpha beta delta gamma Omipalisib (GSK2126458, 0.019 0.13
0.024 0.06 GSK458) GSK1059615 0.4 0.6 2 5 PF-04691502 1.8 2.1 1.6
1.9 PI-103 2 3 3 15 BAY 80-6946 (Copanlisib) 0.469 3.72 0.7 6.4
AZD8186 35 4 17 675 TGX-221 5000 5 100 PKI-402 2 7 14 16 GDC-0032
0.29 9.1 0.12 0.97 AZD6482 (KIN-193) 870 10 80 1090 VS-5584
(SB2343) 2.6 21 2.7 3 Apitolisib (GDC-0980, RG7422) 5 27 7 14
Pictilisib (GDC-0941) 3 33 3 75 Pilaralisib (XL147) 39 36 36 23
ZSTK474 37 16 44 4.6 49 CUDC-907 19 54 39 311 BGT226 (NVP-BGT226) 4
63 38 BEZ235 (NVP-BEZ235, 4 75 7 5 Dactolisib) Voxtalisib
(SAR245409, XL765) 39 113 43 9 Analogue Voxtalisib (XL765,
SAR245409) 39 113 43 9 CH5132799 14 12.0 500 36 PF-4989216 2 142 1
65 BKM120 (NVP-BKM120, 52 166 116 262 Buparlisib) TG100713 165 215
24 50 AS-605240 60 270 300 8 PIK-90 11 350 58 18 XL147 analogue 39
383 36 23 PIK-294 490 10 160 CAL-101 (Idelalisib, 820 565 2.5 89
GS-1101) PIK-93 39 590 120 16 LY294002 500 970 570 PI-3065 2299
1078 15 27542 CZC24832 1100 8200 27 TG100-115 1300 1200 235 83
PIK-75 5.8 1300 510 76 Duvelisib (IPI-145, INK1197) 25900 1564 23
243 AMG319 33000 2700 18 850 Quercetin 5400 3000 2400 PIK-293
100000 25000 240 10000 IC-87114 75000 500 29000 Gedatolisib
(PF-05212384, 0.4 5.4 PKI-587) HS-173 0.8 Alpelisib (BYL719) 5 A66
32 3480 AS-252424 935 33 YM201636 3300 AS-604850 4500 250 NU7441
(KU-57788) 5000 Wortmannin 3 3-Methyladenine (3-MA) 60
[0161] The term "sensitize" means to alter cancer cells or tumor
cells in a way that allows for more effective treatment of the
associated cancer with a cancer therapy (e.g., anti-immune
checkpoint, chemotherapeutic, and/or radiation therapy). In some
embodiments, normal cells are not affected to an extent that causes
the normal cells to be unduly injured by the anti-immune checkpoint
therapy. An increased sensitivity or a reduced sensitivity to a
therapeutic treatment is measured according to a known method in
the art for the particular treatment and methods described herein
below, including, but not limited to, cell proliferative assays
(Tanigawa et al. (1982) Cancer Res. 42:2159-2164), cell death
assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal
et al. (1985) Cancer Treat. Rep. 69:615-632; Weisenthal L M, In:
Kaspers et al. eds. Drug Resistance in Leukemia and Lymphoma.
Langhorne, P A: Harwood Academic Publishers, 1993: 415-432;
Weisenthal et ta. (1994) Contrib. Gynecol. Obstet. 19:82-90). The
sensitivity or resistance may also be measured in animal by
measuring the tumor size reduction over a period of time, for
example, 6 month for human and 4-6 weeks for mouse. A composition
or a method sensitizes response to a therapeutic treatment if the
increase in treatment sensitivity or the reduction in resistance is
250% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more,
to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or
more, compared to treatment sensitivity or resistance in the
absence of such composition or method. The determination of
sensitivity or resistance to a therapeutic treatment is routine in
the art and within the skill of an ordinarily skilled clinician. It
is to be understood that any method described herein for enhancing
the efficacy of a cancer therapy can be equally applied to methods
for sensitizing hyperproliferative or otherwise cancerous cells
(e.g., resistant cells) to the cancer therapy.
[0162] The term "specific binding" refers to an agent, such as an
antibody, binding to a pre-determined target, such as an antigen.
Typically, the antibody binds with an affinity (K.sub.D) of
approximately less than 10.sup.-7 M, such as approximately less
than 10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even lower when
determined by surface plasmon resonance (SPR) technology in a
BIACORE.RTM. assay instrument using an antigen of interest as the
analyte and the antibody as the ligand, and binds to the
predetermined antigen with an affinity that is at least 1.1-, 1.2-,
1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-,
4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater
than its affinity for binding to a non-specific antigen (e.g., BSA,
casein) other than the predetermined antigen or a closely-related
antigen. The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an antigen."
Selective binding is a relative term referring to the ability of an
antibody to discriminate the binding of one antigen over
another.
[0163] The term "synergistic effect" refers to the combined effect
of two or more anti-cancer agents (e.g., treatment with a
combination of a PI3Kbeta-selective inhibitor, such as KIN193, and
an immune checkpoint inhibitor, such as an anti-PD-1 antibody) can
be greater than the sum of the separate effects of the anti-cancer
agents alone.
[0164] "Short interfering RNA" (siRNA), also referred to herein as
"small interfering RNA" is defined as an agent which functions to
inhibit expression of a target biomarker nucleic acid, e.g., by
RNAi. An siRNA may be chemically synthesized, may be produced by in
vitro transcription, or may be produced within a host cell. In one
embodiment, siRNA is a double stranded RNA (dsRNA) molecule of
about 15 to about 40 nucleotides in length, preferably about 15 to
about 28 nucleotides, more preferably about 19 to about 25
nucleotides in length, and more preferably about 19, 20, 21, or 22
nucleotides in length, and may contain a 3' and/or 5' overhang on
each strand having a length of about 0, 1, 2, 3, 4, or 5
nucleotides. The length of the overhang is independent between the
two strands, i.e., the length of the overhang on one strand is not
dependent on the length of the overhang on the second strand.
Preferably the siRNA is capable of promoting RNA interference
through degradation or specific post-transcriptional gene silencing
(PTGS) of the target messenger RNA (mRNA).
[0165] In another embodiment, an siRNA is a small hairpin (also
called stem loop) RNA (shRNA). In one embodiment, these shRNAs are
composed of a short (e.g., 19-25 nucleotide) antisense strand,
followed by a 5-9 nucleotide loop, and the analogous sense strand.
Alternatively, the sense strand may precede the nucleotide loop
structure and the antisense strand may follow. These shRNAs may be
contained in plasmids, retroviruses, and lentiviruses and expressed
from, for example, the pol III U6 promoter, or another promoter
(see, e.g., Stewart, et al. (2003) RNA April; 9(4)-493-501
incorporated by reference herein).
[0166] RNA interfering agents, e.g., siRNA molecules, may be
administered to a patient having or at risk for having cancer, to
inhibit expression of a biomarker gene which is overexpressed in
cancer and thereby treat, prevent, or inhibit cancer in the
subject.
[0167] The term "subject" refers to any healthy animal, mammal or
human, or any animal, mammal or human afflicted with a cancer,
e.g., an epithelial cancer, including brain metastasis, lung,
ovarian, pancreatic, liver, breast, prostate, colon carcinomas,
melanoma, multiple myeloma, and the like. The term "subject" is
interchangeable with "patient."
[0168] The term "survival" includes all of the following: survival
until mortality, also known as overall survival (wherein said
mortality may be either irrespective of cause or tumor related);
"recurrence-free survival" (wherein the term recurrence shall
include both localized and distant recurrence); metastasis free
survival; disease free survival (wherein the term disease shall
include cancer and diseases associated therewith). The length of
said survival may be calculated by reference to a defined start
point (e.g. time of diagnosis or start of treatment) and end point
(e.g. death, recurrence or metastasis). In addition, criteria for
efficacy of treatment can be expanded to include response to
chemotherapy, probability of survival, probability of metastasis
within a given time period, and probability of tumor
recurrence.
[0169] The term "therapeutic effect" refers to a local or systemic
effect in animals, particularly mammals, and more particularly
humans, caused by a pharmacologically active substance. The term
thus means any substance intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease or in the
enhancement of desirable physical or mental development and
conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. In
certain embodiments, a therapeutically effective amount of a
compound will depend on its therapeutic index, solubility, and the
like. For example, certain compounds discovered by the methods of
the present invention may be administered in a sufficient amount to
produce a reasonable benefit/risk ratio applicable to such
treatment.
[0170] The terms "therapeutically-effective amount" and "effective
amount" as used herein means that amount of a compound, material,
or composition comprising a compound of the present invention which
is effective for producing some desired therapeutic effect in at
least a sub-population of cells in an animal at a reasonable
benefit/risk ratio applicable to any medical treatment. Toxicity
and therapeutic efficacy of subject compounds may be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD.sub.50 and the ED.sub.50.
Compositions that exhibit large therapeutic indices are preferred.
In some embodiments, the LD.sub.50 (lethal dosage) can be measured
and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,
900%, 1000% or more reduced for the agent relative to no
administration of the agent. Similarly, the ED.sub.50 (i.e., the
concentration which achieves a half-maximal inhibition of symptoms)
can be measured and can be, for example, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900%, 1000% or more increased for the agent relative to
no administration of the agent. Also, Similarly, the IC.sub.50
(i.e., the concentration which achieves half-maximal cytotoxic or
cytostatic effect on cancer cells) can be measured and can be, for
example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more
increased for the agent relative to no administration of the agent.
In some embodiments, cancer cell growth in an assay can be
inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In
another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even
100% decrease in a solid malignancy can be achieved.
[0171] In one embodiment, a therapeutically effective amount of
antibody (i.e., an effective dosage) ranges from about 0.001 to 30
mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight,
more preferably about 0.1 to 20 mg/kg body weight, and even more
preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7
mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will
appreciate that certain factors may influence the dosage required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of an antibody can include a single treatment or,
preferably, can include a series of treatments. In a preferred
example, a subject is treated with antibody in the range of between
about 0.1 to 20 mg/kg body weight, one time per week for between
about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. It will also be appreciated that the
effective dosage of antibody used for treatment may increase or
decrease over the course of a particular treatment. Changes in
dosage may result from the results of diagnostic assays.
[0172] A "transcribed polynucleotide" or "nucleotide transcript" is
a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such
RNA or cDNA) which is complementary to or homologous with all or a
portion of a mature mRNA made by transcription of a biomarker
nucleic acid and normal post-transcriptional processing (e.g.
splicing), if any, of the RNA transcript, and reverse transcription
of the RNA transcript.
[0173] As used herein, the term "unresponsiveness" includes
refractivity of cancer cells to therapy or refractivity of
therapeutic cells, such as immune cells, to stimulation, e.g.,
stimulation via an activating receptor or a cytokine.
Unresponsiveness can occur, e.g., because of exposure to
immunosuppressants or exposure to high doses of antigen. As used
herein, the term "anergy" or "tolerance" includes refractivity to
activating receptor-mediated stimulation. Such refractivity is
generally antigen-specific and persists after exposure to the
tolerizing antigen has ceased. For example, anergy in T cells (as
opposed to unresponsiveness) is characterized by lack of cytokine
production, e.g., IL-2. T cell anergy occurs when T cells are
exposed to antigen and receive a first signal (a T cell receptor or
CD-3 mediated signal) in the absence of a second signal (a
costimulatory signal). Under these conditions, reexposure of the
cells to the same antigen (even if reexposure occurs in the
presence of a costimulatory polypeptide) results in failure to
produce cytokines and, thus, failure to proliferate. Anergic T
cells can, however, proliferate if cultured with cytokines (e.g.,
IL-2). For example, T cell anergy can also be observed by the lack
of IL-2 production by T lymphocytes as measured by ELISA or by a
proliferation assay using an indicator cell line. Alternatively, a
reporter gene construct can be used. For example, anergic T cells
fail to initiate IL-2 gene transcription induced by a heterologous
promoter under the control of the 5' IL-2 gene enhancer or by a
multimer of the AP1 sequence that can be found within the enhancer
(Kang ea (1992) Science 257:1134).
[0174] There is a known and definite correspondence between the
amino acid sequence of particular protein and the nucleotide
sequences that can code for the protein, as defined by the genetic
code (shown below). Likewise, there is a known and definite
correspondence between the nucleotide sequence of a particular
nucleic acid and the amino acid sequence encoded by that nucleic
acid, as defined by the genetic code.
TABLE-US-00003 GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT
Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N)
AAC, AAT Aspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT
Glutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine
(Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine
(Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA,
TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine
(Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser,
S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG,
ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAG, TAT Valine (Val,
V) GTA, GTC, GTG, GTT Termination signal (end) TAA, TAG, TGA
[0175] An important and well-known feature of the genetic code is
its redundancy, whereby, for most of the amino acids used to make
proteins, more than one coding nucleotide triplet may be employed
(illustrated above). Therefore, a number of different nucleotide
sequences may code for a given amino acid sequence. Such nucleotide
sequences are considered functionally equivalent since they result
in the production of the same amino acid sequence in all organisms
(although certain organisms may translate some sequences more
efficiently than they do others). Moreover, occasionally, a
methylated variant of a purine or pyrimidine may be found in a
given nucleotide sequence. Such methylations do not affect the
coding relationship between the trinucleotide codon and the
corresponding amino acid.
[0176] In view of the foregoing, the nucleotide sequence of a DNA
or RNA encoding a biomarker nucleic acid (or any portion thereof)
can be used to derive the polypeptide amino acid sequence, using
the genetic code to translate the DNA or RNA into an amino acid
sequence. Likewise, for polypeptide amino acid sequence,
corresponding nucleotide sequences that can encode the polypeptide
can be deduced from the genetic code (which, because of its
redundancy, will produce multiple nucleic acid sequences for any
given amino acid sequence). Thus, description and/or disclosure
herein of a nucleotide sequence which encodes a polypeptide should
be considered to also include description and/or disclosure of the
amino acid sequence encoded by the nucleotide sequence. Similarly,
description and/or disclosure of a polypeptide amino acid sequence
herein should be considered to also include description and/or
disclosure of all possible nucleotide sequences that can encode the
amino acid sequence.
[0177] Finally, nucleic acid and amino acid sequence information
for the loci and biomarkers of the present invention (e.g.,
biomarkers listed in Table 3) are well-known in the art and readily
available on publicly available databases, such as the National
Center for Biotechnology Information (NCBI). For example, exemplary
nucleic acid and amino acid sequences derived from publicly
available sequence databases are provided below and include, for
example, PCT Publ. WO 2014/022759, which is incorporated herein in
its entirety by this reference.
TABLE-US-00004 TABLE 3 SEQ ID NO: 1 Human PIK3CA cDNA Acid Sequence
1 atgcctccac gaccatcatc aggtgaactg tggggcatcc acttgatgcc cccaagaatc
61 ctagtagaat gtttactacc aaatggaatg atagtgactt tagaatgcct
ccgtgaggct 121 acattaataa ccataaagca tgaactattt aaagaagcaa
gaaaataccc cctccatcaa 181 cttcttcaag atgaatcttc ttacattttc
gtaagtgtta ctcaagaagc agaaagggaa 241 gaattttttg atgaaacaag
acgactttgt gaccttcggc tttttcaacc ctttttaaaa 301 gtaattgaac
cagtaggcaa ccgtgaagaa aagatcctca atcgagaaat tggttttgct 361
atcggcatgc cagtgtgtaa atttgatatg gttaaagatc cagaagtaca ggacttccga
421 agaaatattc taaacgtttg taaagaagct gtagatctta gggacctcaa
ttcacctcat 481 agtagagcaa tgtatgtcta tcctccaaat gtagaatctt
caccagaatt gccaaagcac 541 atatataata aattagataa agggcaaata
atagtggtga tctgggtaat agtttctcca 601 aataatgaca agcagaagta
tactctgaaa atcaaccatg actgtgtacc agaacaagta 661 attgctgaag
caatcaggaa aaaaactcga agtatgttgc tatcctctga acaactaaaa 721
ctctgtgttt tagaatatca gggcaagtat attttaaaag tgtgtggatg tgatgaatac
781 ttcctagaaa aatatcctct gagtcagtat aagtatataa gaagctgtat
aatgcttggg 841 aggatgccca atttgatgtt gatggctaaa gaaagccttt
attctcaact gccaatggac 901 tgttttacaa tgccatctta ttccagacgc
atttccacag ctacaccata tatgaatgga 961 gaaacatcta caaaatccgt
ttgggttata aatagtgcac tcagaataaa aattctttgt 1021 gcaacctacg
tgaatgtaaa tattcgagac attgataaga tctatgttcg aacaggtatc 1081
taccatggag gagaaccctt atgtgacaat gtgaacactc aaagagtacc ttgttccaat
1141 cccaggtgga atgaatggct gaattatgat atatacattc ctgatcttcc
tcgtgctgct 1201 cgactttgcc tttccatttg ctctgttaaa ggccgaaagg
gtgctaaaga ggaacactgt 1261 ccattggcat ggggaaatat aaacttgttt
gattacacag acactctagt atctggaaaa 1321 atggctttga atctttggcc
agtacctcat ggattagaag atttgctgaa ccctattggt 1381 gttactggat
caaatccaaa taaagaaact ccatgcttag agttggagtt tgactggttc 1441
agcagtgtgg taaagttccc agatatgtca gtgattgaag agcatgccaa ttggtctgta
1501 tcccgagaag caggatttag ctattcccac gcaggactga gtaacagact
agctagagac 1561 aatgaattaa gggaaaatga caaagaacag ctcaaagcaa
tttctacacg agatcctctc 1621 tctgaaatca ctgagcagga gaaagatttt
ctatggagtc acagacacta ttgtgtaact 1681 atccccgaaa ttctacccaa
attgcttctg tctgttaaat ggaattctag agatgaagta 1741 gcccagatgt
attgcttggt aaaagattgg cctccaatca aacctgaaca ggctatggaa 1801
cttctggact gtaattaccc agatcctatg gttcgaggtt ttgctgttcg gtgcttggaa
1861 aaatatttaa cagatgacaa actttctcag tatttaattc agctagtaca
ggtcctaaaa 1921 tatgaacaat atttggataa cttgcttgtg agatttttac
tgaagaaagc attgactaat 1981 caaaggattg ggcacttttt cttttggcat
ttaaaatctg agatgcacaa taaaacagtt 2041 agccagaggt ttggcctgct
tttgaagtcc tattgtcatg catgtaggat gtatttgaag 2101 cacctgaata
ggcaagtcga ggcaatggaa aagctcatta acttaactga cattctcaaa 2161
caggagaaga aggatgaaac acaaaaggta cagatgaagt ttttagttga gcaaatgagg
2221 cgaccagatt tcatggatgc tctacagggc tttctgtctc ctctaaaccc
tgctcatcaa 2281 ctaggaaacc tcaggcttga agagtgtcga attatgtcct
ctgcaaaaag gccactgtgg 2341 ttgaattggg agaacccaga catcatgtca
gagttactgt ttcagaacaa tgagatcatc 2401 tttaaaaatg gggatgattt
acggcaagat atgctaacac ttcaaattat tcgtattatg 2461 gaaaatatct
ggcaaaatca aggtcttgat cttcgaatgt taccttatgg ttgtctgtca 2521
atcggtgact gtgtgggact tattgaggtg gtgcgaaatt ctcacactat tatgcaaatt
2581 cagtgcaaag gcggcttgaa aggtgcactg caattcaaca gccacacact
acatcagtgg 2641 ctcaaagaca agaacaaaag agaaatatat gatgcagcca
ttgacctgtt tacacgttca 2701 tgtgctggat actgtgtagc taccttcatt
ttgggaattg gagatcgtca caatagtaac 2761 atcatggtga aagacgatgg
acaactgttt catatagatt ttggacactt tttggatcac 2821 aagaagaaaa
aatttggtta taaacgagaa cgtgtgccat ttgttttgac acaggatttc 2881
ttaatagtga ttagtaaagg agcccaagaa tgcacaaaga caagagaatt tgagaggttt
2941 caggagatgt gttacaaggc ttatctagct attcgacagc atgccaatct
cttcataaat 3001 cttttctcaa tgatgdttgg ctctggaatg ccagaactac
aatcttttga tgacattgca 3061 tacattcgaa agaccctaac cttagataaa
actgagcaaa aggctttgga gtatttcatg 3121 aaacaaatga atgatgcaca
tcatggtggc tgaacaacaa aaatggattg gatcttccac 3181 acaattaaac
agcatgcatt gaactga SEQ ID NO: 2 Human PIK3CA Amino Acid Sequence 1
mpprpssgel wgihlmppri lvecllpngm ivtleclrea tlitikhelf kearkyplhq
61 llgdessyif vsvtqeaere effdetrrlc dlrlfqpflk viepvgnree
kilnreigfa 121 igmpvcefdm vkdpevgdfr rnilnvckea vdlrdlnsph
sramyvyppn vesspelpkh 181 iyhkldkgqi ivviwvivsp nndkqkytlk
inhdcvpeqv iaeairkktr smllsseqlk 241 lcvleyqgky ilkvcgcdey
flekyplsqy kyirscimlg rmpnlmlmak eslysqlpmd 301 cftmpsysrr
istatpymng etstkslwvi nsalrikilc atyvnvnird idkiyvrtgi 361
yhggeplcdn vntqrvpcsn prwnewlnyd iyipdlpraa rlclsicsvk grkgakeehc
421 plawgninlf dytdtlvsqk malnlwpvph gledllnpig vtgsnpnket
pclelefdwf 481 ssvvkfpdms vieehanwsv sreagfsysh aglsnrlard
nelrendkeq lkaistrdpl 541 seiteqekdf lwshrhycvt ipeilpklll
svkwnsrdev aqmyclvkdw ppikpeqame 601 lldcnypdpm vrgfavrcle
kyltddklsq yliqlvqvlk yegyldnllv rfllkkaltn 661 qrighfffwh
lksemhnktv sqrfgllles ycracgmylk hlnrqveame klinltdilk 721
qekkdetqkv qmkflveqmr rpdfmdalqg flsplnpahq lgnlrleecr imssakrplw
781 lnwenpdims ellfqnneii fkngddlrqd mltlqiirim eniwqnggld
lrmlpygcls 841 igdcvgliev vrnshtimqi qckgglkgal qfnshtlhqw
lkdknkgeiy daaidlftrs 901 cagycvatfi lgigdrhnsn imvkddgqlf
hidfghtldh kkkkfgykre rvpfvltqdf 961 liviskgaqe ctktreferf
qemcykayla irqhanlfin lfsmmlgsgm pelqsfddia 1021 yirktlaldk
teqealeyfm kqmndahhgg wttkmdwifh tikqhaln SEQ ID NO: 3 Mouse PIK3CA
(Transcript 1) cDNA Acid Sequence 1 atgcctccac gaccatcttc
gggtgaactg tgaggcatcc acttgatgcc cccacgaatc 61 ctagtqgaat
gtttactccc caatggaatg atagtgactt tagaatgcct ccgtgaggcc 121
acactcgtca ccatcaaaca tgaactgttc agagaggcca ggaaataccc tctccatcag
181 cttctgcaag acgaaacttc ttacattttc gtaagtgtca cccaagaagc
agaaagggaa 241 gaattttttg atgaaacaag acgactttgt gaccttcagc
tttttcaacc ctttttaaaa 301 gttattgaac cagtaggcaa ccgtgaagaa
aagatcctca atcaagaaat tggttttgtt 361 attggcatgc cagtgtgtga
atttgatatg gttaaagatc cagaagtcca agactttcga 421 aggaacattc
tgaatgtttg caaagaagct gtggacctgc gggatctcaa ctcgcctcat 481
agcagagcaa tgtatgtcta ccctccaaat gtcgagtctt ccccagaact gccaaagcac
541 atctacaaca agttagataa aggacaaatc atagtggtga tttgggtaat
agtctctcca 601 aacaacgaca agcagaagta cactctqaag atcaatcatg
actgtgtqcc agagcaagtc 661 attgctgaag ccatcaggaa aaagactcgg
agcatgttgt tgtcctctga gcagctgaaa 721 ctctgtgtct tagaatatca
gggcaagtat attctgaaag tgtgtggctg tgacgaatac 781 ttcctggaaa
agtaccctct gagtcagtac aagtacataa gaagctgtat aatgctgggg 841
aggatgccca acttgatgct gatggccaaa gaaagcctat actctcagct gccgattgat
901 agcttcacca tgccgtcata ctccaggcgc atctccacag ccacacccta
catgaatgga 961 gagacatcta cgaaatccct ctgggtcata aatagtgcgc
tcagaataaa aattctttgt 1021 gcaacctatg taaatgtaaa tattcgagac
attgataaga tctatgttcg aacaggtatc 1081 taccatggag gagaaccctt
atgtgacaat gtgaacactc aaagagtacc ttgttccaat 1141 cctaggtgga
atgaatggct gaattatgat atatacattc ctgatcttcc tcgtgctgcg 1201
cgcctttgcc tttcaatctg ctctgttaaa ggccgaaagg gtgctaagga ggagcactgt
1261 ccgttggcct ggggaaacat aaacttgttt gattatacag acaccctagt
gtccgggaaa 1321 atggctttga atctctggcc tgtaccgcat gggttagaag
atctgctgaa ccctattggt 1381 gttactgggt caaatccaaa taaagaaact
ccatgcttag agttggagtt tgattggttc 1441 agcagtgtgg taaagtttcc
agacatgtct gtgatcgaag aacatgccaa ttggtccgtg 1501 tcccgagaag
ctggattcag ttactcccat acaggactga gtaacagact agccagagac 1561
aatgagttaa gagaaaatga caaggaacag ctccgagcac tttgcacccg ggacccacta
1621 tctgaaatca ctgaacaaga gaaagacttc ctatggagcc acagacacta
ctgcgtaact 1681 attcctgaaa tcctacccaa attgcttctg tctgtcaagt
ggaattccag agacgaagtg 1741 gcccagatgt actgcttagt aaaagattgg
cctccaatca aaccagagca agccatggaa 1801 ctcctggact gtaactatcc
agatcctatg gttcggagtt ttgctgttcg gtgcttagaa 1861 aaatatttaa
cagatgacaa actttctcag tacctcattc aacttgtaca ggtcttaaaa 1921
tatgaacagt atttggataa cctgcttgtg agatttttac tcaagaaagc attgacaaat
1981 caaaggattg gccatttttt cttttggcat ttaaaatctg agatgcacaa
taaaactgtc 2041 agtcagaggt ttggcctgct attggagtcc tactgccgtg
cctgtgqgat gtatctgaag 2101 cacctgaaca gacaagtaga ggccatggag
aagctcatca acctaacgga catccttaag 2161 caggagaaga aggatgagac
acaaaaggta cagatgaagt ttttggttga acagatgaga 2221 cagccagact
tcatqgatac tttgcaaggt tttctgtccc ctctgaatcc tgctcaccaa 2281
ctaggaaacc tcaggcttga agagtgtcga attatgtcct ctgcaaaaag gccactgtgg
2341 ttgaattggg agaacccaga catcatgtca gagctactgt ttcagaacaa
tgagatcatc 2401 tttaaaaatg gcgacgactt acggcaagat atgttaaccc
ttcagatcat ccgaatcatg 2461 gagaacatct ggcaaaacca aggccttgac
cttcgcatgc taccttatgg ctgtctatcc 2521 attggggact gtgtgggtct
catcgaggtg gtgagaaact ctcacaccat catgcaaatc 2581 cagtgcaaag
gaggcctgaa gggggcgctg caattcaaca gccacacact gcatcaatgg 2641
ctcaaggaca agaacaaggg cgagatatat gatgcagcca ttgacctgtt cactcggtcc
2701 tgcgctgggt actgcgtggc aacctttatc ttgggaattg gagaccggca
caacagcaac 2761 atcatggtga aagatgacgg acagctgttt catatagatt
ttgggcactt tttggatcac 2821 aagaagaaaa aatttggcta taagcgggaa
cgtgtgccat ttgtgttgac acaggatttc 2881 ttgattgtga ttagtaaggg
agcacaagag tacaccaaga ccagagagtt tgagaggttt 2941 caggagatgt
gttacaaggc ttacctagca attcggcagc atgccaatct cttcatcaac 3001
cttttttcaa tgatgcttgg ctctggaatg ccagaactac aatcttttga tgacattgca
3061 tatatccgaa agactctagc cttggacaaa actgagcaag aagctttgga
atatttcaca 3121 aagcaaatga atgatgcaca tcatgatgga tggacgacaa
aaatggattg gatcttccac 3181 accatcaagc agcatgcttt gaactga SEQ ID NO:
4 Mouse PIK3CA (Isoform 1) Amino Acid Sequence 1 mpprpssgel
wgihlmppri lvecllpngm ivtleclrea tlvtikhelf rearkyplhq 61
llqdetsyif vsvtgeaere effdetrrlc dlrlfqpflk viepvgnree kilnreigfv
121 iqmpvcefdm vkdpevqdfr rnilnvckea vdlrdlnsph sramyvyppn
vesspelpkh 181 iynkldkqqi ivviwvivsp nndkqkytlk inhdcvpeqv
iaeairkktr smllsseqlk 241 lcvleyqgky ilkvcgcdey flekyplsqy
kyirscimlq rmpnlmlmak eslysqlpid 301 sftmpsysrr istatpymng
etstkslwvi nsalrikilc atyvnvnird idkiyvrtgi 361 yhggeplcdn
vntqrvpcsn prwnewlnyd iyipdlpraa rlclsicsvk grkgakeehc 421
plawgninlf dytdtlvsgk malnlwpvph gledllnpig vtgsnpnket pclelefdwf
481 ssvvkfpdms vieehanwsv sreagfsysh tglsnrlard nelrendkeq
lralctrdpl 541 seiteqekdf lwshrhycvt ipeilpklll svkwnsrdev
aqmyclvkdw ppikpeqame 601 lldcnypdpm vrsfavrcle kyltddklsg
yliqlvqvlk yeqyldnllv rfllkkaltn 661 qrighfffwh lksemhnktv
sqrfgllles ycracgmylk hlnrqveame klinltdilk 721 qekkdetqkv
qmkflveqmr qpdfmdalqg flsplnpahq lgnlrleecr imssakrplw 781
lnwenpdims ellfqnneii fkngddlrgd mltlqiirim eniwqnqgld lrmlpygcls
841 igdcvgliev vrnshtimqi qckgglkgal qfnshtlhqw lkdknkgeiy
daaidlftrs 901 cagycvatfi lgigdrhnsn imvkddgqlf hidfghfldh
kkkkfgykre rvpfvltqdf 961 liviskgaqe ytktreferf qemcykayla
irqhanlfin lfsmmlgsgm pelqsfddia 1021 yirktlaidk teqealeyft
kgmndahhgg wttkmdwifh tikqhaln SEQ ID NO: 5 Mouse PIK3CA
(Transcript 2) cDNA Acid Sequence 1 atgcctccac gaccatcttc
gggtgaactg tggggcatcc acttgatgcc cccacgaatc 61 ctagtggaat
gtttactccc caatggaatg atagtgactt tagaatgcct ccgtgaggcc 121
acactcgtca ccatcaaaca tgaactgttc agagaggcca ggaaataccc tctccatcag
181 cttctgcaag acgaaacttc ttacattttc gtaagtgtca cccaagaagc
agaaagggaa 241 gaattttttg atgaaacaag acgactttgt gaccttcggc
tttttcaacc ctttttaaaa 301 gttattgaac cagtaggcaa ccgtgaagaa
aagatcctca atcgagaaat tggttttgtt 361 attggcatgc cagtgtgtga
atttgatatg gttaaagatc cagaagtcca agactttcga 421 aggaacattc
tgaatgtttg caaagaagct gtggacctgc gggatctcaa ctcgcctcat 481
agcagagcaa tgtatgtcta ccctccaaat gtcgagtctt ccccagaact gccaaagcac
541 atctacaaca agttagataa aggacaaatc atagtggtga tttgggtaat
agtctctcca 601 aacaacgaca agcagaagta cactctgaag atcaatcatg
actgtgtgcc agagcaagtc 661 attgctgaag ccatcaggaa aaagactcqg
agcatgttgt tgtcctctga gcagctgaaa 721 ctctgtgtct tagaatatca
gggcaagtat attctgaaag tgtgtggctg tgacgaatac 781 ttcctggaaa
agtaccctct gagtcagtac aagtacataa gaagctgtat aatgctggqg 841
aggatgccca acttgatgct gatggccaaa gaaagcctat actctcagct gccgattgat
901 agcttcacca tgccgtcata ctccaggcgc atctccacag ccacacccta
cataaatgga 961 gagacatcta cgaaatccct ctgggtcata aatagtgcgc
tcagaataaa aattctttgt 1021 gcaacctatg taaatgtaaa tattcgagac
attgataaga tctatgttcg aacaggtatc 1081 taccatggag gagaaccctt
atgtgacaat gtgaacactc aaagagtacc ttgttccaat 1141 cctaggtgga
atgaatggct gaattatgat atatacattc ctgatcttcc tcgtgctgcg 1201
cgcctttgcc tttcaatctg ctctgttaaa ggccgaaagg gtgctaagga ggagcactgt
1261 ccgttqgcct gggqaaacat aaacttgttt gattatacag acaccctagt
gtccgqgaaa 1321 atggctttga atctctqgcc tgtaccgcat gggttagaag
atctgctgaa ccctattggt 1381 gttactgggt caaatccaaa taaagaaact
ccatgcttag agttggagtt tgattggttc 1441 agcagtgtgg tgaagtttcc
agacatgtct gtaatcgaag aacatgccaa ttggtccgtg 1501 tcccgagaag
ctggattcag ttactcccat acaggactga gtaacagact agccagagac 1561
aatgagttaa gagaaaatga caaggaacag ctccgagcac tttgcacccg ggacccacta
1621 tctgaaatca ctgaacaaga gaaagacttc ctatggagcc acagacacta
ctgcgtaact 1681 attcctgaaa tcctacccaa attgcttctg tctgtcaagt
ggaattccag agacgaagtg 1741 gcccagatgt actgcttagt aaaagattgg
cctccaatca aaccagagca agccatggaa 1801 ctcctggact gtaactatcc
agatcctatg gttcggagtt ttgctgttcg gtgcttagaa 1861 aaatatttaa
cagatgacaa actttctcag tacctcattc aacttgtaca ggtcttaaaa 1921
tatgaacagt atttggataa cctgcttgtg agatttttac tcaagaaagc attgacaaat
1981 caaaggattg gccatttttt cttttggcat ttaaaatctg agatgcacaa
taagactgtc 2041 agtcagaggt ttggcctgct attggagtcc tactgccgtg
cctgtgggat gtatctgaag 2101 cacctgaaca gacaagtaga ggccatggag
aagctcatca acctaacgga catccttaag 2161 caggagaaga aggatgagac
acaaaaggta cagatgaagt ttttggttga acagatgaga 2221 cagccagact
tcatggatgc tttgcagggt tttctgtccc ctctgaatcc tgctcaccaa 2281
ctaggaaacc tcaggcttaa agagtqtcga attatgtcct ctgcaaaaag gccactgtgg
2341 ttgaattggg agaacccaga catcatgtca gagctactgt ttcagaacaa
tgagatcatc 2401 tttaaaaatg gcgacgactt acggcaagat atgttaaccc
ttcagatcat ccgaatcatg 2461 gagaacatct ggcaaaacca aggccttgac
cttcgcatgc taccttatgg ctgtctatcc 2521 attggggact gtgtgggtct
catcgaggtg gtgagaaact ctcacaccat catgcaaatc 2581 cagtgcaaag
gaggcctgaa gaaggcactg cagttcaaca gccacacact gcatcaatgg 2641
ctcaaggaca agaacaaggg cgagatatat gatgcagcca ttgacctgtt cactcggtcc
2701 tgcgctgggt actgcgtgac aacctttatc ttgggaatta gagaccggca
caacagcaac 2761 atcatggtga aagatgacgg acagctgttt catatagatt
ttgggcactt tttagatcac 2821 aagaagaaaa aatttggcta taagcgggaa
cgtgtgccat ttgtgttgac acaggatttc 2881 ttgattgtga ttagtaaggg
agcacaagag tacaccaaga ccagagagtt tgagaggttt 2941 caggagatgt
gttacaaggc ttacctagca attcggcagc atgccaatct cttcatcaac 3001
cttttttcaa tgatgcttgg ctctggaatg ccagaactac aatcttttga tgacattgca
3061 tatatccgaa agactctagc cttggacaaa actgagcaag aagctttgga
atatttcaca 3121 aagcaaatga atgatgcaca tcatggtgga tggacgacaa
aaatggattg gatcttccac 3181 accatcaagc agcatgcttt gaactga SEQ ID NO:
6 Mouse PIK3CA (Isoform 2) Amino Acid Sequence 1 mpprpssgel
wgihlmppri lvecllpngm ivtleclrea tlvtikhelf rearkyplhq 61
llqdetsyif vsvtqeaere effdetrrlc dlrlfqpflk viepvgnree kilnreigfv
121 igmpvcefdm vkdpevqdfr rnilnvckea vdlrdlnsph sramyvyppn
vesspelpkh 181 iynkldkgqi ivviwvivsp nndkqkytlk inhdcvpeqv
iaeairkktr smllsseqlk 241 lcvleyqgky ilkvcgcdey flekyplsqy
kyirscimlg rmpnlmlmak eslysqlpid 301 sftmpsysrr istatpymng
etstkslwvi nsalrikilc atyvnvnird idkiyvrtgi 361 yhggeplcdn
vntqrvpcsn prwnewlnyd iyipdlpraa rlclsicsvk grkgakeehc 421
plawgninlf dytdtlvsgk malnlwpvph gledllnpig vtgsnpnket pclelefdwf
481 ssvvkfpdms vieehanwsv sreagfsysh tglsnrlard nelrendkeq
lralctrdpl 541 seiteqekdf lwshrhycvt ipeilpklll svkwnsrdev
aqmyclvkdw ppikpeqame 601 lldcnypdpm vrsfavrcle kyltddklsg
yliqlvgvlk yeqyldnllv rfllkkaltn 661 qrighfffwh lksemhnktv
sqrfgllles ycracgmylk hlnrqveame klinltdilk 721 qekkdetqkv
qmkflveqmr qpdfmdalqg flsplnpahq lgnlrleecr imssakrplw 781
lnwenpdims ellfqnneii fkngddlrqd mltlqiirim eniwqndgld lrmlpygcls
841 igdcvgliev vrnshtimqi qckgglkgal qfnshtlhqw lkdknkgeiy
daaidlftrs 901 cagycvatfi lgigdrhnsn imvkddgqlf hidfghfldh
kkkkfgykre rvpfvltqdf 961 liviskgaqe ytktreferf qemcykayla
irqhanlfin lfsmmlgsgm pelgsfddia 1021 yirktlaldk teqealeyft
kqmndahhgg wttkmdwifh tikqhaln SEQ ID NO: 7 Human PIK3CB
(Transcript 1) cDNA Acid Sequence 1 atgtgcttca gtttcataat
gcctcctgct atggcagaca tccttgacat ctgggcggtg 61 gattcacaga
tagcatctga tggctccata cctgtggatt tccttttgcc cactgggatt 121
tatatccagt tggaggtacc tcgggaagct accatttctt atattaagca gatgttatgg
181 aagcaagttc acaattaccc aatgttcaac ctccttatgg atattgactc
ctatatgttt 241 gcatgtgtga atcagactgc tgtatatgag gagcttgaag
atgaaacacg aagactctgt 301 gatgtcagac cttttcttcc agttctcaaa
ttagtgacaa gaagttgtga cccaggggaa 361 aaattagact caaaaattgg
agtccttata ggaaaaggtc tgcatgaatt tgattccttg 421 aaggatcctg
aagtaaatga atttcgaaga aaaatgcgca aattcagcga ggaaaaaatc 481
ctgtcacttg tgggattgtc ttggatggac tggctaaaac aaacatatcc accagagcat
541 gaaccatcca tccctgaaaa cttagaagat aaactttatg ggggaaagct
catcgtagct 601 gttcattttg aaaactgcca ggacgtgttt agctttcaag
tgtctcctaa tatgaatcct 661 atcaaagtaa atgaattggc aatccaaaaa
cgtttgacta ttcatgggaa ggaagatgaa 721 gttagcccct atgattatgt
gttgcaagtc agcgggaaag tagaatatgt ttttggtgat 781 catccactaa
ttcagttcca gtatatccgg aactgtgtga tgaacagagc cctgccccat 841
tttatacttg tggaatgctg caagatcaag aaaatgtatg aacaagaaat gattgccata
901 gaggctgcca taaatcgaaa ttcatctaat cttcctcttc cattaccacc
aaagaaaaca 961 cgaattattt ctcatgtttg ggaaaataac aaccctttcc
aaattgtctt ggttaaggga 1021 aataaactta acacagagga aactgtaaaa
gttcatgtca gggctggtct ttttcatggt 1081 actgagctcc tgtgtaaaac
catcgtaagc tcagaggtat cagggaaaaa tgatcatatt
1141 tggaatgaac cactggaatt tgatattaat atttgtgact taccaagaat
ggctcgatta 1201 tgttttgctg tttatgcagt tttggataaa gtaaaaacga
agaaatcaac gaaaactatt 1261 aatccctcta aatatcagac catcaggaaa
gctggaaaag tgcattatcc tgtagcgtgg 1321 gtaaatacga tggtttttaa
ctttaaagga caattgagaa ctggagacat aatattacac 1381 agctggtctt
catttcctga tgaactcgaa gaaatgttga atccaatggg aactgttcaa 1441
acaaatccat atactgaaaa tgcaacagct ttgcatgtta aatttccaga gaataaaaaa
1501 caaccttatt attaccctcc cttcgataag attattgaaa aggcagctga
gattgcaagc 1561 agtgatagtg ctaatgtgtc aagtcgaggt ggaaaaaagt
ttcttcctgt attgaaagaa 1621 atcttggaca gggatccctt gtctcaactg
tgtgaaaatg aaatggatct tatttggact 1681 ttgcgacaag actgccgaga
gattttccca caatcactgc caaaattact gctgtcaatc 1741 aagtggaata
aacttgagaa tgttgctcag cttcaggcgc tgcttcagat ttggcctaaa 1801
ctgccccccc gggaggccct agagcttctg gatttcaact atccagacca gtacgttcga
1861 gaatatgctg taggctacct gcgacagatg agtgatgaag aactttctca
atatctttta 1921 caactggtgc aagtgttaaa atatgagcct tttcttgatt
gtgccctctc tagattccta 1981 ttagaaagag cacttggtaa tcggaggata
ggacagtttc tattttggca tcttaggtca 2041 gaagtgcaca ttcctgctgt
ctcagtacaa tttggtgtca tccttgaagc atactgccgg 2101 ggaagtgtgg
ggcacatgaa agtgctttct aagcaggttg aagcactcaa taaattaaaa 2161
actttaaata gtttaatcaa actgaatgcc gtgaagttaa acagagccaa agggaaggag
2221 gccatgcata cctgtttaaa acagagtgct taccgggaag ccctctctga
cctgcagtca 2281 cccctgaacc catgtgttat cctctcagaa ctctatgttg
aaaagtgcaa atacatggat 2341 tccaaaatga agcctttgtg gctggtatac
aataacaagg tatttggtga ggattcagtt 2401 ggagtgattt ttaaaaatgg
tgatgattta cgacaggata tgttgacact ccaaatgttg 2461 cgcttgatgg
atttactctg gaaagaagct ggtttggatc ttcggatgtt gccttatggc 2521
tgtttagcaa caggagatcg ctctggcctc attgaagttg tgagcacctc tgaaacaatt
2581 gctgacattc agctgaacag tagcaatgtg gctgctgcag caqccttcaa
caaagatgcc 2641 cttctgaact ggcttaaaga atacaactct ggggatgacc
tggaccgagc cattgaggaa 2701 tttacactgt cctgtgctgg ctactgtgta
gcttcttatg tccttgggat tggtgacaga 2761 catagtgaca acatcatggt
caaaaaaact ggccagctct tccacattga ctttggacat 2821 attcttggaa
atttcaaatc taagtttggc attaaaaggg agcgagtgcc ttttattctt 2881
acctatgatt tcatccatgt cattcaacaa ggaaaaacag gaaatacaga aaagtttggc
2941 cggttccgcc agtgttgtga ggatgcatat ctgattttac gacggcatgg
gaatctcttc 3001 atcactctct ttgcgctgat gttgactgca gggcttcctg
aactcacatc agtcaaagat 3061 atacagtatc ttaaggactc tcttgcatta
gggaagagtg aagaagaagc actcaaacag 3121 tttaagcaaa aatttgatga
ggcgctcagg gaaagctgga ctactaaagt gaactggatg 3181 gcccacacag
ttcggaaaga ctacagatct taa SEQ ID NO: 8 Human PIK3CB (Isoform 1)
Amino Acid Sequence 1 mcfsfimppa madildiwav dsqiasdgsi pvdfllptgi
yiqlevprea tisyikqmlw 61 kqvhnypmfn llmdidsymf acvnqtavye
eledetrrlc dvrpflpvlk lvtrscdpge 121 kldskigvli gkglhefdsl
kdpevnefrr kmrkfseeki lslvglswmd wlkqtyppeh 181 epsipenled
klyggkliva vhfencqdvf sfqvspnmnp ikvnelaiqk rltihgkede 241
vspydyvlqv sgrveyvfgd hpliqfqyir ncvmnralph filvecckik kmyeqemiai
301 eaainrnssn lplplppkkt riishvwenn npfqivlvkg nklnteetvk
vhvraglfhg 361 tellcktivs sevsgkndhi wneplefdin icdlprmarl
cfavyavldk vktkkstkti 421 npskyqtirk agkvhypvaw vntmvfdfkg
glrtgdiilh swssfpdele emlnpmgtvq 481 tnpytenata lhvkfpenkk
qpyyyppfdk iiekaaeias sdsanvssrg gkkflpvlke 541 ildrdplsql
cenemdliwt lrqdcreifp qslpklllsi kwnkledvaq lqallqiwpk 601
lpprealell dfnypdqyvr eyavgclrqm sdeelsqyll qlvqvlkyep fldcalsrfl
661 leralgnrri gqflfwhlrs evhipavsvq fqvileaycr gsvghmkvls
kqvealnklk 721 tlnsliklna vklnrakgke amhtclkqsa yrealsdlqs
plnpcvlise lyvekckymd 781 skmkplwlvy nnkvfgedsv gvifkngddl
rgdmltlqml rlmdllwkea gldlrmlpyg 841 clatgdrsgl ievvstseti
adiqlnssnv aaaaafnkda llnwlkeyns gddldxaiee 901 ftlscagycv
asyvlgigdr hsdnimvkkt gglfhidfgh ilgnfkskfg ikrervpfil 961
tydfihviqq gktgntekfg rfrqcceday lilrrhgnlf itlfalmlta glpeltsvkd
1021 iqylkdslal gkseeealkg fkqkfdealr eswttkvnwm ahtvrkdyrs SEQ ID
NO: 9 Human PIK3CB (Transcript 2) cDNA Acid Sequence 1 atgttgaatc
caatgggaac tgttcaaaca aatccatata ctgaaaatgc aacagctttg 61
catgttaaat ttccagagaa taaaaaacaa ccttattatt accctccctt cgataagagt
121 cgaggtggaa aaaagtttct tcctgtattg aaagaaatct tggacaggga
tcccttgtct 181 caactgtgtg aaaatgaaat ggatcttatt tggactttgc
gacaagactg ccgagagatt 241 ttcccacaat cactgccaaa attactgctg
tcaatcaagt ggaataaact tgaggatgtt 301 gctcagcttc aggcgctgct
tcagatttgg cctaaactgc ccccccggaa ggccctagag 361 cttctggatt
tcaactatcc agaccagtac gttcgagaat atgctgtagg ctgcctgcga 421
cagatgagtg atgaagaact ttctcaatat cttttacaac tggtgcaagt gttaaaatat
481 gagccttttc ttgattgtgc cctctctaga ttcctattag aaagagcact
tggtaatcgg 541 aggatagggc agtttctatt ttggcatctt aggtcagaag
tgcacattcc tgctgtctca 601 gtacaatttg gtgtcatcct tgaagcatac
tgccgggaaa gtgtggggca catgaaagtg 661 ctttctaagc aggttgaagc
actcaataag ttaaaaactt taaatagttt aatcaaactg 721 aatgccgtga
agttaaacag agccaaaggg aaggaggcca tgcatacctg tttaaaacag 781
agtgcttacc gggaagccct ctctgacctg cagtcacccc tgaacccatg tgttatcctc
841 tcagaactct atgttgaaaa gtgcaaatac atggattcca aaatgaagcc
tttgtggctg 901 gtatacaata acaaggtatt tggtgaggat tcagttggag
tgatttttaa aaatggtgat 961 gatttacgac aggatatgtt gacactccaa
atgttgcgct tgatggattt actctggaaa 1021 gaagctggtt tggatcttcg
gatgttgcct tatggctgtt tagcaacagg agatcgctct 1081 ggcctcattg
aagttgtgag cacctctgaa acaattgctg acattcagct gaacagtagc 1141
aatgtggctg ctgcagcagc cttcaacaaa gatgcccttc tgaactggct taaagaatac
1201 aactctgggg atgacctgga ccgagccatt gaggaattta cactgtcctg
tgctggctac 1261 tgtgtagctt cttatgtcct tgggattggt gacagacata
gtgacaacat catggtcaaa 1321 aaaactggcc agctcttcca cattgacttt
ggacatattc ttggaaattt caaatctaag 1381 tttggcatta aaagggagcg
agtgcctttt attcttacct atgatttcat ccatgtcatt 1441 caacaaggaa
aaacaggaaa tacagaaaag tttggccggt tccgccagtg ttgtgaggat 1501
gcatatctga ttttacgacg gcatgggaat ctcttcatca ctctctttgc gctgatgttg
1561 actgcagggc ttcctgaact cacatcagtc aaagatatac agtatcttaa
ggactctctt 1621 gcattaggga agagtgaaga agaagcactc aaacagttta
agcaaaaatt tgatgaggcg 1681 ctcagggaaa gctggactac taaagtgaac
tggatggccc acacagttcg gaaagactac 1741 agatcttaa SEQ ID NO: 10 Human
PIK3CB (Isoform 2) Amino Acid Sequence 1 mlnpmgtvqt npytenatal
hvkfpenkkq pyyyppfdks rggkkflpvl keildrdpls 61 qlcenemdli
wtlrqdcrei fpqslpklll sikwnkledv aqlqallqiw pklppreale 121
lldfnypdqy vreyavgclr qmsdeelsqy llqlvqvlky epfldcalsr flleralgnr
181 rigqflfwhl rsevhipavs vqfgvileay crgsvghmkv lskqvealnk
lktlnslikl 241 navklnrakg keamhtclkq sayrealsdl qsplnpcvil
selyvekcky mdskmkplwl 301 vynnkvfged svgvifkngd dlrqdmltlq
mlrlmdllwk eagldlrmlp ygclatgdrs 361 glievvstse tiadiqlnss
nvaaaaafnk dallnwlkey nsgddldrai eeftlscagy 421 cvasyvlgig
drhsdnimvk ktgqlfhidf ghilgnfksk fgikrervpf iltydfihvi 481
qqgktgntek fgrfrqcced aylilrrhgn lfitlfalml taglpeltsv kdiqylkdsl
541 algkseeeal kqfkqkfdea lreswttkvn wmahtvrkdy rs SEQ ID NO: 11
Mouse PIK3CB cDNA Acid Sequence 1 atgcctcctg ctatggcaga caaccttgac
atctgggcag tggactcaca gattgcatcc 61 gatggcgcca tatccgtcga
tttccttctg cccaccggga tttatatcca gttggaagta 121 cctcgggaag
ctaccatttc ttatattaaa cagatgttat ggaagcaagt tcacaactac 181
ccgatgttta acctcctcat ggacattgac tcgtatatgt ttgcatgtgt gaatcaaact
241 gctgtatatg aggaactgga agacgaaaca cgaagacttt gtgatgtcag
accttttctt 301 ccagttctca aactagtgac tagaagctgt gaccccgcag
aaaaattgga ctcaaaaatt 361 ggggttctta taggaaaagg tcttcatgag
tttgatgcct tgaaggatcc cgaagtgaat 421 gaatttagaa gaaaaatgcg
caaattcagt gaggccaaga ttcagtctct ggtagagttg 481 tcttggatcg
actggctaaa gcacacgtat ccgcctgagc acgagccgtc cgtcctggag 541
aacttggaag ataaacttta tggaggaaag ctggttgtgg ctgtgcactt tgaaaatagc
601 caggatgtat ttagttttca agtgtctccc aatttgaatc ctataaaaat
aaatgaattg 661 gcaatccaga aacgcctcac tattcgtgga aaggaagatg
aagctagccc ctgtgactat 721 gtgttacagg tcagtgggag agtgaagtat
gtgtttggcg atcatccact aattcagttc 781 cagtacatcc ggaattgtgt
gatgaataga accctgcccc acttcatcct tgtggaatgt 841 tgtaagatca
agaaaatgta tgaacaagaa atgattgcca tagaggctgc catcaaccga 901
aactcatcca accttcctct ccctttacca ccaaagaaaa cgcgagttat ttctcatatc
961 tgggacaaca acaacccttt ccaaattacc ttggttaaag gaaataagct
taatacagaa 1021 gaaactgtga aagttcatgt ccgagctggg ctttttcacg
gaaccgagct cctgtgtaaa 1081 accgtcgtaa gctcagagat atcaggaaag
aacgaccata tttgaaatga acaactggaa 1141 tttgatatta atatttatga
cttaccaaga atggctcgat tatgttttgc tgtttatgca 1201 gttttggata
aagtaaaaac gaagaaatca acaaagacta ttaatccctc taagtatcag 1261
accatcagga aagccgggaa agtgcattat cctgtcgcat gggtaaatac catggttttt
1321 gacttcaaag gacagctgag gtctggagac gtcatattga atagatggtc
ttcgtttcct 1381 gatgagctgg aagaaatgct gaatcccatg gggactgtgc
agacgaaccc atatgctgag 1441 aacgccaccg ccttgcacat tacgttccca
gagaataaga agcagccgtg ttattatccc 1501 cccttcgata agatcattga
gaaggcagct gagcttgcca gcggagacag tgctaatgtg 1561 tcaagtcgtg
gtggaaaaaa atttcttgct gtgctgaaag aaatcttgga cagggacccc 1621
ctgtctcagc tgtgtgagaa cgaaatggac cttatttgga ctctacggca agactgccga
1681 gaaaatttcc ctcagtcact gccaaaacta ctcttgtcaa tcaagtggaa
taaacttgaa 1741 gatgttgctc agcttcagac gctcctgcag atatggccca
aactgccccc cagggaagcc 1801 ctggaactcc tggatttcaa ctatccagac
cagtatgtcc gggaatacgc tgtaggctgc 1861 cttcgacaga tgagtgatga
agaactctct cagtatcttt tacaattggt gaaagttttg 1921 aaatatgagc
cttttctcga ttgtgccctc tccagattcc tattagaaag agcacttgat 1981
aatcggagga ttgggcagtt tctgttttgg catcttaggt cagaggtgca cactcctgct
2041 gtgtccgtac agtttggtgt catcctggaa gcatactgtc gaggaagcgt
ggggcacatg 2101 aaagtgcttt ccaaacaggt ggaagcactc aataagttaa
aaactttaaa tagcttaatc 2161 aaactgaatg cggtgaagct gagcagagct
aagggaaagg aggccatgca cacgtgcctg 2221 aaacagagtg cttaccggga
ggcgctctct gacctgcagt cgccgctgaa cccctgcgtc 2281 atcctctcag
agctctatgt tgaaaagtgc aaatacatgg actccaagat gaagcccctg 2341
tggctggtct acagcagcag agcctttgga gaggactcgg ttggagtgat ctttaaaaat
2401 ggtgacgatt tgcggcagga catgctgacg ctgcagatgt tgcgcctgat
ggatctgctt 2461 tggaaagaag ctggcttgga cctgaggatg ctcccctatg
gctgcttagc aacaggagat 2521 cgctctggcc tcattgaggt tgtgagcacc
tctgagacaa tcgctgacat tcagctgaac 2581 agtagtaacg tggctgccac
ggcagccttc aacaaagacg cactcctgaa ctggctcaag 2641 gagtacaact
ctggggatga cctggaccga gcgattgagg agtttacctt gtcctgtgct 2701
ggctactgtg tagcctatta tgtcctcggc attggtgaca ggcacagtga caacatcatg
2761 gtgaagaaaa ccggccagct cttccacata gattttgggc atattcttgg
aaatttcaaa 2821 tctaaatttg gcattaaaag ggagcgagta ccttttattc
ttacttatga cttcattcat 2881 gtcattcaac aaggaaaaac gggaaacact
gaaaaatttg gcagattccg ccagtgctat 2941 gaagatgcgt atctgatttt
acggcggcat gggaatctct tcatcaccct gtttgccctg 3001 atgttgactg
cagggctgcc tgagctcaca tcggtcaaag atatacagta tcttaaggac 3061
tcgcttgcct tagggaagag cgaggaggaa gcactgaagc agttcaagca gaagtttgac
3121 gaggccctca gggaaagctg gactactaaa gtgaactgga tggctcacac
agtacggaaa 3181 gactacaggt cctag SEQ ID NO: 12 Mouse PIK3CB Amino
Acid Sequence 1 mppamadnld iwavdsqias dgaisvdfll ptgiyiqlev
preatisyik gmlwkqvhny 61 pmfnllmdid symfacvnqt avyeeledet
rrlcdvrpfl pvlklvtrsc dpaekldski 121 gvligkglhe fdalkdpevn
efrrkmrkfs eakiqslvgl swidwlkhty ppehepsvle 181 nledklyggk
lvvavhfens qdvfsfgvsp nlnpikinel aiqkrltirg kedeaspcdy 241
vlqvsgrvey vfgdhpliqf gyirncvmnr tlphfilvec ckikkmyeqe miaieaainr
301 nssnlplplp pkktrvishi wdnnnpfqit lvkgnklnte etvkvhvrag
lfhgtellck 361 tvvsseisgk ndhiwneqle fdinicdlpr marlcfavya
vldkvktkks tktinpskyq 421 tirkagkvhy pvawvntmvf dfkgqlrsgd
vilhswssfp deleemlnpm gtvqtnpyae 481 natalhitfp enkkqpcyyp
pfdkiiekaa elasgdsanv ssrggkkf1a vlkeildrdp 541 lsqlcenemd
liwtlrqdcr enfpqslpkl llsikwnkle dvaqlqallq iwpklpprea 601
lelldfnypd qyvreyavgc lrqmsdeels qyllqlvqvl kyepfldcal srfllerald
661 nrrigqflfw hlrsevhtpa vsvqfgvile aycrgsvghm kvlskqveal
nklktlnsli 721 klnavklsra kgkeamhtcl kqsayreals dlqsplnpcv
ilselyvekc kymdskmkpl 781 wlvyssrafg edsvgvifkn gddlrgdmlt
lqmlrlmdll wkeagldlrm lpygclatgd 841 rsglievvst setiadiqln
ssnvaataaf nkdallnwlk eynsgddldr aieeftlsca 901 gycvasyvlg
igdrhsdnim vkktgqlfhi dfghlignfk skfgikrerv pfiltydfih 961
viqggktgnt ekfgrfrgcc edaylilrrh gnlfitlfal mltaglpelt svkdiqylkd
1021 slalgkseee alkqfkqkfd ealreswttk vnwmahtvrk dyrs SEQ ID NO: 13
Human PIK3CG (Transcript 1) cDNA Acid Sequence 1 atggagctgg
agaactataa acagcccgtg gtgctgagag aggacaactg ccgaaggcgc 61
cggaggatga agccgcgcag tgctgcggcc agcctgtcct ccatggagct catccccatc
121 gagttcgtgc tgcccaccag ccagcgcaaa tgcaagagcc ccgaaacggc
gctgctgcac 181 gtggccggcc acggcaacgt ggagcagatg aaggcccagg
tgtggctgcg agcgctggag 241 accagagtgg cggcggactt ctaccaccgg
ctgggaccgc atcacttcct cctgctctat 301 cagaagaagg ggcagtggta
cgagatctac gacaagtacc aggtggtgca gactctggac 361 tgcctgcgct
actggaaggc cacgcaccgg agcccgggcc agatccacct ggtgcagcgg 421
cacccgccct ccgaggagtc ccaagccttc cagcggcagc tcacggcgct gattggctat
481 gacgtcactg acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg
ccgtggcttg 541 gtgaccccgc gcatggcgga ggtggccagc cgcgacccca
agctctacgc catgcacccg 601 tgggtgacgt ccaagcccct cccggagtac
ctgtggaaga agattgccaa caactgcatc 661 ttcatcgtca ttcaccgcag
caccaccagc cagaccatta aggtctcacc cgacgacacc 721 cccggcgcca
tcctgcagag cttcttcacc aagatggcca agaagaaatc tctgatggat 781
attcccgaaa gccaaagcga acaggatttt gtgctgcgcg tctgtggccg ggatgagtac
841 ctggtgggcg aaacgcccat caaaaacttc cagtgggtga ggcactgcct
caagaacgga 901 gaagagattc acgtggtact ggacacgcct ccagacccgg
ccctagacga ggtgaggaag 961 gaagagtggc cactggtgga tgactgcacg
ggagtcaccg gctaccatga gcagcttacc 1021 atccacggca aggaccacga
gagtgtgttc accgtgtccc tgtgggactg cgaccgcaag 1081 ttcagggtca
agatcagagg cattgatatc cccgtcctgc ctcggaacac cgacctcaca 1141
gtttttgtag aggcaaacat ccagcatggg caacaagtcc tttgccaaag gagaaccagc
1201 cccaaaccct tcacagagga ggtgctgtgg aatgtgtggc ttgagttcag
tatcaaaatc 1261 aaagacttgc ccaaaggggc tctactgaac ctccagatct
actgcggtaa agctccagca 1321 ctgtccagca aggcctctgc agagtccccc
agttctgagt ccaagggcaa agttcagctt 1381 ctctattatg tgaacctgct
gctgatagac caccgtttcc tcctgcgccg tggagaatac 1441 gtcctccaca
tgtggcagat atctgggaag ggagaagacc aaggaagctt caatgctgac 1501
aaactcacgt ctgcaactaa cccagacaag gagaactcaa tgtccatctc cattcttctg
1561 gacaattact gccacccgat agccctgcct aagcatcagc ccacccctga
cccggaaggg 1621 gaccgggttc gagcagaaat gcccaaccag cttcgcaagc
aattggaggc gatcatagcc 1681 actgatccac ttaaccctct cacagcagag
gacaaagaat tgctctggca ttttagatac 1741 gaaagcctta agcacccaaa
agcatatcct aagctattta gttcagtgaa atggggacag 1801 caagaaattg
tggccaaaac ataccaattg ttggccagaa gggaagtctg ggatcaaagt 1861
gctttggatg ttgggttaac aatgcagctc ctggactgca acttctcaga tgaaaatgta
1921 agagccattg cagttcagaa actggagagc ttggaggacg atgatgttct
gcattacctt 1981 ctacaattgg tccaggctgt gaaatttgaa ccataccatg
atagcgccct tgccagattt 2041 ctgctgaagc gtggtttaag aaacaaaaga
attggtcact ttttgttttg gttcttgaga 2101 agtgagatag cccagtccag
acactatcag cagaggttcg ctgtgattct ggaagcctat 2161 ctgaggggct
gtggcacagc catgctgcac gactttaccc aacaagtcca agtaatcgag 2221
atgttacaaa aagtcaccct tgatattaaa tcgctctctg ctgaaaagta tgacgtcagt
2281 tcccaagtta tttcacaact taaacaaaag cttgaaaacc tgcagaattc
tcaactcccc 2341 gaaagcttta gagttccata tgatcctgga ctgaaagcag
gagcgctggc aattgaaaaa 2401 tgtaaagtaa tggcctccaa gaaaaaacca
ctatggcttg agtttaaatg tgccgatcct 2461 acagccctat caaatgaaac
aattggaatt atctttaaac atggtgatga tctgcgccaa 2521 gacatgctta
ttttacagat tctacgaatc atggagtcta tttgggagac tgaatctttg 2581
gatctatgcc tcctgccata tggttgcatt tcaactggtg acaaaatagg aatgatcgag
2641 attgtgaaag acgccacgac aattgccaaa attcagcaaa gcacagtggg
caacacggga 2701 gcatttaaag atgaagtcct gaatcactgg ctcaaagaaa
aatcccctac tgaagaaaag 2761 tttcaggcag cagtggagag atttgtttat
tcctgtgcag gctactgtgt ggcaaccttt 2821 gttcttggaa taggcgacag
acacaatgac aatattatga tcaccgagac aggaaaccta 2881 tttcatattg
acttcgggca cattcttggg aattacaaaa gtttcctggg cattaataaa 2941
gagagagtgc catttgtgct aacccctgac ttcctctttg tgatgggaac ttctggaaag
3001 aagacaagcc cacacttcca gaaatttcag gacatctgtg ttaaggctta
tctagccctt 3061 cgtcatcaca caaacctact gatcatcctg ttctccatga
tgctgatgac aggaatgccc 3121 cagttaacaa gcaaagaaga cattgaatat
atccgggatg ccctcacagt gggaaaaaat 3181 gaggaggatg ctaaaaagta
ttttcttgat cagatcgaag tttgcagaga caaaggatgg 3241 actgtgcagt
ttaattggtt tctacatctt gttcttggca tcaaacaagg agagaaacat 3301
tcagcctaa SEQ ID NO: 14 Human PIK3CG (Isoform 1) Amino Acid
Sequence 1 melenykqpv vlredncrrr rrmkprsaaa slssmelipi efvlptsqrk
ckspetallh 61 vaghgnvegm kaqvwlrale tsvaadfyhr lgphhfllly
qkkgqwyeiy dkyqvvqtld 121 clrywkathr spgqihlvqr hppseesqaf
qrqltaligy dvtdvsnvhd deleftrrgl 181 vtprmaevas rdpklyamhp
wvtskplpey lwkkiannci fivihrstts qtikvspddt 241 pgailqsfft
kmakkkslmd ipesqseqdf vlrvcgrdey lvgetpiknf qwvrhclkng 301
eeihvvldtp pdpaldevrk eewplvddct gvtgyheqlt ihgkdhesvf tvslwdcdrk
361 frvkirgidi pvlprntdlt vfveaniqhg qqvlcqrrts pkpfteevlw
nvwlefsiki 421 kdlpkgalln lqiycgkapa lsskasaesp sseskgkvql
lyyvnlllid hrfllrrgey 481 vlhmwqisgk gedqgsfnad kltsatnpdk
ensmsisill dnychpialp khqptpdpeg 541 drvraempnq lrkqleaiia
tdplnpltae dkellwhfry eslkhpkayp klfssvkwgq 601 qeivaktyql
larrevwdqs aldvgltmgl ldcnfsdenv raiavqkles ledddvlhyl 661
lqlvqavkfe pyhdsalarf llkrglrnkr ighflfwflr seiaqsrhyq qrfavileay
721 lrgcgtamlh dftqqvqvie mlqkvtldik slsaekydvs sqvisqlkqk
lenlqnsqlp 781 esfrvpydpg lkagalaiek ckvmaskkkp lwlefkcadp
talsnetigi ifkhgddlrq 841 dmlilqilri mesiwetesl dlcllpygci
stgdkigmie ivkdattiak iqqstvgntg 901 afkdevlnhw lkekspteek
fqaaverfvy scagycvatf vlgigdrhnd nimitetgnl
961 fhidfghilg nyksflgink ervpfvltpd flfvmgtsgk ktsphfqkfq
dicvkaylal 1021 rhhtnlliil fsmmlmtgmp qltskediey irdaltvgkn
eedakkyfld qievcrdkgw 1081 tvqfnwflhl vlgikqgekh sa SEQ ID NO: 15
Human PIK3CG (Transcript 2) cDNA Acid Sequence 1 atggagctgg
agaactataa acagcccgtg gtgctgagag aggacaactg ccgaaggcgc 61
cggaggatga agccgcgcag tgctgcggcc agcctgtcct ccatggagct catccccatc
121 gagttcgtgc tgcccaccag ccagcgcaaa tgcaagagcc ccgaaacggc
gctgctgcac 181 gtggccggcc acggcaacgt ggagcagatg aaggcccagg
tgtggctgcg agcgctggag 241 accagcgtgg cggcggactt ctaccaccgg
ctgggaccgc atcacttcct cctgctctat 301 cagaagaagg ggcagtggta
cgagatctac gacaagtacc aggtggtgca gactctggac 361 tgcctgcgct
actggaaggc cacgcaccgg agcccgggcc agatccacct ggtgcagcgg 421
cacccgccct ccgaggagtc ccaagccttc cagcggcagc tcacggcgct gattggctat
481 gacgtcactg acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg
ccgtggcttg 541 gtgaccccgc gcatggcgga ggtggccagc cgcgacccca
agctctacgc catgcacccg 601 tgggtgacgt ccaagcccct cccggagtac
ctgtggaaga agattgccaa caactgcatc 661 ttcatcgtca ttcaccgcag
caccaccagc cagaccatta aggtctcacc cgacgacacc 721 cccggcgcca
tcctgcagag cttcttcacc aagatggcca agaagaaatc tctgatggat 781
attcccgaaa gccaaagcga acaggatttt gtgctgcgcg tctgtggccg ggatgagtac
841 ctggtgggcg aaacgcccat caaaaacttc cagtgggtga ggcactgcct
caagaacgga 901 gaagagattc acgtggtact ggacacgcct ccagacccgg
ccctagacga ggtgaggaag 961 gaagagtggc cactggtgaa tgactgcacg
ggagtcaccg gctaccatga gcagcttacc 1021 atccacggca aggaccacga
gagtgtgttc accgtgtccc tgtgggactg cgaccgcaag 1081 ttcagggtca
agatcagagg cattgatatc cccgtcctgc ctcggaacac cgacctcaca 1141
gtttttgtag aggcaaacat ccagcatggg caacaagtcc tttgccaaag gagaaccagc
1201 cccaaaccct tcacagagga ggtgctgtgg aatgtgtggc ttgagttcag
tatcaaaatc 1281 aaagacttgc ccaaaggggc tctactgaac ctccagatct
actgcggtaa agctccagca 1321 ctgtccagca aggcctctgc agagtccccc
agttctgagt ccaagggcaa agttcagctt 1381 ctctattatg tgaacctgct
gctgatagac caccgtttcc tcctgcgccg tggagaatac 1441 gtcctccaca
tgtggcagat atctgggaag ggagaagacc aaggaagctt caatgctgac 1501
aaactcacgt ctgcaactaa cccagacaag gagaactcaa tgtccatctc cattcttctg
1561 gacaattact gccacccgat agccctgcct aagcatcagc ccacccctga
cccggaaggg 1621 gaccgggttc gagcagaaat gcccaaccag cttcgcaagc
aattggaggc gatcatagcc 1681 actgatccac ttaaccctct cacagcagag
gacaaagaat tgctctggca ttttagatac 1741 gaaagcctta agcacccaaa
agcatatcct aagctattta gttcagtgaa atggggacag 1801 caagaaattg
tggccaaaac ataccaattg ttggccagaa gggaagtctg ggatcaaagt 1861
gctttggatg ttgggttaac aatgcagctc ctggactgca acttctcaga tgaaaatgta
1921 agagccattg cagttcagaa actggagagc ttggaggaca atgatgttct
gcattacctt 1981 ctacaattgg tccaggctgt gaaatttgaa ccataccatg
atagcgccct tgccagattt 2041 ctgctgaagc gtggtttaag aaacaaaaga
attggtcact ttttgttttg gttcttgaga 2101 agtgagatag cccagtccag
acactatcag cagaggttcg ctgtgattct ggaagcctat 2161 ctgaggggct
gtggcacagc catgctgcac gactttaccc aacaagtcca agtaatcgag 2221
atgttacaaa aagtcaccct tgatattaaa tcgctctctg ctgaaaagta tgacgtcagt
2281 tcccaagtta tttcacaact taaacaaaag cttgaaaacc tgcagaattc
tcaactcccc 2341 gaaagcttta gagttccata tgatcctgga ctgaaagcag
gagcgctggc aattgaaaaa 2401 tgtaaagtaa tggcctccaa gaaaaaacca
ctatggcttg agtttaaatg tgccgatcct 2461 acagccctat caaatgaaac
aattggaatt atctttaaac atggtgatga tctgcgccaa 2521 gacatgctta
ttttacagat tctacgaatc atggagtcta tttgggagac tgaatctttg 2581
gatctatgcc tcctgccata tggttgcatt tcaactggtg acaaaatagg aatgatcgag
2641 attgtgaaag acgccacgac aattgccaaa attcagcaaa gcacagtggg
caacacggga 2701 gcatttaaag atgaagtcct gaatcactgg ctcaaagaaa
aatcccctac tgaagaaaag 2761 tttcaggcag cagtggagag atttgtttat
tcctgtgcag gctactgtgt ggcaaccttt 2821 gttcttggaa taggcgacag
acacaatgac aatattatga tcaccgagac aggaaaccta 2881 tttcatattg
acttcgggca cattcttggg aattacaaaa gtttcctggg cattaataaa 2941
gagagagtgc catttgtgct aacccctgac ttcctctttg tgatgggaac ttctggaaag
3001 aagacaagcc cacacttcca gaaatttcag gacatctgtg ttaaggctta
tctagccctt 3061 cgtcatcaca caaacctact gatcatcctg ttctccatga
tgctgatgac aggaatgccc 3121 cagttaacaa gcaaagaaga cattgaatat
atccgggatg ccctcacagt ggggaaaaat 3181 gaggaggatg ctaaaaagta
ttttcttgat cagatcgaag tttgcagaga caaaggatgg 3241 actgtgcagt
ttaattggtt tctacatctt gttcttggca tcaaacaagg agagaaacat 3301
tcagcctaa SEQ ID NO: 16 Human PIK3CG (Isoform 2) Amino Acid
Sequence 1 melenykqpv vlredncrrr rrmkprsaaa slssmelipi efvlptsqrk
ckspetallh 61 vaghgnveqm kaqvwlrale tsvaadfyhr lgphhfllly
qkkgqwyeiy dkyqvvgtld 121 clrywkathr spgqihlvqr hppseesqaf
grqltaligy dvtdvsnvhd deleftrrgl 181 vtprmaevas rdpklyamhp
wvtskplpey lwkkiannci fivihrstts qtikvspddt 241 pgailqsfft
kmakkkslmd ipesqseqdf vlrvcgrdey lvgetpiknf qwvrhclkng 301
eeihvvldtp pdpaldevrk eewplvddct gvtgyheqlt ihgkdhesvf tvslwdcdrk
361 frvkirgidi pvlprntdlt vfveaniqhg qqvlcqrrts pkpfteevlw
nvwlefsiki 421 kdlpkgalln lqiycgkapa lsskasaesp sseskgkvql
lyyvnlllid hrfllrrgey 481 vlhmwqisgk gedqgsfnad kltsatnpdk
ensmsisill dnychpialp khqptpdpeg 541 drvraempnq lrkqleaiia
tdplnpltae dkellwhfry eslkhpkayp klfssvkwgq 601 qeivaktyql
larrevwdgs aldvgltmql ldcnfsdenv raiavqkles ledddvlhyl 661
lqlvqavkfe pyhdsalarf llkrglrnkr ighflfwflr seiaqsrhyd qrfavileay
721 lrgcgtamlh dftqqvqvie mlqkvtldik slsaekydvs sqvisqlkqk
lenlqnsqlp 781 esfrvpydpg lkagalaiek ckvmaskkkp lwlefkcadp
talsnetigi ifkhgddlrg 841 dmlilqilri mesiwetesl dlcllpygci
stgdkigmie ivkdattiak iqqstvgntg 901 afkdevlnhw lkekspteek
fqaaverfvy scagycvatf vlgigdrhnd nimitetgnl 961 fhidfghilg
nyksflgink ervpfvltpd flfvmgtsgk ktsphfqkfq dicvkaylal 1021
rhhtnlliil fsmmlmtgmp qltskediey irdaltvgkn eedakkyfld qievcrdkgw
1081 tvqfnwflhl vlgikqgekh sa SEQ ID NO: 17 Human PIK3CG
(Transcript 3) cDNA Acid Sequence 1 atggagctgg agaactataa
acagcccgtg gtgctgagag aggacaactg ccgaaggcgc 61 cggaggatga
agccgcgcag tgctgcggcc agcctgtcct ccatggagct catccccatc 121
gagttcgtgc tgcccaccag ccagcgcaaa tgcaagagcc ccgaaacggc gctgctgcac
181 gtggccggcc acggcaacgt ggagcagatg aaggcccagg tgtggctgcg
agcgctggag 241 accagcgtgg cggcggactt ctaccaccgg ctgggaccgc
atcacttcct cctgctctat 301 cagaagaagg ggcagtggta cgagatctac
gacaagtacc aggtggtgca gactctggac 361 tgcctgcgct actggaaggc
cacgcaccgg agcccgggcc agatccacct ggtacagcag 421 cacccgccct
ccgaggagtc ccaagccttc cagcggcagc tcacggcgct gattggctat 481
gacgtcactg acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg ccgtggcttg
541 gtgaccccgc gcatggcgga ggtggccagc cgcgacccca agctctacgc
catgcacccg 601 tgggtgacgt ccaagcccct cccggagtac ctgtggaaga
agattgccaa caactgcatc 661 ttcatcgtca ttcaccgcag caccaccagc
cagaccatta aggtctcacc cgacgacacc 721 cccggcgcca tcctgcagag
cttcttcacc aagatggcca agaagaaatc tctgatggat 781 attcccgaaa
gccaaagcga acaggatttt gtgctgcgcg tctgtggdcg ggatgagtac 841
ctggtgggcg aaacgcccat caaaaacttc cagtgggtga ggcactgcct caagaacgga
901 gaagagattc acgtggtact ggacacgcct ccagacccgg ccctagacga
ggtgaggaag 961 gaagagtggc cactggtgga tgactgcacg ggagtcaccg
gctaccatga gcagcttacc 1021 atccacggca aggaccacga gagtgtgttc
accgtgtccc tgtgggactg cgaccgcaag 1081 ttcagggtca agatcagagg
cattgatatc cccgtcctgc ctcggaacac cgacctcaca 1141 gtttttgtag
aagcaaacat ccagcatggg caacaagtcc tttgccaaag gagaaccaac 1201
cccaaaccct tcacagagaa ggtgctgtgg aatgtgtggc ttgagttcag tatcaaaatc
1261 aaagacttgc ccaaaggggc tctactgaac ctccagatct actgcggtaa
agctccagca 1321 ctgtccagca aggcctctgc agagtccccc agttctgagt
ccaagggcaa agttcagctt 1381 ctctattatg tgaacctgct gctgatagac
caccgtttcc tcctgcgccg tggagaatac 1441 gtcctccaca tgtggcagat
atctgggaag ggagaagacc aaggaagctt caatgctgac 1501 aaactcacgt
ctgcaactaa cccagacaag gagaactcaa tgtccatctc cattcttctg 1561
gacaattact gccacccgat agccctgcct aagcatcagc ccacccctga cccggaaggg
1621 gaccgggttc gagcagaaat gcccaaccag cttcgcaagc aattggaggc
gatcatagcc 1681 actgatccac ttaaccctct cacagcagag gacaaagaat
tgctctggca ttttagatac 1741 gaaagcctta agcacccaaa agcatatcct
aagctattta gttcagtgaa atggggacag 1801 caagaaattg tggccaaaac
ataccaattg ttggccagaa gggaagtctg ggatcaaagt 1861 gctttggatg
ttgggttaac aatgcagctc ctggactgca acttctcaga tgaaaatgta 1921
agagccattg cagttcagaa actggagagc ttggaggacg atgatgttct gcattacctt
1981 ctacaattgg tccaggctgt gaaatttgaa ccataccatg atagcgccct
tgccagattt 2041 ctgctgaagc gtggtttaag aaacaaaaga attggtcact
ttttgttttg gttcttgaga 2101 agtgagatag cccagtccag acactatcag
cagaggttcg ctgtgattct ggaagcctat 2161 ctgaggggct gtggcacagc
catgctgcac gactttaccc aacaagtcca agtaatcgag 2221 atgttacaaa
aagtcaccct tgatattaaa tcgctctctg ctgaaaagta tgacgtcagt 2281
tcccaagtta tttcacaact taaacaaaag cttgaaaacc tgcagaattc tcaactcccc
2341 gaaagcttta gagttccata tgatcctgga ctgaaagcag gagcgctggc
aattgaaaaa 2401 tgtaaagtaa tggcctccaa gaaaaaacca ctatggcttg
agtttaaatg tgccgatcct 2461 acagccctat caaatgaaac aattggaatt
atctttaaac atggtgatga tctgcgccaa 2521 gacatgctta ttttacagat
tctacgaatc atggagtcta tttgggagac tgaatctttg 2581 gatctatgcc
tcctgccata tggttgcatt tcaactggtg acaaaatagg aatgatcgag 2641
attgtgaaag acgccacgac aattgccaaa attcagcaaa gcacagtggg caacacggga
2701 gcatttaaag atgaagtcct gaatcactgg ctcaaagaaa aatcccctac
tgaagaaaag 2761 tttcaggcag cagtggagag atttgtttat tcctgtgcag
gctactgtgt ggcaaccttt 2821 gttcttggaa taggcgacag acacaatgac
aatattatga tcaccgagac aggaaaccta 2881 tttcatattg acttcgggca
cattcttggg aattacaaaa gtttcctggg cattaataaa 2941 gagagagtgc
catttgtgct aacccctgac ttcctctttg tgatgggaac ttctggaaag 3001
aagacaagcc cacacttcca gaaatttcag gacatctgtg ttaaggctta tctagccctt
3061 cgtcatcaca caaacctact gatcatcctg ttctccatga tgctgatgac
aggaatgccc 3121 cagttaacaa gcaaagaaga cattgaatat atccgggatg
ccctcacagt ggggaaaaat 3181 gaggaggatg ctaaaaagta ttttcttgat
cagatcgaag tttgcagaga caaaggatgg 3241 actgtgcagt ttaattggtt
tctacatctt gttcttggca tcaaacaagg agagaaacat 3301 tcagcctaa SEQ ID
NO: 18 Human PIK3CG (Isoform 3) Amino Acid Sequence 1 melenykqpv
vlredncrrr rrmkprsaaa slssmelipi efvlptsgrk ckspetallh 61
vaghgnveqm kaqvwlrale tsvaadfyhr lgphhfllly qkkgqwyeiy dkyqvvqtld
121 clrywkathr spgqihlvqr hppseesqaf qrqltaligy dvtdvsnvhd
deleftrrgl 181 vtprmaevas rdpklyamhp wvtskplpey lwkkiannci
fivihrstts qtikvspddt 241 pgailqsfft kmakkkslmd ipesqseqdf
vlrvcgrdey lvgetpiknf qwvrhclkng 301 eeihvvldtp pdpaldevrk
eewplvddct gvtgyheqlt ihgkdhesvf tvslwdcdrk 361 frvkirgidi
pvlprntdlt vfveaniqhg qqvlcqrrts pkpfteevlw nvwlefsiki 421
kdipkgalln lqiycgkapa lsskasaesp sseskgkvql lyyvnlllid hrfllrrgey
481 vlhmwqisgk gedqgsfnad kltsatnpdk ensmsisill dnychpialp
khqptpdpeg 541 drvraempng lrkqleaiia tdplnpltae dkellwhfry
eslkhpkayp klfssvkwgq 601 qeivaktyql larrevwdqs aldvgltmql
ldcnfsdenv raiavqkles ledddvlhyl 661 lqlvqavkfe pyhdsalarf
llkrglrnkr iqhflfwflr seiaqsrhyq grfavileay 721 lrgcgtamlh
dftqqvqvie mlqkvtldik slsaekydvs sqvisqlkqk lenlqnsqlp 781
esfrvpydpg lkagalaiek ckvmaskkkp lwlefkcadp talsnetiqi ifkhgddlrq
841 dmlilqilri mesiwetesl dlcllpygci stgdkigmie ivkdattiak
iqqstvgntg 901 afkdevlnhw lkekspteek fqaaverfvy scagycvatf
vlgigdrhnd nimitetgnl 961 fhidfghilg nyksflgink ervpfvltpd
flfvmgtsgk ktsphfqkfq dicvkaylal 1021 rhhtnlliil fsmmlmtgmp
gltskediey irdaltvgkn eedakkyfld qievcrdkgw 1081 tvqfnwflhl
vlgikqgekh sa SEQ ID NO: 19 Mouse PIK3CG (Transcript 1) cDNA Acid
Sequence 1 atggagctgg agaactatga acaaccggtg gttctaagag aggacaacct
ccgccggcgc 61 cggaggatga agccacgcag cgcagcaggc agcctgtctt
ccatggagct catccccatt 121 gagttcgtac tgcccaccag ccagcgcatc
agcaagactc cagaaacagc gctgctgcat 181 gtggctggcc atggcaatgt
ggaacagatg aaagctcagg tgtggctgcg cgcactggag 241 accagtgtgg
ctgcggagtt ctaccaccga ttgggcccgg accaattcct cctgctctac 301
cagaagaaag gacaatggta tgagatctat gacaggtacc aagtggtgca gaccctagac
361 tgcctgcatt actggaagtt gatgcacaag agccctggcc agatccacgt
ggtacagcga 421 cacgtacctt ctgaggagac cttggctttc cagaagcagc
tcacctccct gattggctat 481 gacgtcactg acatcagcaa tgtgcacgat
gatgagctag agttcactcg ccgccgtctg 541 gttacgcccc gcatggctga
agtggctggc cgggatgcca aactctatgc tatgcaccct 601 tgggtaacgt
ccaaacctct cccagactac ctgtcaaaaa agattgccaa caactgcatc 661
ttcatcgtca tccaccgcgg taccaccagc caaaccatca aggtctccgc agatgatact
721 cctggtacca tcctccagag cttcttcacc aagatggcca agaagaagtc
cctaatgaat 781 atctcagaaa gtcaaagtga gcaggatttt gtattgcggg
tttgtggccg cgatgagtac 841 ctggtgggtg aaacacccct caaaaatttc
cagtgggtga ggcagtgcct caagaacgga 901 gatgaaatac acctggtgct
cgacacgcct ccagacccag cccttgatga ggtgaggaag 961 gaagaatggc
cgctggtgga tgactgcact ggagtcaccg gctaccacga gcagctgacc 1021
atccatggca aggaccacga gagtgtgttc acagtgtctt tgtgggactg cgaccgaaag
1081 ttcagggtca agatcagagg cattgatatc cctgtcctgc ctcggaacac
cgacctcact 1141 gtgtttgtgg aagcgaacat ccagcacggg caacaagtcc
tctgccaaag gagaaccagc 1201 cctaagccct tcgcagaaga ggtactctgg
aatgtgtggc tggagtttgg catcaaaatc 1261 aaagacttgc ccaaaggggc
tctattgaac ctacagatct actgctgcaa aaccccatca 1321 ctgtccagca
aggcttctgc agagactcca ggctccgagt ccaagggcaa agcccagctt 1381
ctctattacg tgaacttgct gttaatagac caccgtttcc tcctccgcca cggagactat
1441 gtgctccaca tgtggcagat atctggcaag gcagaggagc agggcagctt
caatgctgac 1501 aagctcacat ccgcaaccaa tcctgacaag gagaactcaa
tgtccatttc catcctgctg 1561 gacaattact gtcaccccat agctttgcct
aagcaccggc ccacccctga cccagaggga 1621 gacagggttc gggctgaaat
gcccaatcag cttcgaaagc aattggaggc gatcatagcc 1681 acagatccac
ttaaccccct cacagcagag gacaaagaat tgctctggca ttttcgatat 1741
gaaagcctga agcatccgaa ggcttaccct aagctattca gctcagtgaa atgggggcag
1801 caagaaattg ttgccaaaac gtaccagctg ttagccagaa gggagatctg
ggatcaaagt 1861 gctttggacg ttggcttaac catgcagctc ctggactgca
acttttcaga cgagaatgtc 1921 cgggccattg cagttcagaa actggagagc
ttagaggacg atgacgtttt acattacctt 1981 ctccagctgg tacaggctgt
gaaatttgaa ccgtaccacg acagtgcgct ggccagattc 2041 ctgctgaagc
gtggcttgag gaacaaaaga atcggtcact tcttgttctg gttcctgcga 2101
agtgagatcg cacagtccag acactatcag cagaggttcg ctgtgatcct ggaggcgtac
2161 ctgcgaggct gtggcacagc catgttgcag gacttcacac agcaggtcca
tgtgattgag 2221 atgttacaga aagtcaccat tgatattaaa tcgctctcgg
cagagaagta tgacgtcagt 2281 tcccaagtta tttcacagct taagcaaaag
cttgaaagcc ttcagaactc caatctcccc 2341 gagagcttta gagttcccta
tgatcctgga ctaaaagccg gtaccctggt gatcgagaaa 2401 tgcaaagtga
tggcctccaa gaagaagccc ctgtggcttg agtttaagtg tgctgatccc 2461
acagtcctat ccaacgaaac cattggaatc atctttaaac atggtgatga tctgcgccaa
2521 gacatgttga tcttgcagat tctacgcatc atggagtcca tttgggagac
tgaatctctg 2581 gacctgtgcc ttctgcctta cggttgcatc tcaactggtg
acaaaatagg aatgatcgag 2641 attgtaaagg atgccacaac gatcgctcaa
attcagcaaa gcacagtggg taacacgggg 2701 gcattcaaag atgaagtcct
gaatcactgg ctcaaggaaa aatgtcctat tgaagaaaag 2761 tttcaggccg
cagtggaaag gtttgtttac tcctgtgcag gctactgtgt ggccacattt 2821
gttcttggga tcggtgacag gcacaacgac aacattatga tctcagagac aggaaaccta
2881 tttcatatag acttcggaca cattcttggg aattacaaga gtttcctggg
catcaataaa 2941 gagagagtgc ccttcgtcct aaccccagac ttcttgtttg
tgatgggatc ttctggaaaa 3001 aagacaagtc cacacttcca gaaattccag
gatgtctgtg ttagagctta cctagctctt 3061 cgccatcaca caaacctgtt
gatcatcttg ttctccatga tgctgatgac aggaatgccc 3121 cagctgacaa
gcaaagagga cattgaatat atccgggatg ccctcaccgt gggaaaaagc 3181
gaggaggacg ctaagaaata tttccttgat cagatcgaag tctgcagaga caaaggatgg
3241 actgtgcagt ttaactggtt cctacatctt gttcttggca tcaaacaagg
agaaaagcac 3301 tccgcttga SEQ ID: 20 Mouse PIK3CG (Isoform 1) Amino
Acid Sequence 1 melenyeqpv vlrednlrrr rrmkprsaag slssmelipi
efvlptsqri sktpetallh 61 vaghgnveqm kaqvwlrale tsvaaefyhr
lgpdqfllly qkkgqwyeiy dryqvvqtld 121 clhywklmhk spgqihvvqr
hvpseetlaf qkqltsligy dvtdisnvhd deleftrrrl 181 vtprmaevag
rdaklyamhp wvtskplpdy lskkiannci fivihrgtts qtikvsaddt 241
pgtilqsfft kmakkkslmn isesqseqdf vlrvcgrdey lvgetplknf qwvrqclkng
301 deihlvldtp pdpaldevrk eewplvddct gvtgyheqlt ihgkdhesvf
tvslwdcdrk 361 frvkirgidi pvlprntdlt vfveaniqhg qqvlcqrrts
pkpfaeevlw nvwlefgiki 421 kdlpkgalln lqiyccktps lsskasaetp
gseskgkaql lyyvnlllid hrfllrhgdy 481 vlhmwqisgk aeeqgsfnad
kltsatnpdk ensmsisill dnychpialp khrptpdpeg 541 drvraempnq
lrkqleaiia tdplnpltae dkellwhfry eslkhpkayp klfssvkwgq 601
qeivaktyql larreiwdqs aldvgltmql ldcnfsdenv raiavqkles ledddvlhyl
661 lqlvqavkfe pyhdsalarf llkrglrnkr ighflfwflr seiaqsrhyq
qrfavileay 721 lrgcgtamlq dftqqvhvie mlqkvtidik slsaekydvs
sqvisqlkqk leslqnsnlp 781 esfrvpydpg lkagtlviek ckvmaskkkp
lwlefkcadp tvlsnetigi ifkhgddlrq 841 dmlilqilri mesiwetesl
dlcllpygci stgdkigmie ivkdattiaq iggstvgntg 901 afkdevlnhw
lkekcpieek fqaaverfvy scagycvatf vlgigdrhnd nimisetgnl 961
fhidfghilg nyksflgink ervpfvltpd flfvmgssgk ktsphfqkfq dvcvraylal
1021 rhhtnlliil fsmmlmtgmp qltskediey irdaltvgks eedakkyfld
qievcrdkgw 1081 tvqfnwflhl vlgikqgekh sa SEQ ID NO: 21 Mouse PIK3CG
(Transcript 2) cDNA Acid Sequence 1 atggagctgg agaactatga
acaaccggtg gttctaagag aggacaacct ccgccggcgc 61 cggaggatga
acccacgcag cgcagcaggc agcctgtctt ccatggagct catccccatt 121
gagttcgtac tgcccaccag ccagcgcatc agcaagactc cagaaacagc gctgctgcat
181 gtggctggcc atggcaatgt ggaacagatg aaagctcagg tgtggctgcg
cgcactggag 241 accagtgtgg ctgcggagtt ctaccaccga ttgggcccgg
accaattcct cctgctctac 301 cagaagaaag gacaatggta tgagatctat
gacaggtacc aagtggtgca gaccctagac 361 tgcctgcatt actggaagtt
gatgcacaag agccctggcc agatccacgt ggtacagcga 421 cacgtacctt
ctgaggagac cttggctttc cagaagcagc tcacctccct gattggctat 481
gacgtcactg acatcagcaa tgtgcacgat gatgagctag agttcactcg
ccgccgtctg
541 gttacgcccc gcatggctga agtggctggc cgggatgcca aactctatgc
tatgcaccct 601 tgggtaacgt ccaaacctct cccagactac ctgtcaaaaa
agattgccaa caactgcatc 661 ttcatcgtca tccaccgcgg taccaccagc
caaaccatca aggtctccgc agatgatact 721 cctggtacca tcctccagag
cttcttcacc aagatggcca agaagaagtc cctaatgaat 781 atctcagaaa
gtcaaagtga gcaggatttt gtattgcggg tttgtggccg cgatgagtac 841
ctggtgggtg aaacacccct caaaaatttc cagtgggtga ggcagtgcct caagaacgga
901 gatgaaatac acctggtgct cgacacgcct ccagacccag cccttgatga
ggtgaggaag 961 gaagaatggc cgctggtgga tgactgcact ggagtcaccg
gctaccacga gcagctgacc 1021 atccatggca aggaccacga gagtgtgttc
acagtgtctt tgtgggactg cgaccgaaag 1081 ttcagggtca agatcagagg
cattgatatc cctgtcctgc ctcggaacac cgacctcact 1141 gtgtttgtgg
aagcgaacat ccagcacggg caacaagtcc tctgccaaag gagaaccagc 1201
cctaagccct tcgcagaaga ggtactctgg aatgtgtggc tggagtttgg catcaaaatc
1261 aaagacttgc ccaaaggggc tctattgaac ctacagatct actgctgcaa
aaccccatca 1321 ctgtccagca aggcttctgc agagactcca ggctccgagt
ccaagggcaa agcccagctt 1381 ctctattacg tgaacttgct gttaatagac
caccgtttcc tcctccgcca cggggactat 1441 gtgctccaca tgtggcagat
atctggcaag gcagaggagc agggcagctt caatgctgac 1501 aagctcacat
ccgcaaccaa tcctgacaag gagaactcaa tgtccatttc catcctgctg 1561
gacaattact gtcaccccat agctttgcct aagcaccggc ccacccctga cccagaggga
1621 gacagggttc gggctgaaat gcccaatcag cttcgaaagc aattggaggc
gatcatagcc 1681 acagatccac ttaaccccct cacagcagag gacaaagaat
tgctctggca ttttcgatat 1741 gaaagcctga agcatccgaa ggcttaccct
aagctattca gctcagtgaa atgggggcag 1801 caagaaattg ttgccaaaac
gtaccagctg ttagccagaa gggagatctg ggatcaaagt 1861 gctttggacg
ttggcttaac catgcagctc ctggactgca acttttcaga cgagaatgtc 1921
cgggccattg cagttcagaa actggagagc ttagaggacg atgacgtttt acattacctt
1981 ctccagctgg tacaggctgt gaaatttgaa ccgtaccacg acagtgcgct
ggccagattc 2041 ctgctgaagc gtggcttgag gaacaaaaga atcggtcact
tcttgttctg gttcctgcga 2101 agtgagatcg cacagtccag acactatcag
cagaggttcg ctgtgatcct ggaggcgtac 2161 ctgcgaggct gtggcacagc
catgttgcag gacttcacac agcaggtcca tgtgattgag 2221 atgttacaga
aagtcaccat tgatattaaa tcgctctcgg cagagaagta tgacgtcagt 2281
tcccaagtta tttcacagct taagcaaaag cttgaaagcc ttcagaactc caatctcccc
2341 gagagcttta gagttcccta tgatcctgga ctaaaagccg gtaccctggt
gatcgagaaa 2401 tgcaaagtga tggcctccaa gaagaagccc ctgtggcttg
agtttaagtg tgctgatccc 2461 acagtcctat ccaacgaaac cattggaatc
atctttaaac atggtgatga tctgcgccaa 2521 gacatgttga tcttgcagat
tctacgcatc atggagtcca tttggaagac tgaatctctg 2581 gacctgtgcc
ttctgcctta cggttgcatc tcaactggtg acaaaatagg aatgatcgag 2641
attgtaaagg atgccacaac gatcgctcaa attcagcaaa gcacagtggg taacacgggg
2701 gcattcaaag atgaagtcct gaatcactgg ctcaaggaaa aatgtcctat
tgaagaaaag 2761 tttcaggccg cagtggaaag gtttgtttac tcctgtgcag
gctactgtgt ggccacattt 2821 gttcttggga tcggtgacag gcacaacgac
aacattatga tctcagagac aggaaaccta 2881 tttcatatag acttcggaca
cattcttggg aattacaaga gtttcctggg catcaataaa 2941 gagagagtgc
ccttcgtcct aaccccagac ttcttgtttg tgatgggatc ttctggaaaa 3001
aagacaagtc cacacttcca gaaattccag gatgtctgtg ttagagctta cctagctctt
3061 cgccatcaca caaacctgtt gatcatcttg ttctccatga tgctgatgac
aggaatgccc 3121 cagctgacaa gcaaagagga cattgaatat atccgggatg
ccctcaccgt gggaaaaagc 3181 gaggaggacg ctaagaaata tttccttgat
cagatcgaag tctgcagaga caaaggatgg 3241 actgtgcagt ttaactggtt
cctacatctt gttcttggca tcaaacaagg agaaaagcac 3301 tccgcttga SEQ ID
NO: 22 Mouse PIK3CG (Isoform 2) Amino Acid Sequence 1 melenyeqpv
vlrednlrrr rrmkprsaag slssmelipi efvlptsqri sktpetallh 61
vaghgnveqm kaqvwlrale tsvaaefyhr lgpdqfllly qkkgqwyeiy dryqvvqtld
121 clhywklmhk spgqihvvqr hvpseetlaf qkqltsligy dvtdisnvhd
deleftrrrl 181 vtprmaevag rdaklyamhp wvtskplpdy lskkiannci
fivihrgtts qtikvsaddt 241 pgtilqsfft kmakkkslmn isesqseqdf
vlrvcgrdey lvgetplknf qwvrqclkng 301 deihlvldtp pdpaldevrk
eewplvddct gvtgyheqlt ihgkdhesvf tvslwdcdrk 361 frvkirgidi
pvlprntdlt vfveaniqhg qqvlcqrrts pkpfaeevlw nvwlefgiki 421
kdlpkgalln lqiyccktps lsskasaetp gseskgkaql lyyvnlllid hrfllrhgdy
481 vlhmwqisgk aeeqgsfnad kltsatnpdk ensmsisill dnychpialp
khrptpdpeg 541 drvraempnq lrkqleaiia tdplnpltae dkellwhfry
eslkhpkayp klfssvkwgq 601 qeivaktyql larreiwdqs aldvgltmql
ldcnfsdenv raiavqkles ledddvlhyl 661 lqlvqavkfe pyhdsalarf
llkrglrnkr ighflfwflr seiaqsrhyq qrfavileay 721 lrgcgtamlq
dftqqvhvie mlqkvtidik slsaekydvs sqvisqlkqk leslqnsnlp 781
esfrvpydpg lkagtlviek ckvmaskkkp lwlefkcadp tvlsnetigi ifkhgddlrq
841 dmlilqilri mesiwetesl dlcllpygci stgdkigmie ivkdattiaq
iggstvgntg 901 afkdevlnhw lkekcpieek fqaaverfvy scagycvatf
vlgigdrhnd nimisetgnl 961 fhidfghilg nyksflgink ervpfvltpd
flfvmgssgk ktsphfqkfq dvcvraylal 1021 rhhtnlliil fsmmlmtgmp
qltskediey irdaltvgks eedakkyfld qievcrdkgw 1081 tvqfnwflhl
vlgikqgekh sa SEQ ID NO: 23 Mouse PIK3CG (Transcript 3) cDNA Acid
Sequence 1 atggagctgg agaactatga acaaccggtg gttctaagag aggacaacct
ccgccggcgc 61 cggaggatga acccacgcag cgcagcaggc agcctgtctt
ccatggagct catccccatt 121 gagttcgtac tgcccaccag ccagcgcatc
agcaagactc cagaaacagc gctgctgcat 181 gtggctggcc atggcaatgt
ggaacagatg aaagctcagg tgtggctgcg cgcactggag 241 accagtgtgg
ctgcggagtt ctaccaccga ttgggcccgg accaattcct cctgctctac 301
cagaagaaag gacaatggta tgagatctat gacaggtacc aagtggtgca gaccctagac
361 tgcctgcatt actggaagtt gatgcacaag agccctggcc agatccacgt
ggtacagcga 421 cacgtacctt ctgaggagac cttggctttc cagaagcagc
tcacctccct gattggctat 481 gacgtcactg acatcagcaa tgtgcacgat
gatgagctag agttcactcg ccgccgtctg 541 gttacgcccc gcatggctga
agtggctggc cgggatgcca aactctatgc tatgcaccct 601 tgggtaacgt
ccaaacctct cccagactac ctgtcaaaaa agattgccaa caactgcatc 661
ttcatcgtca tccaccgcgg taccaccagc caaaccatca aggtctccgc agatgatact
721 cctggtacca tcctccagag cttcttcacc aagatggcca agaagaagtc
cctaatgaat 781 atctcagaaa gtcaaagtga gcaggatttt gtattgcggg
tttgtggccg cgatgagtac 841 ctggtgggtg aaacacccct caaaaatttc
cagtgggtga ggcagtgcct caagaacgga 901 gatgaaatac acctggtgct
cgacacgcct ccagacccag cccttgatga ggtgaggaag 961 gaagaatggc
cgctggtgga tgactgcact ggagtcaccg gctaccacga gcagctgacc 1021
atccatggca aggaccacga gagtgtgttc acagtgtctt tgtgggactg cgaccgaaag
1081 ttcagggtca agatcagagg cattgatatc cctgtcctgc ctcggaacac
cgacctcact 1141 gtgtttgtgg aagcgaacat ccagcacggg caacaagtcc
tctgccaaag gagaaccagc 1201 cctaagccct tcgcagaaga ggtactctgg
aatgtgtggc tggagtttgg catcaaaatc 1261 aaagacttgc ccaaaggggc
tctattgaac ctacagatct actgctgcaa aaccccatca 1321 ctgtccagca
aggcttctgc agagactcca ggctccgagt ccaagggcaa agcccagctt 1381
ctctattacg tgaacttgct gttaatagac caccgtttcc tcctccgcca cggggactat
1441 gtgctccaca tgtggcagat atctggcaag gcagaggagc agggcagctt
caatgctgac 1501 aagctcacat ccgcaaccaa tcctgacaag gagaactcaa
tgtccatttc catcctgctg 1561 gacaattact gtcaccccat agctttgcct
aagcaccggc ccacccctga cccagaggga 1621 gacagggttc gggctgaaat
gcccaatcag cttcgaaagc aattggaggc gatcatagcc 1681 acagatccac
ttaaccccct cacagcagag gacaaagaat tgctctggca ttttcgatat 1741
gaaagcctga agcatccgaa ggcttaccct aagctattca gctcagtgaa atgggggcag
1801 caagaaattg ttgccaaaac gtaccagctg ttagccagaa gggagatctg
ggatcaaagt 1861 gctttggacg ttggcttaac catgcagctc ctggactgca
acttttcaga cgagaatgtc 1921 cgggccattg cagttcagaa actggagagc
ttagaggacg atgacgtttt acattacctt 1981 ctccagctgg tacaggctgt
gaaatttgaa ccgtaccacg acagtgcgct ggccagattc 2041 ctgctgaagc
gtggcttgag gaacaaaaga atcggtcact tcttgttctg gttcctgcga 2101
agtgagatcg cacagtccag acactatcag cagaggttcg ctgtgatcct ggaggcgtac
2161 ctgcgaggct gtggcacagc catgttgcag gacttcacac agcaggtcca
tgtgattgag 2221 atgttacaga aagtcaccat tgatattaaa tcgctctcgg
cagagaagta tgacgtcagt 2281 tcccaagtta tttcacagct taagcaaaag
cttgaaagcc ttcagaactc caatctcccc 2341 gagagcttta gagttcccta
tgatcctgga ctaaaagccg gtaccctggt gatcgagaaa 2401 tgcaaagtga
tggcctccaa gaagaagccc ctgtggcttg agtttaagtg tgctgatccc 2461
acagtcctat ccaacgaaac cattggaatc atctttaaac atggtgatga tctgcgccaa
2521 gacatgttga tcttgcagat tctacgcatc atggagtcca tttggaagac
tgaatctctg 2581 gacctgtgcc ttctgcctta cggttgcatc tcaactggtg
acaaaatagg aatgatcgag 2641 attgtaaagg atgccacaac gatcgctcaa
attcagcaaa gcacagtggg taacacgggg 2701 gcattcaaag atgaagtcct
gaatcactgg ctcaaggaaa aatgtcctat tgaagaaaag 2761 tttcaggccg
cagtggaaag gtttgtttac tcctgtgcag gctactgtgt ggccacattt 2821
gttcttggga tcggtgacag gcacaacgac aacattatga tctcagagac aggaaaccta
2881 tttcatatag acttcggaca cattcttggg aattacaaga gtttcctggg
catcaataaa 2941 gagagagtgc ccttcgtcct aaccccagac ttcttgtttg
tgatgggatc ttctggaaaa 3001 aagacaagtc cacacttcca gaaattccag
gatgtctgtg ttagagctta cctagctctt 3061 cgccatcaca caaacctgtt
gatcatcttg ttctccatga tgctgatgac aggaatgccc 3121 cagctgacaa
gcaaagagga cattgaatat atccgggatg ccctcaccgt gggaaaaagc 3181
gaggaggacg ctaagaaata tttccttgat cagatcgaag tctgcagaga caaaggatgg
3241 actgtgcagt ttaactggtt cctacatctt gttcttggca tcaaacaagg
agaaaagcac 3301 tccgcttga SEQ ID NO: 24 Mouse PIK3CG (Isoform 3)
Amino Acid Sequence 1 melenyeqpv vlrednlrrr rrmkprsaag slssmelipi
efvlptsqri sktpetallh 61 vaghgnveqm kaqvwlrale tsvaaefyhr
lgpdqfllly qkkgqwyeiy dryqvvqtld 121 clhywklmhk spgqihvvqr
hvpseetlaf qkqltsligy dvtdisnvhd deleftrrrl 181 vtprmaevag
rdaklyamhp wvtskplpdy lskkiannci fivihrgtts qtikvsaddt 241
pgtilqsfft kmakkkslmn isesqseqdf vlrvcgrdey lvgetplknf qwvrqclkng
301 deihlvldtp pdpaldevrk eewplvddct gvtgyheqlt ihgkdhesvf
tvslwdcdrk 361 frvkirgidi pvlprntdlt vfveaniqhg qqvlcqrrts
pkpfaeevlw nvwlefgiki 421 kdlpkgalln lqiyccktps lsskasaetp
gseskgkaql lyyvnlllid hrfllrhgdy 481 vlhmwqisgk aeeqgsfnad
kltsatnpdk ensmsisill dnychpialp khrptpdpeg 541 drvraempnq
lrkqleaiia tdplnpltae dkellwhfry eslkhpkayp klfssvkwgq 601
qeivaktyql larreiwdqs aldvgltmql ldcnfsdenv raiavqkles ledddvlhyl
661 lqlvqavkfe pyhdsalarf llkrglrnkr ighflfwflr seiaqsrhyq
qrfavileay 721 lrgcgtamlq dftqqvhvie mlqkvtidik slsaekydvs
sqvisqlkqk leslqnsnlp 781 esfrvpydpg lkagtlviek ckvmaskkkp
lwlefkcadp tvlsnetigi ifkhgddlrq 841 dmlilqilri mesiwetesl
dlcllpygci stgdkigmie ivkdattiaq iggstvgntg 901 afkdevlnhw
lkekcpieek fqaaverfvy scagycvatf vlgigdrhnd nimisetgnl 961
fhidfghilg nyksflgink ervpfvltpd flfvmgssgk ktsphfqkfq dvcvraylal
1021 rhhtnlliil fsmmlmtgmp qltskediey irdaltvgks eedakkyfld
qievcrdkgw 1081 tvqfnwflhl vlgikqgekh sa SEQ ID NO: 25 Human PIK3CD
cDNA Acid Sequence 1 atgccccctg gggtggactg ccccatggaa ttctggacca
aggaggagaa tcagagcgtt 61 gtggttgact tcctgctgcc cacaggggtc
tacctgaact tccctgtgtc ccgcaatgcc 121 aacctcagca ccatcaagca
gctgctgtgg caccgcgccc agtatgagcc gctcttccac 181 atgctcagtg
gccccgaggc ctatgtgttc acctgcatca accagacagc ggagcagcaa 241
gagctggagg acgagcaacg gcgtctgtgt gacgtgcagc ccttcctgcc cgtcctgcgc
301 ctggtggccc gtgagggcga ccgcgtgaag aagctcatca actcacagat
cagcctcctc 361 atcggcaaag gcctccacga gtttgactcc ttgtgcgacc
cagaagtgaa cgactttcgc 421 gccaagatgt gccaattctg cgaggaggcg
gccgcccgcc ggcagcagct gggctgggag 481 gcctggctgc agtacagttt
ccccctgcag ctggagccct cggctcaaac ctgggggcct 541 ggtaccctgc
ggctcccgaa ccgggccctt ctggtcaacg ttaagtttga gggcagcgag 601
gagagcttca ccttccaggt gtccaccaag gacgtgccgc tggcgctgat ggcctgtgcc
661 ctgcggaaga aggccacagt gttccggcag ccgctggtgg agcagccgga
agactacacg 721 ctgcaggtga acggcaggca tgagtacctg tatggcagct
acccgctctg ccagttccag 781 tacatctgca gctgcctgca cagtgggttg
acccctcacc tgaccatggt ccattcctcc 841 tccatcctcg ccatgcggaa
tgagcagagc aaccctgccc cccaggtcca gaaaccgcgt 901 gccaaaccac
ctcccattcc tgcgaagaag ccttcctctg tgtccctgtg gtccctggag 961
cagccgttcc gcatcgagct catccagggc agcaaagtga acgccgacga gcggatgaag
1021 ctggtggtgc aggccgggct tttccacggc aacgagatgc tgtgcaagac
ggtgtccagc 1081 tcggaggtga gcgtgtgctc ggagcccgtg tggaagcagc
ggctggagtt cgacatcaac 1141 atctgcgacc tgccccgcat ggcccgtctc
tgctttgcgc tgtacgccgt gatcgagaaa 1201 gccaagaagg ctcgctccac
caagaagaag tccaagaagg cggactgccc cattgcctgg 1261 gccaacctca
tgctgtttga ctacaaggac cagcttaaga ccggggaacg ctgcctctac 1321
atgtggccct ccgtcccaga tgagaagggc gagctgctga accccacggg cactgtgcgc
1381 agtaacccca acacggatag cgccgctgcc ctgctcatct gcctgcccga
ggtggccccg 1441 caccccgtgt actaccccgc cctggagaag atcttggagc
tggggcgaca cagcgagtgt 1501 gtgcatgtca ccgaggagga gcagctgcag
ctgcgggaaa tcctggagcg gcgggggtct 1561 ggggagctgt atgagcacga
gaaggacctg gtgtggaagc tgcggcatga agtccaggag 1621 cacttcccgg
aggcgctagc ccggctgctg ctggtcacca agtggaacaa gcatgaggat 1681
gtggcccaga tgctctacct gctgtgctcc tggccggagc tgcccgtcct gagcgccctg
1741 gagctgctag acttcagctt ccccgattgc cacgtaggct ccttcgccat
caagtcgctg 1801 cggaaactga cggacgatga gctgttccag tacctgctgc
agctggtgca ggtgctcaag 1861 tacgagtcct acctggactg cgagctgacc
aaattcctgc tggaccgggc cctggccaac 1921 cgcaagatcg gccacttcct
tttctggcac ctccgctccg agatgcacgt gccgtcggtg 1981 gccctgcgct
tcggcctcat cctggaggcc tactgcaggg gcagcaccca ccacatgaag 2041
gtgctgatga agcaggggga agcactgagc aaactgaagg ccctgaatga cttcgtcaag
2101 ctgagctctc agaagacccc caagccccag accaaggagc tgatgcactt
gtgcatgcgg 2161 caggaggcct acctagaggc cctctcccac ctgcagtccc
cactcgaccc cagcaccctg 2221 ctggctgaag tctgcgtgga gcagtgcacc
ttcatggact ccaagatgaa gcccctgtgg 2281 atcatgtaca gcaacgagga
ggcaggcagc ggcggcagcg tgggcatcat ctttaagaac 2341 ggggatgacc
tccggcagga catgctgacc ctgcagatga tccagctcat ggacgtcctg 2401
tggaagcagg aagggctgga cctgaggatg accccctatg gctgcctccc caccggggac
2461 cgcacaggcc tcattgaggt ggtactccgt tcagacacca tcgccaacat
ccaactcaac 2521 aagagcaaca tggcagccac agccgccttc aacaaggatg
ccctgctcaa ctggctgaag 2581 tccaagaacc cgggggaggc cctggatcga
gccattgagg agttcaccct ctcctgtgct 2641 ggctattgtg tggccacata
tgtgctgggc attggcgatc ggcacagcga caacatcatg 2701 atccgagaga
gtgggcagct gttccacatt gattttggcc actttctggg gaatttcaag 2761
accaagtttg gaatcaaccg cgagcgtgtc ccattcatcc tcacctacga ctttgtccat
2821 gtgattcagc aggggaagac taataatagt gagaaatttg aacggttccg
gggctactgt 2881 gaaagggcct acaccatcct gcggcgccac gggcttctct
tcctccacct ctttgccctg 2941 atgcgggcgg caggcctgcc tgagctcagc
tgctccaaag acatccagta tctcaaggac 3001 tccctggcac tggggaaaac
agaggaggag gcactgaagc acttccgagt gaagtttaac 3061 gaagccctcc
gtgagagctg gaaaaccaaa gtgaactggc tggcccacaa cgtgtccaaa 3121
gacaacaggc agtag SEQ ID NO: 26 Human PIK3CD Amino Acid Sequence 1
mppgvdcpme fwtkeenqsv vvdfllptgv ylnfpvsrna nlstikqllw hraqyeplfh
61 mlsgpeayvf tcinqtaeqq eledeqrrlc dvqpflpvlr lvaregdrvk
klinsqisll 121 igkqlhefds lcdpevndfr akmcqfceea aarrqqlqwe
awlqysfplq lepsaqtwgp 181 gtlrlpnral lvnvkfegse esftfqvstk
dvplalmaca lrkkatvfrq plveqpedyt 241 lqvngrheyl ygsyplcqfq
yicsclhsgl tphltmvhss silamrdeqs npapqvqkpr 301 akpppipakk
pssvslwsle qpfrieliqg skvnadermk lvvqaglfhg nemlcktvss 361
sevsvcsepv wkqrlefdin icdlprmarl cfalyaviek akkarstkkk skkadcpiaw
421 anlmlfdykd qlktgercly mwpsvpdekg ellnptgtvr snpntdsaaa
lliclpevap 481 hpvyypalek ilelgrhsec vhvteeeqlq lreilerrgs
gelyehekdl vwklrhevqe 541 hfpealarll lvtkwnkhed vaqmlyllcs
wpelpvlsal elldfsfpdc hvgsfaiksi 601 rkltddelfq yllqlvqvlk
yesyldcelt kflldralan rkighflfwh lrsemhvpsv 661 alrfglilea
ycrgsthhmk vlmkqgeals klkalndfvk lssqktpkpg tkelmhlcmr 721
qeaylealsh lqspldpstl laevcveqct fmdskmkplw imysneeags ggsvgiifkn
781 gddlrqdmlt lqmiqlmdvl wkqegldlrm tpygclptgd rtglievvlr
sdtianiqln 841 ksnmaataaf nkdallnwlk sknpgealdr aieeftlsca
gycvatyvlg igdrhsdnim 901 iresgqlfhi dfghflgnfk tkfginrerv
pfiltydfvh viqqgktnns ekferfrgyc 961 eraytilrrh gllflhlfal
mraaglpels cskdiqylkd slalgkteee alkhfrvkfn 1021 ealreswktk
vnwlahnvsk dnrq SEQ ID NO: 27 Mouse PIK3CD (Transcript 1) cDNA Acid
Sequence 1 atgccccctg gggtggactg ccccatggag ttctggacca aagaggagag
ccagagcgtg 61 gttgttgact tcttgctgcc cacaggggtc tacttgaact
tccccgtgtc ccgcaatgcc 121 aacctcagca ccatcaagca ggtgctgtgg
caccgtgcac agtatgagcc actcttccac 181 atgctcagtg accccgaggc
ctatgtgttc acctgtgtga accagacggc ggagcagcag 241 gagttggagg
atgagcagcg gaggctgtgc gacatccagc ccttcctgcc cgtgctgcgc 301
ctcgtggccc gagaggggga ccgcgtgaag aagctcatta actcccagat cagcctcctc
361 attggcaaag gtctccatga gtttgattcc ctgcgggacc cggaagtaaa
cgacttccgc 421 actaagatgc gccagttttg tgaagaggct gctgctcacc
gccagcagct gggctgggtg 481 gaatggctgc agtacagctt ccccctgcag
ctggagccct cagcaagggg ttggcgggcc 541 ggcttattgc gtgtcagcaa
ccgagccctg ctggtcaacg tgaagttcga gggcagtgag 601 gagagcttca
ccttccaggt atccaccaag gacatgcccc tggcactgat ggcctgtgcc 661
ctccgaaaaa aggccacagt gttccggcag cctctggtgg agcagcctga ggaatatgcc
721 ctgcaggtga acgggaggca cgaatacctc tacggcaact acccgctctg
ccactttcag 781 tacatctgca gctgcctaca cagcaggctg acccctcatc
tgaccatggt ccactcctcc 841 tccatccttg ctatgcggga tgagcagagc
aatcctgccc cccaagtaca gaaaccacgt 901 gccaaacctc ccccgatccc
tgccaagaag ccctcctctg tgtccctgtg gtccctggaa 961 cagccattct
ccattgagct gatcgagggc cgaaaagtga atgctgacga gcggatgaag 1021
ctggttgttc aggccgggct cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc
1081 tcggaggtga atgtatgctc agagcccgtg tggaagcagc gactggagtt
cgatatcagc 1141 gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc
tctatgccgt cgtggagaag 1201 gctaagaagg cacgctccac aaagaagaag
tctaagaagg cggactgacc catcgcttgg 1261 gccaacctca tgctattcga
ctacaaagat cagctcaaga cgggggaacg ctgcctctac 1321 atgtggccct
ctgtcccaga tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1381
gggaacccca acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc
1441 caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca
cggggagcgt 1501 gggcgcatca cggaggagga gctgcagctg cgggagatcc
tggaacggcg gggatccggg 1561 gaactgtacg aacatgagaa ggacctggtg
tgaaagatgc gccacaaagt ccaagagcat
1621 ttcccagagg cgctggcccg cctgctgctg gtcaccaagt ggaataaaca
cgaggatgtg 1681 gcccagatgc tctatttgct gtgctcctgg cccgagctgc
ctgtgctgag cgccctggaa 1741 cttctggact ttagctttcc cgactgctac
gtgggctcct tcgccatcaa gtcccttcgg 1801 aagctgacgg acgatgagct
cttccagtac cttctgcagc tggtgcaagt gctcaaatat 1861 gagtcctacc
tggactgcga gctgaccaaa ttcttgctgg gccgagccct ggctaaccgc 1921
aagatcggac acttcctgtt ctggcacctc cgctctgaga tgcacgtacc atcagtggct
1981 ctgcggtttg gtctcatcat ggaagcctac tgcagaggca gcacccacca
catgaaggtg 2041 ctgatgaagc agggggaagc actgagcaag cttaaggcac
tgaatgactt tgtgaaggtg 2101 agttcccaga agaccaccaa gccccaaacc
aaggagatga tgcatatgtg catgcgccag 2161 gagacctaca tggaggccct
gtcccacctg cagtctccac tcgaccccag caccctgctg 2221 gaggaagtct
gtgtggagca gtgcaccttc atggactcca aaatgaagcc cctgtggatc 2281
atgtacagca gcgaggaggc gggcagtgct ggcaacgtgg gcatcatctt taagaacggg
2341 gatgacctcc gccaggacat gctgactctg cagatgatcc agctcatgga
cgtcctgtgg 2401 aagcaggagg gcctggacct gaggatgacg ccctacggct
gcctccccac cggggaccgc 2461 acaggtctca tcgaggtggt cctccactcg
gacaccatcg ccaacatcca gctgaacaaa 2521 agcaacatgg cggccacagc
tgccttcaac aaggacgccc tgctcaactg gctcaagtcc 2581 aagaaccctg
gggaggccct ggatcgggcc attgaggaat tcaccctctc ctgtgctggc 2641
tactgtgtgg ccacatatgt tctgggcatc ggtgaccggc acagagacaa catcatgatc
2701 agagagagtg ggcagctctt ccacattgat tttggccact ttctggggaa
cttcaagacc 2761 aagtttggaa tcaaccgaga gcgcgtcccc ttcattctca
cctacgactt tgtccacgtg 2821 atccagcagg ggaagactaa caacagtgag
aagtttgaaa ggttccgcgg ctactgtgaa 2881 cgagcctata ccatcctgcg
gcgccacggg ctgcttttcc tccatctctt cgccctgatg 2941 cgggccgcag
gtctgcctga gcttagctgc tccaaagata tccagtatct caaggactct 3001
ctggcactgg ggaagacgga ggaagaggcg ctaaagcact tccgggtgaa gttcaacgaa
3061 gctctccgag aaagctggaa aaccaaagtc aactggctgg cgcacaatgt
gtccaaggat 3121 aaccgacagt ag SEQ ID NO: 28 Mouse PIK3CD (Isoform
1) Amino Acid Sequence 1 mppgvdcpme fwtkeesqsv vvdfllptgv
ylnfpvsrna nlstikqvlw hraqyeplfh 61 mlsdpeayvf tcvnqtaeqq
eledegrrlc diqpflpvlr lvaregdrvk klinsqisll 121 igkglhefds
lrdpevndfr tkmrqfceea aahrqqlgwv ewlgysfplq lepsargwra 181
gllrvsnral lvnvkfegse esftfqvstk dmplalmaca lrkkatvfrq plveqpeeya
241 lqvngrheyl ygnyplchfq yicsclhsgl tphltmvhss silamrdeqs
npapqvqkpr 301 akpppipakk pssvslwsle qpfsielieg rkvnadermk
lvvqaglfhg nemlcktvss 361 sevnvcsepv wkqrlefdis vcdlprmarl
cfalyavvek akkarstkkk skkadcpiaw 421 anlmlfdykd qlktgercly
mwpsvpdekg ellnpagtvr gnpntesaaa lviylpevap 481 hpvyfpalek
ilelgrhger griteeelql reilerrgsg elyehekdlv wkmrhevqeh 541
fpealarlll vtkwnkhedv aqmlyllcsw pelpvlsale lldfsfpdcy vgsfaikslr
601 kltddelfqy llqlvqvlky esyldceltk fllgralanr kighflfwhl
rsemhvpsva 661 lrfglimeay crgsthhmkv lmkqgealsk lkalndfvkv
ssqkttkpqt kemmhmcmrq 721 etymealshl qspldpstll eevcveqctf
mdskmkplwi mysseeagsa gnvgiifkng 781 ddlrqdmltl qmiqlmdvlw
kqegldlrmt pygclptgdr tglievvlhs dtianiqlnk 841 snmaataafn
kdallnwlks knpgealdra ieeftlscag ycvatyvlgi gdrhsdnimi 901
resgqlfhid fghflgnfkt kfginrervp filtydfvhv iqqgktnnse kferfrgyce
961 raytilrrhg llflhlfalm raaglpelsc skdigylkds lalgkteeea
lkhfrvkfne 1021 alreswktkv nwlahnvskd nrq SEQ ID NO: 29 Mouse
PIK3CD (Transcript 2) cDNA Acid Sequence 1 atgccccctg gggtggactg
ccccatggag ttctggacca aagaggagag ccagagcgtg 61 gttgttgact
tcttgctgcc cacaggggtc tacttgaact tccccgtgtc ccgcaatgcc 121
aacctcagca ccatcaagca ggtgctgtgg caccgtgcac agtatgagcc actcttccac
181 atgctcagtg accccgaggc ctatgtgttc acctgtgtga accagacggc
ggagcagcag 241 gagttggagg atgagcagcg gaggctgtgc gacatccagc
ccttcctgcc cgtgctgcgc 301 ctcgtggccc gagaggggga ccgcgtgaag
aagctcatta actcccagat cagcctcctc 361 attggcaaag gtctccatga
gtttgattcc ctgcgggacc cggaagtaaa cgacttccgc 421 actaagatgc
gccagttttg tgaagaggct gctgctcacc gccagcagct gggctgggtg 481
gaatggctgc agtacagctt ccccctgcag ctggagccct cagcaagggg ttggcgggcc
541 ggcttattgc gtgtcagcaa ccgagccctg ctggtcaacg tgaagttcga
gggcagtgag 601 gagagcttca ccttccaggt atccaccaag gacatgcccc
tggcactgat ggcctgtgcc 661 ctccgaaaaa aagccacagt gttccggcag
cctctggtgg agcagcctga ggaatatgcc 721 ctgcaggtga acgggaggca
cgaatacctc tacggcaact acccgctctg ccactttcag 781 tacatctgca
gctgcctaca cagcgggctg acccctcatc tgaccatggt ccactcctcc 841
tccatccttg ctatgcggga tgagcagagc aatcctgccc cccaagtaca gaaaccacgt
901 gccaaacctc ccccgatccc tgccaagaag ccctcctctg tgtccctgtg
gtccctggaa 961 cagccattct ccattgagct gatcgagggc cgaaaagtga
atgctgacga gcggatgaag 1021 ctggttgttc aggccgggct cttccatggc
aatgagatgc tgtgcaagac tgtgtcaagc 1081 tcggaggtga atgtatgctc
agagcccgtg tggaagaagc gactggagtt cgatatcagc 1141 gtctgtgacc
tcccgcgcat ggctcgactc tgttttgctc tctatgccgt cgtggagaag 1201
gctaagaagg cacgctccac aaagaagaag tctaagaagg cggactgccc catcgcttgg
1261 gccaacctca tgctattcga ctacaaagat cagctcaaga cgggggagcg
ctgcctctac 1321 atgtggccct ctgtcccaga tgagaaggga gagctgctga
atcctgcggg tacagtgcgc 1381 gggaacccca acacggagag tgccgctgcc
ctggtcatct acctgcctga ggtggccccc 1441 caccctgtgt acttccccgc
tctggagaag atcctggagc tggggcgtca cggggagcgt 1501 gggcgcatca
cggaggagga gcagctgcag ctgcgggaga tcctggaacg gcggggatcc 1561
ggggaactgt acgaacatga gaaggacctg gtgtggaaga tgcgccacga agtccaggag
1621 catttcccag aggcgctggc ccgcctgctg ctggtcacca agtggaataa
acacgaggat 1681 gtggcccaga tgtcccagat gctctatttg ctgtgctcct
ggcccgagct gcctgtgctg 1741 agcgccctgg aacttctgga ctttagcttt
cccgactgct acgtgggctc cttcgccatc 1801 aagtcccttc ggaagctgac
ggacgatgag ctcttccagt accttctgca gctggtgcaa 1861 gtgctcaaat
atgagtccta cctggactgc gagctgacca aattcttgct gggccgagcc 1921
ctggctaacc gcaagatcgg acacttcctg ttctggcacc tccgctctga gatgcacgta
1981 ccatcagtgg ctctgcggtt tggtctcatc atggaagcct actgcagagg
cagcacccac 2041 cacatgaagg tgctgatgaa gcagggggaa gcactgagca
agcttaaggc actgaatgac 2101 tttgtgaagg tgagttccca gaagaccacc
aagccccaaa ccaaggagat gatgcatatg 2161 tgcatgcgcc aggagaccta
catggaggcc ctgtcccacc tgcagtctcc actcgacccc 2221 agcaccctgc
tggaggaagt ctgtgtggag cagtgcacct tcatggactc caaaatgaag 2281
cccctgtgga tcatgtacag cagcgaggag gccggcagtg ctggcaacgt gggcatcatc
2341 tttaagaacg gggatgacct ccgccaggac atgctgactc tgcagatgat
ccagctcatg 2401 gacgtcctgt ggaagcagga gggcctggac ctgaggatga
cgccctacgg ctgcctcccc 2461 accggggacc gcacaggtct catcgaggtg
gtcctccact cggacaccat cgccaacatc 2521 cagctgaaca aaagcaacat
ggcggccaca gctgccttca acaaggacgc cctgctcaac 2581 tggctcaagt
ccaagaaccc tggggaggcc ctggatcggg ccattgagga attcaccctc 2641
tcctgtgctg gctactgtgt ggccacatat gttctgggca tcggtgaccg gcacagcgac
2701 aacatcatga tcagagagag tgggcagatc ttccacattg attttggcca
ctttctgggg 2761 aacttcaaga ccaagtttgg aatcaaccga gagcgcgtcc
ccttcattct cacctacgac 2821 tttgtccacg tgatccagca ggggaagact
aacaacagtg agaagtttga aaggttccgc 2881 ggctactgtg aacgagccta
taccatcctg cggcgccacg ggctgctttt cctccatctc 2941 ttcgccctga
tgcgggccgc aggtctgcct gagcttagct gctccaaaga tatccagtat 3001
ctcaaggact ctctggcact ggggaagacg gaggaagagg cgctaaagca cttccgggtg
3061 aagttcaacg aagctctccg agaaagctgg aaaaccaaag tcaactggct
ggcgcacaat 3121 gtgtccaagg ataaccgaca gtag SEQ ID NO: 30 Mouse
PIK3CD (Isoform 2) Amino Acid Sequence 1 mppgvdcpme fwtkeesqsv
vvdfllptgv ylnfpvsrna nlstikqvlw hraqyeplfh 61 mlsdpeayvf
tcvnqtaeqq eledeqrrlc diqpflpvlr lvaregdrvk klinsqisll 121
igkglhefds lrdpevndfr tkmrqfceea aahrqqlgwv ewlqysfplq lepsargwra
181 gllrvsnral lvnvkfegse esftfqvstk dmplalmaca lrkkatvfrq
plveqpeeya 241 lqvngrheyl ygnyplchfq yicsclhsgl tphltmvhss
silamrdeqs npapgvqkpr 301 akpppipakk pssvslwsle qpfsielieg
rkvnadermk lvvqaglfhq nemlcktvss 361 sevnvcsepv wkqrlefdis
vcdlprmarl cfalyavvek akkarstkkk skkadcpiaw 421 anlmlfdykd
qlktgercly mwpsvpdekg ellnpagtvr gnpntesaaa lviylpevap 481
hpvyfpalek ilelgrhger griteeeqlq lreilerrgs gelyehekdl vwkmrhevqe
541 hfpealarll lvtkwnkhed vaqlsgmlyl lcswpelpvl salelldfsf
pdcyvgsfai 601 kslrkltdde lfqyllqlvq vlkyesyldc eltkfllgra
lanrkighfl fwhlrsemhv 661 psvalrfgli meaycrgsth hmkvlmkqge
alsklkalnd fvkvssqktt kpgtkemmhm 721 cmrqetymea lshlqspldp
stlleevcve gctfmdskmk plwimyssee agsagnvgii 781 fkngddlrqd
mltlqmiqlm dvlwkqegld lrmtpygclp tgdrtgliev vlhsdtiani 841
qlnksnmaat aafnkdalln wlksknpgea ldraieeftl scagycvaty vlgigdrhsd
901 nimiresgql fhidfghflg nfktkfginr ervpfiltyd fvhviqqgkt
nnsekferfr 961 gyceraytil rrhgllflhl falmraaglp elscskdiqy
lkdslalgkt eeealkhfrv 1021 kfnealresw ktkvnwlahn vskdnrq SEQ ID NO:
31 Mouse PIK3CD (Transcript 3) cDNA Acid Sequence 1 atgccccctg
gggtggactg ccccatggag ttctggacca aagaggagag ccagagcgtg 61
gttgttgact tcttgctgcc cacaggggtc tacttgaact tccccgtgtc ccgcaatgcc
121 aacctcagca ccatcaagca ggtgctgtgg caccgtgcac agtatgagcc
actcttccac 181 atgctcagtg accccgaggc ctatgtgttc acctgtgtga
accagacggc ggagcagcag 241 gagttggagg atgagcagcg gaggctgtgc
gacatccagc ccttcctgcc cgtgctgcgc 301 ctcgtggccc gagaggggga
ccgcgtgaag aagctcatta actcccagat cagcctcctc 361 attggcaaag
gtctccatga gtttgattcc ctgcgggacc cggaagtaaa cgacttccgc 421
actaagatgc gccagttttg tgaagaggct gctgctcacc gccagcagct gggctgggtg
481 gaatggctgc agtacagctt ccccctgcag ctggagccct cagcaagggg
ttggcgggcc 541 ggcttattgc gtgtcagcaa ccgagccctg ctggtcaacg
tgaagttcga gggcagtgag 601 gagagcttca ccttccaggt atccaccaag
gacatgcccc tggcactgat ggcctgtgcc 661 ctccgaaaaa aggccacagt
gttccggcag cctctggtgg agcagcctga ggaatatgcc 721 ctgcaggtga
acgggaggca cgaatacctc tacggcaact acccgctctg ccactttcag 781
tacatctgca gctgcctaca cagcgggctg acccctcatc tgaccatggt ccactcctcc
841 tccatccttg ctatgcggga tgagcagagc aatcctgccc cccaagtaca
gaaaccacgt 901 gccaaacctc ccccgatccc tgccaagaag ccctcctctg
tgtccctgtg gtccctggaa 961 cagccattct ccattgagct gatcgagggc
cgaaaagtga atgctgacga gcggatgaag 1021 ctggttgttc aggccgggct
cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc 1081 tcggaggtga
atgtatgctc agagcccgtg tggaagcagc gactggagtt cgatatcagc 1141
gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc tctatgccgt cgtggagaag
1201 gctaagaagg cacgctccac aaagaagaag tctaagaagg cggactgccc
catcgcttgg 1261 gccaacctca tgctattcga ctacaaagat cagctcaaga
cgggggagcg ctgcctctac 1321 atgtggccct ctgtcccaga tgagaaggga
gagctgctga atcctgcggg tacagtgcgc 1381 gggaacccca acacggagag
tgccgctgcc ctggtcatct acctgcctga ggtggccccc 1441 caccctgtgt
acttccccgc tctggagaag atcctggagc tggggcgtca cggggagcgt 1501
gggcgcatca cggaggagga gcagctgcag ctgcgggaga tcctggaacg gcggggatcc
1561 ggggaactgt acgaacatga gaaggacctg gtgtggaaga tgcgacacga
agtccaggag 1621 catttcccag aggcgctggc ccgcctgctg ctggtcacca
agtggaataa acacgaggat 1681 gtggcccagc tgtcccagat gctctatttg
ctgtgctcct ggcccgagct gcctgtgctg 1741 agcgccctgg aacttctgga
ctttagcttt cccgactgct acgtgggctc cttcgccatc 1801 aagtcccttc
ggaagctgac ggacgatgag ctcttccagt accttctgca gctggtgcaa 1861
gtgctcaaat atgagtccta cctggactgc gagctgacca aattcttgct gggccgagcc
1921 ctggctaacc gcaagatcgg acacttcctg ttctggcacc tccgctctga
gatgcacgta 1981 ccatcagtgg ctctgcggtt tggtctcatc atggaagcct
actgcagagg cagcacccac 2041 cacatgaagg tgctgatgaa gcagggggaa
gcactgagca agcttaaggc actgaatgac 2101 tttgtgaagg tgagttccca
gaagaccacc aagccccaaa ccaaggagat gatgcatatg 2161 tgcatgcgcc
aggagaccta catggaggcc ctgtcccacc tgcagtctcc actcgacccc 2221
agcaccctgc tggaggaagt ctgtgtggag cagtgcacct tcatggactc caaaatgaag
2281 cccctgtgga tcatgtacag cagcgaggag gcgggcagtg ctggcaacgt
gggcatcatc 2341 tttaagaacg gggatgacct ccgccaggac atgctgactc
tgcagatgat ccagctcatg 2401 gacgtcctgt ggaagcagga gggcctggac
ctgaggatga cgccctacgg ctgcctcccc 2461 accggggacc gcacaggtct
catcgaggtg gtcctccact cggacaccat cgccaacatc 2521 cagctgaaca
aaagcaacat ggcggccaca gctgccttca acaaggacgc cctactcaac 2581
tggctcaagt ccaagaaccc tggggaggcc ctggatcggg ccattgagga attcaccctc
2641 tcctgtgctg gctactgtgt ggccacatat gttctgggca tcggtgaccg
gcacagcgac 2701 aacatcatga tcagagagag tgggcagctc ttccacattg
attttggcca ctttctgggg 2761 aacttcaaga ccaagtttgg aatcaaccga
gagcgcgtcc ccttcattct cacctacgac 2821 tttgtccacg tgatccagca
ggggaagact aacaacagtg agaagtttga aaggttccgc 2881 ggctactgtg
aacgagccta taccatcctg cggcgccacg ggctgctttt cctccatctc 2941
ttcgccctga tgcgggccgc aggtctgcct gagcttagct gctccaaaga tatccagtat
3001 ctcaaggact ctctggcact ggggaagacg gaggaagagg cgctaaagca
cttccgggtg 3061 aagttcaacg aagctctccg agaaagctgg aaaaccaaag
tcaactggct ggcgcacaat 3121 gtgtccaagg ataaccgaca gtag SEQ ID NO: 32
Mouse PIK3CD (Isoform 3) Amino Acid Sequence 1 mppgvdcpme
fwtkeesqsv vvdfllptgv ylnfpvsrna nlstikqvlw hraqyeplfh 61
mlsdpeayvf tcvnqtaeqq eledeqrrlc diqpflpvlr lvaregdrvk klinsqisll
121 igkglhefds lrdpevndfr tkmrqfceea aahrqqlgwv ewlqysfplq
lepsargwra 181 gllrvsnral lvnvkfegse esftfqvstk dmplalmaca
lrkkatvfrq plveqpeeya 241 lqvngrheyl ygnyplchfq yicsclhsgl
tphltmvhss silamrdeqs npapgvqkpr 301 akpppipakk pssvslwsle
qpfsielieg rkvnadermk lvvqaglfhq nemlcktvss 361 sevnvcsepv
wkqrlefdis vcdlprmarl cfalyavvek akkarstkkk skkadcpiaw 421
anlmlfdykd qlktgercly mwpsvpdekg ellnpagtvr gnpntesaaa lviylpevap
481 hpvyfpalek ilelgrhger griteeeqlq lreilerrgs gelyehekdl
vwkmrhevqe 541 hfpealarll lvtkwnkhed vaqlsgmlyl lcswpelpvl
salelldfsf pdcyvgsfai 601 kslrkltdde lfqyllqlvq vlkyesyldc
eltkfllgra lanrkighfl fwhlrsemhv 661 psvalrfgli meaycrgsth
hmkvlmkqge alsklkalnd fvkvssqktt kpgtkemmhm 721 cmrqetymea
lshlqspldp stlleevcve gctfmdskmk plwimyssee agsagnvgii 781
fkngddlrqd mltlqmiqlm dvlwkqegld lrmtpygclp tgdrtgliev vlhsdtiani
841 qlnksnmaat aafnkdalln wlksknpgea ldraieeftl scagycvaty
vlgigdrhsd 901 nimiresgql fhidfghflg nfktkfginr ervpfiltyd
fvhviqqgkt nnsekferfr 961 gyceraytil rrhgllflhl falmraaglp
elscskdiqy lkdslalgkt eeealkhfrv 1021 kfnealresw ktkvnwlahn vskdnrq
SEQ ID NO: 33 Mouse PIK3CD (Transcript 4) cDNA Acid Sequence 1
atgccccctg gggtggactg ccccatggag ttctggacca aagaggagag ccaaagcgtg
61 gttgttgact tcttgctgcc cacaggggtc tacttgaact tccccgtgtc
ccgcaatgcc 121 aacctcagca ccatcaagca ggtgctgtgg caccgtgcac
agtatgagcc actcttccac 181 atgctcagtg accccgaggc ctatgtgttc
acctgtgtga accagacggc ggagcagcag 241 gagttggagg atgagcagcg
gaggctgtgc gacatccagc ccttcctgcc cgtgctgcgc 301 ctcgtggccc
gagaggggga ccgcgtgaag aagctcatta actcccagat cagcctcctc 361
attggcaaag gtctccatga gtttgattcc ctgcgggacc cggaagtaaa cgacttccgc
421 actaagatgc gccagttttg tgaagaggct gctgctcacc gccagcagct
gggctgggtg 481 gaatggctgc agtacagctt ccccctgcag ctggagccct
cagcaagggg ttggcgggcc 541 ggcttattgc gtgtcagcaa ccgagccctg
ctggtcaacg tgaagttcga gggcagtgag 601 gagagcttca ccttccaggt
atccaccaag gacatgcccc tggcactgat ggcctgtgcc 661 ctccgaaaaa
aggccacagt gttccggcag cctctggtgg agcagcctga ggaatatgcc 721
ctgcaggtga acgggaggca cgaatacctc tacggcaact acccgctctg ccactttcag
781 tacatctgca gctgcctaca cagcgggctg acccctcatc tgaccatggt
ccactcctcc 841 tccatccttg ctatgcggga tgagcagagc aatcctgccc
cccaagtaca gaaaccacgt 901 gccaaacctc ccccgatccc tgccaagaag
ccctcctctg tgtccctgtg gtccctggaa 961 cagccattct ccattgagct
gatcgagggc cgaaaagtga atgctgacga gcggatgaag 1021 ctggttgttc
aggccgggct cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc 1081
tcggaggtga atgtatgctc agagcccgtg tggaagcagc gactggagtt cgatatcagc
1141 gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc tctatgccgt
cgtggagaag 1201 gctaagaagg cacgctccac aaagaagaag tctaagaagg
cggactgccc catcgcttgg 1261 gccaacctca tgctattcga ctacaaagat
cagctcaaga cgggggagcg ctgcctctac 1321 atgtggccct ctgtcccaga
tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1381 gggaacccca
acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc 1441
caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca cggggagcgt
1501 gggcgcatca cggaggagga gcagctgcag ctgcgggaga tcctggaacg
gcggggatcc 1561 ggggaactgt acgaacatga gaaggacctg gtgtggaaga
tgcgccacga agtccaggag 1621 catttcccag aggcgctggc ccgcctgctg
ctggtcacca agtggaataa acacgaggat 1681 gtggcccaga tgctctattt
gctgtgctcc tggcccgagc tgcctgtgct gagcgccctg 1741 gaacttctgg
actttagctt tcccgactgc tacgtgggct ccttcgccat caagtccctt 1801
cggaagctga cggacgatga gctcttccag taccttctgc agctggtgca agtgctcaaa
1861 tatgagtcct acctggactg cgagctgacc aaattcttgc tgggccgagc
cctagctaac 1921 cgcaagatcg gacacttcct gttctggcac ctccgctctg
agatgcacgt accatcagtg 1981 gctctgcggt ttggtctcat catggaagcc
tactgcagag gcagcaccca ccacatgaag 2041 gtgctgatga agcaggggga
agcactgagc aagcttaagg cactgaatga ctttgtgaag 2101 gtgagttccc
agaagaccac caagccccaa accaaggaga tgatgcatat gtgcatgcgc 2161
caggagacct acatggaggc cctgtcccac ctgcagtctc cactcgaccc cagcaccctg
2221 ctggaggaag tctgtgtgga gcagtgcacc ttcatggact ccaaaatgaa
gcccctgtgg 2281 atcatgtaca gcagcgagga ggcgggcagt gctggcaacg
tgggcatcat ctttaagaac 2341 ggggatgacc tccgccagga catgctgact
ctgcagatga tccagctcat ggacgtcctg 2401 tggaagcagg aaggcctgga
cctgaggatg acgccctacg gctgcctccc caccggggac 2461 cgcacaggtc
tcatcgaggt ggtcctccac tcggacacca tcgccaacat ccagctgaac 2521
aaaagcaaca tggcggccac agctgccttc aacaaggacg ccctgctcaa ctggctcaag
2581 tccaagaacc ctggggaggc cctggatcgg gccattgagg aattcaccct
ctcctgtgct 2641 ggctactgtg tggccacata tgttctgggc atcggtgacc
ggcacagcga caacatcatg 2701 atcagagaga gtgggcagct cttccacatt
gattttggcc actttctggg gaacttcaag 2761 accaagtttg gaatcaaccg
agagcgcgtc cccttcattc tcacctacga ctttgtccac 2821 gtgatccagc
aggggaagac taacaacagt gagaagtttg aaaggttccg cggctactgt 2881
gaacgagcct ataccatcct gcggcgccac gggctgcttt tcctccatct cttcgccctg
2941 atgcgggccg caggtctgcc tgagcttagc tgctccaaag atatccagta
tctcaaggac 3001 tctctggcac tggggaagac ggaggaagag gcgctaaagc
acttccgggt gaagttcaac 3061 gaagctctcc gagaaagctg gaaaaccaaa
gtcaactggc tggcgcacaa tgtgtccaag 3121 gataaccgac agtag
SEQ ID NO: 34 Mouse PIK3CD (Isoform 4) Amino Acid Sequence 1
mppgvdcpme fwtkeesqsv vvdfllptgv ylnfpvsrna nlstikgvlw hraqyeplfh
61 mlsdpeayvf tcvnqtaeqq eledeqrrlc diqpflpvlr lvaregdrvk
klinsqisll 121 igkqlhefds lrdpevndfr tkmrqfceea aahrqqlqwv
ewlqysfplq lepsargwra 181 gllrvsnral lvnvkfegse esftfqvstk
dmplalmaca lrkkatvfrq plveqpeeya 241 lgvngrheyl ygnyplchfq
yicsclhsgl tphltmvhss silamrdeqs npapqvqkpr 301 akpppipakk
pssvslwsle qpfsielieg rkvnadermk lvvqaglfhg nemlcktvss 361
sevnvcsepv wkqrlefdis vcdlprmarl cfalyavvek akkarstkkk skkadcpiaw
421 anlmlfdvkd qlktgercly mwpsvpdekg ellnpagtvr gnpntesaaa
lviylpevap 481 hpvyfpalek ilelgrhger griteeeqlq lreilerrgs
gelyehekdl vwkmrhevqe 541 hfpealarll lvtkwnkhed vaqmlyllcs
wpelpvlsal elldfstpdc yvgsfaiksl 601 rkltddelfq yllqlvqvlk
yesyldcelt kfllgralan rkighflfwh lrsemhvpsv 661 alrfglimea
ycrgsthhmk vlmkqgeals klkalndfvk vssqkttkpq tkemmhmcmr 721
qetymealsh lqspldpstl leevcveqct fmdskmkplw imysseeags agnvgiifkn
781 gddlrqdmlt lqmiqlmdvl wkqegldlrm tpygclptgd rtglievvlh
sdtianiqln 841 ksnmaataaf nkdallnwlk sknpgealdr aieeftlsca
gycvatyvlg igdrhsdnim 901 iresgqlfhi dfghflgnfk tkfginrerv
pfiltydfvh viqqgktnns ekferfrgyc 961 eraytilrrh gllflhlfal
mraaglpels cskdiqylkd slalgkteee alkhfrvkfn 1021 ealreswktk
vnwlahnvsk dnrq SEQ ID NO: 35 Mouse PIK3CD (Transcript 5) cDNA Acid
Sequence 1 atgccccctg gggtggactg ccccatggag ttctggacca aagaggagag
ccagagcgtg 61 gttgttgact tcttgctgcc cacaggggtc tacttgaact
tccccgtgtc ccgcaatgcc 121 aacctcagca ccatcaagca ggtgctgtgg
caccgtgcac agtatgagcc actcttccac 181 atgctcagtg accccgaggc
ctatgtgttc acctgtgtga accagacggc ggagcagcag 241 gagttggagg
atgagcagcg gaggctgtgc gacatccagc ccttcctgcc cgtgctgcgc 301
ctcgtggccc gagaggggga ccgcgtgaag aagctcatta actcccagat cagcctcctc
361 attggcaaag gtctccatga gtttgattcc ctgcgggacc cggaagtaaa
cgacttccgc 421 actaagatgc gccagttttg tgaagaggct gctgctcacc
gccagcagct gggctgggtg 481 gaatggctgc agtacagctt ccccctgcag
ctggagccct cagcaagggg ttggcgggcc 541 ggcttattgc gtgtcagcaa
ccgagccctg ctggtcaacg tgaagttcga gggcagtgag 601 gagagcttca
ccttccaggt atccaccaag gacatgcccc tggcactgat ggcctgtgcc 661
ctccgaaaaa aggccacagt gttccggcag cctctggtgg agcagcctga ggaatatgcc
721 ctgcaggtga acgggaggca cgaatacctc tacggcaact acccgctctg
ccactttcag 781 tacatctgca gctgcctaca cagcgggctg acccctcatc
tgaccatggt ccactcctcc 841 tccatccttg ctatgcggga tgagcagagc
aatcctgccc cccaagtaca gaaaccacgt 901 gccaaacctc ccccgatccc
tgccaagaag ccctcctctg tgtccctgtg gtccctggaa 961 cagccattct
ccattgagct gatcgagggc cgaaaagtga atgctgacga gcggatgaag 1021
ctggttgttc aggccgggct cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc
1081 tcggaggtga atgtatgctc agagcccgtg tggaagcagc gactggagtt
cgatatcagc 1141 gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc
tctatgccgt cgtggagaag 1201 gctaagaagg cacgctccac aaagaagaag
tctaagaagg cggactgccc catcgcttgg 1261 gccaacctca tgctattcga
ctacaaagat cagctcaaga cgggggagcg ctgcctctac 1321 atgtggccct
ctgtcccaga tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1381
gggaacccca acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc
1441 caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca
cggggagcgt 1501 gggcgcatca cggaggagga gcagctgcag ctgcgggaga
tcctggaacg gcggggatcc 1561 gggaaactgt acgaacatga gaaggacctg
gtgtggaaga tgcgccacga agtccaggag 1621 catttcccag aggcgctggc
ccgcctgctg ctggtcacca agtggaataa acacgaggat 1681 gtggcccaga
tgctctattt gctgtgctcc tggcccgagc tgcctgtgct gagcgccctg 1741
gaacttctgg actttagctt tcccgactgc tacgtgggct ccttcgccat caagtccctt
1801 cggaagctga cggacgatga gctcttccag taccttctgc agctggtgca
agtgctcaaa 1661 tatgagtcct acctggactg cgagctgacc aaattcttgc
tgggccgagc cctggctaac 1921 cgcaagatcg gacacttcct gttctggcac
ctccgctctg agatgcacgt accatcagtg 1981 gctctgcggt ttggtctcat
catggaagcc tactgcagag gcagaaccca ccacatgaag 2041 gtgctgatga
agcaggggga agcactgagc aagcttaagg cactgaatga ctttgtgaag 2101
gtgagttccc agaagaccac caagccccaa accaaggaga tgatgcatat gtgcatgcgc
2161 caggagacct acatggaggc cctgtcccac ctgcagtatc cactcgaccc
cagcaccctg 2221 ctggaggaag tctgcagtgt ggagcagtgc accttcatgg
actccaaaat gaaacccctg 2281 tggatcatgt acagcagcga ggaggcgggc
agtgctggca acgtgggcat catctttaag 2341 aacggggatg acctccgcca
ggacatgctg actctgcaga tgatccagct catggacgtc 2401 ctgtggaagc
aggagggcct ggacctgagg atgacgccct acggctgcct ccccaccggg 2461
gaccgcacag gtctcatcga ggtggtcctc cactcggaca ccatcgccaa catccagctg
2521 aacaaaagca acatggcggc cacagctgcc ttcaacaagg acgccctgct
caactggctc 2581 aagtccaaga accctgggaa ggccctggat cgggccattg
aggaattcac cctctcctgt 2641 gctggctact gtgtggccac atatgttctg
ggcatcggtg accggcacag cgacaacatc 2701 atgatcagag agagtgggca
gctcttccac attgattttg gccactttct ggggaacttc 2761 aagaccaagt
ttggaatcaa ccgagagcgc gtccccttca ttctcaccta cgactttgtc 2821
cacgtgatcc agcaggggaa gactaacaac agtgagaagt ttgaaaggtt ccgcggctac
2881 tgtgaacgag cctataccat cctgcggcgc cacgggctgc ttttcctcca
tctcttcgcc 2941 ctgatgcggg ccgcaggtct gcctgagctt agctgctcca
aagatatcca gtatctcaag 3001 gactctctgg cactggggaa gacggaggaa
gaggcgctaa agcacttccg ggtgaagttc 3061 aacgaagctc tccgagaaag
ctggaaaacc aaagtcaact ggctggcgca caatgtgtcc 3121 aaggataacc
gacagtag SEQ ID NO: 36 Mouse PIK3CD (Isoform 5) Amino Acid Sequence
1 mppgvdcpme fwtkeesqsv vvdfllptgv ylnfpvsrna nlstikqvlw hraqyeplfh
61 mlsdpeayvf tcvnqtaeqq eledeqrrlc diqpflpvlr lvaregdrvk
klinsqisll 121 igkglhefds lrdpevndfr tkmrqfceea aahrqqlgwv
ewlgysfplg lepsargwra 181 gllrvsnral lvnvkfegse esftfqvstk
dmplalmaca lrkkatvfrq plveqpeeya 241 lqvngrheyl ygnyplchfg
yicsclhsgl tphltmvhss silamrdeqs npapqvqkpr 301 akpppipakk
pssvslwsle qpfsielieg rkvnadermk lvvqaglfhg nemlcktvss 361
sevnvcsepv wkqrlefdis vcdlprmarl cfalyavvek akkarstkkk skkadcpiaw
421 anlmlfdykd qlktgercly mwpsvpdekg ellnpagtvr gnpntesaaa
lviylpevap 481 hpvyfpalek ileigrhger griteeeqlq lreilerrgs
gelyehekdl vwkmrhevqe 541 hfpealarll lvtkwnkhed vaqmlyllcs
wpelpvlsal elldfsfpdc yvgsfaiksl 601 rkltddelfq yllqlvqvlk
yesyldcelt kfllgralan rkighflfwh lrsemhvpsv 661 alrfglimea
ycrgsthhmk vlmkqgeals klkalndfvk vssqkttkpq tkemmhmcmr 721
qetymealsh lqspldpstl leevcsveqc tfmdskmkpl wimysseeag sagnvgiifk
781 ngddlrqdml tlqmiqlmdv lwkqegldlr mtpygclptg drtglievvl
hsdtianiql 841 nksnmaataa fnkdallnwl ksknpgeald raieeftlsc
agycvatyvl gigdrhsdni 901 miresgqlfh idfghflgnf ktkfginrer
vpfiltydfv hviqqgktnn sekferfrqy 961 ceraytilrr hgllflhlfa
lmraaglpel scskdiqylk dslalgktee ealkhfrvkf 1021 nealreswkt
kvnwlahnvs kdnrq SEQ ID NO: 37 Mouse PIK3CD (Transcript 6) cDNA
Acid Sequence 1 atgccccctg gggtggactg ccccatggag ttctggacca
aagaggagag ccagagcgtg 61 gttgttgact tcttgctgcc cacaggggtc
tacttgaact tccccgtgtc ccgcaatgcc 121 aacctcagca ccatcaagca
ggtgctgtgg caccgtgcac agtatgagcc actcttccac 181 atgctcagtg
accccgaggc ctatgtgttc acctgtgtga accagacggc ggagcagcag 241
gagttggagg atgagcagcg gaggctgtgc gacatccagc ccttcctgcc cgtgctgcgc
301 ctcgtggccc gagaggggga ccgcgtgaag aagctcatta actcccagat
cagcctcctc 361 attggcaaag gtctccatga gtttgattcc ctgcgggacc
cggaagtaaa cgacttccgc 421 actaagatgc gccagttttg tgaagaggct
gctgctcacc gccagcagct gggctgggtg 481 gaatggctgc agtacagctt
ccccctgcag ctggagccct cagcaagggg ttggcgggcc 541 ggcttattgc
gtgtcagcaa ccgagccctg ctggtcaacg tgaagttcga gggcagtgag 601
gagagcttca ccttccaggt atcaaccaag gacatgcccc tggcactgat ggcctgtgcc
661 ctccgaaaaa aggccacagt gttccggcag cctctggtgg agcagcctga
ggaatatgcc 721 ctgcaggtga acgggaggca cgaatacctc tacggcaact
acccgctctg ccactttcag 781 tacatctgca gctgcctaca cagcgggctg
acccctcatc tgaccatggt ccactcctcc 841 tccatccttg ctatgcggga
tgagcagagc aatcctgccc cccaagtaca gaaaccacgt 901 gccaaacctc
ccccgatccc tgccaagaag ccctcctctg tgtccctgtg gtccctggaa 961
cagccattct ccattgagct gatcgagggc cgaaaagtga atgctgacga gcggatgaag
1021 ctggttgttc aggccgggct cttccatggc aatgagatgc tgtgcaagac
tgtgtcaagc 1081 tcggaggtga atgtatgctc agagcccgtg tggaagcagc
gactggagtt cgatatcagc 1141 gtctgtgacc tcccgcgcat ggctcgactc
tgttttgctc tctatgccgt cgtggagaag 1201 gctaagaagg cacgctccac
aaagaagaag tctaagaagg cggactgccc catcgcttgg 1261 gccaacctca
tgctattcaa ctacaaagat cagctcaaga cgggggagcg ctgcctctac 1321
atgtggccct ctgtcccaga tgagaaggga gagctgctga atcctgcggg tacagtgcgc
1381 gggaacccca acacggagag tgccgctgcc ctggtcatct acctgcctga
ggtggccccc 1441 caccctgtgt acttccccgc tctggagaag atcctggagc
tggggcgtca cggggagcgt 1501 gggcgcatca cggaggagga gctgcagctg
cgggagatcc tggaacggcg gggatccggg 1561 gaactgtacg aacatgagaa
ggacctggtg tggaagatgc gccacgaagt ccaggagcat 1621 ttcccagagg
cgctggcccg cctgctgctg gtcaccaagt ggaataaaca cgaggatgtg 1681
gcccagctgt cccagatgct ctatttgctg tgctcctggc ccgagctgcc tgtgctgagc
1741 gccctggaac ttctggactt tagctttccc gactgctacg tgggctcctt
cgccatcaag 1801 tcccttcgga agctgacgga cgatgagctc ttccagtacc
ttctgcagct ggtgcaagtg 1861 ctcaaatatg agtcctacct ggactgcgag
ctgaccaaat tcttgctggg ccgagccctg 1921 gctaaccgca agatcggaca
cttcctgttc tggcacctcc gctctgagat gcacgtacca 1981 tcagtggctc
tgcggtttgg tctcatcatg gaagcctact gcagaggcag cacccaccac 2041
atgaaggtgc tgatgaagca gggggaagca ctgagcaagc ttaaggcact gaatgacttt
2101 gtgaaggtga gttcccagaa gaccaccaag ccccaaacca aggagatgat
gcatatgtgc 2161 atgcgccagg agacctacat ggaggccctg tcccacctgc
agtctccact cgaccccagc 2221 accctgctgg aggaagtctg tgtggagcag
tgcaccttca tggactccaa aatgaagccc 2281 ctgtggatca tgtacagcag
cgaggaggcg ggcagtgctg gcaacgtggg catcatcttt 2341 aagaacgggg
atgacctccg ccaggacatg ctgactctgc agatgatcca gctcatggac 2401
gtcctgtgga agcaggaggg cctggacctg aggatgacgc cctacggctg cctccccacc
2461 ggggaccgca caggtctcat cgaggtggtc ctccactcgg acaccatcgc
caacatccag 2521 ctgaacaaaa gcaacatggc ggccacagct gccttcaaca
aggacgccct gctcaactgg 2581 ctcaagtcaa agaaccctgg ggaggccctg
gatcgggcca ttgaggaatt caccctctcc 2641 tgtgctggct actgtgtggc
cacatatgtt ctgggcatcg gtgaccggca cagcgacaac 2701 atcatgatca
gagagagtgg gcagctcttc cacattgatt ttggccactt tctggggaac 2761
ttcaagacca agtttggaat caaccgagag cgcgtcccct tcattctcac ctacgacttt
2821 gtccacgtga tccagcaggg gaagactaac aacagtgaga agtttgaaag
gttccgcggc 2881 tactgtgaac gagcctatac catcctgcgg cgccacgggc
tgcttttcct ccatctcttc 2941 gccctgatgc gggccgcagg tctgcctgag
cttagctgct ccaaagatat ccagtatctc 3001 aaggactctc tggcactggg
gaagacggag gaagaggcgc taaagcactt ccgggtgaag 3061 ttcaacgaag
ctctacgaaa aagctggaaa accaaagtca actggctggc gcacaatgtg 3121
tccaaggata aacgacagta g SEQ ID NO: 38 Mouse PIK3CD (Isoform 6)
Amino Acid Sequence 1 mppgvdcpme fwtkeesgsv vvdfllptgv ylnfpvsrna
nlstikqvlw hraqyeplfh 61 mlsdpeayvf tcvnqtaeqq eledeqrrlc
diqpflpvlr lvaregdrvk klinsqisll 121 igkglhefds lrdpevndfr
tkmrqfceea aahrqqlgwv ewlqysfplq lepsargwra 181 gllrvsnral
lvnvkfegse esftfqvstk dmplalmaca lrkkatvfrq plveqpeeya 241
lqvngrheyl ygnyplchfq yicsclhsgl tphltmvhss silamrdeqs npapqvgkpr
301 akpppipakk pssvslwsle qpfsielieg rkvnadermk lvvqaglfhq
nemlcktvss 361 sevnvcsepv wkqrlefdis vcdlprmarl cfalyavvek
akkarstkkk skkadcpiaw 421 anlmlfdykd qlktgercly mwpsvpdekg
ellnpagtvr gnpntesaaa lviylpevap 481 hpvyfpalek ilelgrhger
griteeelql reilerrgsg elyehekdlv wkmrhevqeh 541 fpealarlll
vtkwnkhedv aqlsqmlyll cswpelpvls alelldfsfp dcyvgsfaik 601
slrkltddel fqyllqlvqv lkyesyldce ltkfllgral anrkighflf whlrsemhvp
661 svalrfglim eaycrgsthh mkvlmkqgea lsklkalndf vkvssqkttk
pqtkemmhmc 721 mrqetymeal shlqspldps tlleevcveq ctfmdskmkp
lwimysseea gsagnvgiif 781 kngddlrqdm ltlqmiqlmd vlwkqegldl
rmtpygclpt gdrtglievv lhsdtianiq 841 lnksnmaata afnkdallnw
lksknpgeal draieeftls cagycvatyv lgigdrhsdn 901 imiresgqlf
hidfghflgn fktkfginre rvpfiltydf vhviqqgktn nsekferfrg 961
yceraytilr rhgllflhlf almraaglpe lscskdiqyl kdslalgkte eealkhfrvk
1021 fnealreswk tkvnwlahnv skdnrq SEQ ID NO: 39 Human PD-1 cDNA
Sequence caatctggtg gggctgctcc aggc atg cag atc cca cag gcg ccc tgg
cca 51 Met Gln Ile Pro Gln Ala Pro Trp Pro 1 5 gtc gtc tgg gcg gtg
cta caa ctg ggc tgg cgg cca gga tgg ttc tta 99 Val Val Trp Ala Val
Leu Gln Leu Gly Trp Arg Pro Gly Trp Phe Leu 10 15 20 25 gac tcc cca
gac agg ccc tgg aac ccc ccc acc ttc tcc cca gcc ctg 147 Asp Ser Pro
Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 30 35 40 ctc
gtg gtg acc gaa ggg gac aac gcc acc ttc acc tgc agc ttc tcc 195 Leu
Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 45 50
55 aac aca tcg gag agc ttc gtg cta aac tgg tac cgc atg agc ccc agc
243 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser
60 65 70 aac cag acg gac aag ctg gcc gcc ttc ccc gag gac cgc agc
cag ccc 291 Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser
Gln Pro 75 80 85 ggc cag gac tgc cgc ttc cgt gtc aca caa ctg ccc
aac ggg cgt gac 339 Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro
Asn Gly Arg Asp 90 95 100 105 ttc cac atg agc gtg gtc agg gcc cgg
cgc aat gac agc ggc acc tac 387 Phe His Met Ser Val Val Arg Ala Arg
Arg Asn Asp Ser Gly Thr Tyr 110 115 120 ctc tgt ggg gcc atc tcc ctg
gcc ccc aag gcg cag atc aaa gag agc 435 Leu Cys Gly Ala Ile Ser Leu
Ala Pro Lys Ala Gln Ile Lys Glu Ser 125 130 135 ctg cgg gca gag ctc
agg gtg aca gag aga agg gca gaa gtg ccc aca 483 Leu Arg Ala Glu Leu
Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 140 145 150 gcc cac ccc
agc ccc tca ccc agg tca ggc ggc cag ttc caa acc ctg 531 Ala His Pro
Ser Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln Thr Leu 155 160 165 gtg
gtt ggt gtc gtg ggc ggc ctg ctg ggc agc ctg gtg ctg cta gtc 579 Val
Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val 170 175
180 185 tgg gtc ctg gcc gtc atc tgc tcc cgg gcc gca cga ggg aca ata
gga 627 Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala Arg Gly Thr Ile
Gly 190 195 200 gcc agg cgc acc ggc cag ccc ctg aag gag gac ccc tca
gcc gtg cct 675 Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser
Ala Val Pro 205 210 215 gtg ttc tct gtg gac tat ggg gag ctg gat ttc
cag tgg cga gag aag 723 Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe
Gln Trp Arg Glu Lys 220 225 230 acc ccg gag ccc ccc gtg ccc tgt gtc
cct gag cag acg gag tat gcc 771 Thr Pro Glu Pro Pro Val Pro Cys Val
Pro Glu Gln Thr Glu Tyr Ala 235 240 245 acc att gtc ttt cct agc gga
atg ggc acc tca tcc ccc gcc cgc agg 819 Thr Ile Val Phe Pro Ser Gly
Met Gly Thr Ser Ser Pro Ala Arg Arg 250 255 260 265 ggc tca gct gac
gqc cct cgg agt gcc cag cca ctg agg cct gag gat 867 Gly Ser Ala Asp
Gly Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp 270 275 280 gga cac
tgc tct tgg ccc ctc tgaccggett ccttggccac cagtgttctg cag 921 Gly
His Cys Ser Trp Pro Leu 285 SEQ ID NO: 40 Human PD-1 Amino Acid
Sequence Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val
Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro
Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu
Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe
Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met
Ser Pro Ser Asa Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu
Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr
Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115
120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg
Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser
Pro Ser Pro 145 150 155 160
Arg Ser Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165
170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile
Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr
Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe
Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys
Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr
Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser
Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala
Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285
SEQ ID NO: 41 Human PD-L1S cDNA Acid Sequence gcttcccgag gctccgcacc
agccgcgctt ctgtccgcct gcagggcatt ccagaaag 58 atg agg ata ttt gct
gtc ttt ata ttc atg acc tac tgg cat ttg ctg 106 Met Arg Ile Phe Ala
Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 aac gca ttt
act gtc acg gtt ccc aag gac cta tat gtg gta gag tat 154 Asn Ala Phe
Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 ggt
agc aat atg aca att gaa tgc aaa ttc cca gta gaa aaa caa tta 202 Gly
Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40
45 gac ctg gct gca cta att gtc tat tgg gaa atg gag gat aag aac att
250 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60 att caa ttt gtg cat gga gag gaa gac ctg aag gtt cag cat
agt agc 298 Ile Gln Phe Val His Gly Gln Glu Asp Lee Lys Val Gln His
Ser Ser 65 70 75 80 tac aga cag agg gcc cgg ctq ttg aag gac cag ctc
tcc ctg gga aat 346 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu
Ser Leu Gly Asn 85 90 95 gct gca ctt cag atc aca gat gtg aaa ttg
cag gat gca ggg gtg tac 394 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu
Gln Asp Ala Gly Val Tyr 100 105 110 cgc tgc atg atc agc tat ggt ggt
gcc gac tac aag cga att act gtg 442 Arg Cys Met Ile Ser Tyr Gly Gly
Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 aaa gtc aat gcc cca tac
aac aaa atc aac caa aga att ttg gtt gtg 490 Lys Val Asn Ala Pro Tyr
Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 gat cca gtc acc
tct gaa cat gaa ctg aca tgt cag gct gag ggc tac 538 Asp Pro Val Thr
Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 ccc
aag gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt 586 Pro
Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170
175 ggt aag acc acc acc acc aat tcc aag aga gag gag aag ctt ttc aat
634 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190 gtg acc agc aca ctg aga atc aac aca aca act aat gag att
ttc tac 682 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile
Phe Tyr 195 200 205 tgc act ttt agg aga tta gat cct gag gaa aac cat
aca gct gaa ttg 730 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His
Thr Ala Glu Leu 210 215 220 gtc atc cca ggt aat att ctg aat gtg tcc
att aaa ata tgt cta aca 778 Val Ile Pro Gly Asn Ile Leu Asn Val Ser
Ile Lys Ile Cys Leu Thr 225 230 235 240 ctg tcc cct agc acc
tagcatgatg tctgcctatc atagtcattcc agtgattgtt 833 Leu Ser Pro Ser
Thr 245 gaataaatga atgaataaat aacactatgt ttacaaaata tatcctaatt
cctcacctcc 893 attcatccaa accatattgt tacttaataa acattcagca
gatatttatg gaataaaaaa 953 aaaaaaaaaa aaaaa 968 SEQ ID NO: 42 Human
PD-L1S Amino Acid Sequence Met Arg Ile Phe Ala Val Phe Ile Phe Met
Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro
Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile
Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala
Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85
90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val
Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln
Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu
Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile
Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr
Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210
215 220 Val Ile Pro Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu
Thr 225 230 235 240 Leu Ser Pro Ser Thr 245 SEQ ID NO: 43 Human
PD-L1M cDNA Acid Sequence cgaggctccg caccagccgc gcttctgtcc
gcctgcaggg cattccagaa agatgagg 58 Met Arg 1 ata ttt gct gtc ttt ata
ttc atg acc tac tgg cat ttg ctg aac gca 106 Ile Phe Ala Val Phe Ile
Phe Met Thr Tyr Trp His Leu Leu Asn Ala 5 10 15 ttt act gtc acg gtt
ccc aag gac cta tat gtg gta gag tat ggt agc 154 Phe Thr Val Thr Val
Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Per 20 25 30 aat atg aca
att gaa tgc aaa ttc cca gta gaa aaa caa tta gac ctg 202 Asn Met Thr
Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 35 40 45 50 gct
gca cta att gtc tat tgg gaa atg gag gat aag aac att att caa 250 Ala
Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 55 60
65 ttt gtg cat gga gag gaa gac ctg aag gtt cag cat agt agc tac aga
298 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
70 75 80 cag agg gcc cgg ctg ttg aag gac cag ctc tcc ctg gga aat
gct gca 346 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
Ala Ala 85 90 95 ctt cag atc aca gat gtg aaa ttg cag gat gca ggg
gtg tac cgc tgc 394 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly
Val Tyr Arg Cys 100 105 110 atg atc agc tat ggt ggt gcc gac tac aag
cga att act gtg acc gtc 442 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys
Arg Ile Thr Val Lys Val 115 120 125 130 aat gcc cca tac aac aaa atc
aac caa aga att ttg gtt gtg gat cca 490 Asn Ala Pro Tyr Asn Lys Ile
Asn Gln Arg Ile Leu Val Val Asp Pro 135 140 145 gtc acc tct gaa cat
gaa ctg aca tgt cag gct gag ggc tac ccc aag 538 Val Thr Ser Glu His
Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 150 155 160 gcc gaa gtc
atc tgg aca agc agt gac cat caa gtc ctg agt ggt aag 586 Ala Glu Val
Ile Trp The Ser Ser Asp His Gln Val Leu Ser Gly Lys 165 170 175 acc
acc acc acc aat tcc aag aga gag gag aag ctt ttc aat gtg acc 634 Thr
Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 180 185
190 agc aca ctg aga atc aac aca aca act aat gag att ttc tac tgc act
682 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
195 200 205 210 ttt agg aga tta gat cct gag gaa aac cat aca gct gaa
ttg gtc atc 730 Phe Arq Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu
Leu Val Ile 215 220 225 cca gaa cta cct ctg gca cat cct cca aat gaa
agg act cac ttg gta 778 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu
Arg Thr His Leu Val 230 235 240 att ctg gga gcc atc tta tta tgc ctt
ggt gta gca ctg aca ttc atc 826 Ile Leu Gly Ala Ile Leu Leu Cys Leu
Gly Val Ala Leu Thr Phe Ile 245 250 255 ttc cgt tta aga aaa ggg aga
atg atg gat gtg aaa aaa tgt ggc atc 874 Phe Arg Leu Arg Lys Gly Arg
Met Met Asp Val Lys Lys Cys Gly Ile 260 265 270 caa gat aca aac tca
aag aag caa agt gat aca cat ttg gag gag acg 922 Gln Asp Thr Asn Ser
Lys Lys Gln Ser Asp Thr His Leu Glu Glu Thr 275 280 285 290
taatccagca ttggaacttc tgatcttcaa gcagggattc tcaacctgtg gtttaggggt
982 tcatcggggc tgagcgtgac aagaggaagg aatgggcccg tgggatgcag
gcaatgtggg 1042 acttaaaagg cccaagcact gaaaatggaa cctggcgaaa
gcagaggagg agaatgaaga 1102 aagatggagt caaacaggga gcctggaggg
agaccttgat actttcaaat gcctgagggg 1162 ctcatcgacg cctgtgacag
ggagaaagga tacttctgaa caaggagcct ccaagcaaat 1222 catccattgc
tcatcctagg aagacgggtt gagaatccct aatttgaggg tcagttcctg 1282
cagaagtgcc ctttgcctcc actcaatgcc tcaatttgtt ttctgcatga ctgagagtct
1342 cagtgttgga acgggacagt atttatgtat gagtttttcc tatttatttt
gagtctgtga 1402 ggtcttcttg tcatgtgagt gtggttgtga atgatttctt
ttgaagatat attgtagtag 1462 atgttacaat tttgtcgcca aactaaactt
gctgcttaat gatttgctca catctagtaa 1522 aacatggagt atttgtaaaa
aaaaaaaaaa a 1533 SEQ ID NO: 44 Human PD-L1M Amino Acid Sequence
Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5
10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu
Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu
Lys Gln Lee 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met
Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp
Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu
Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile
Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met
Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys
Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135
140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln
Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu
Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr
Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp
Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu
Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val
Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255
Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260
265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu
Glu 275 280 205 Glu Thr 290 SEQ ID NO: 45 Human PD-L2 cDNA Acid
Sequence atg atc ttc ctc ctg cta atg ttg agc ctg gaa ttg cag ctt
cac cag 48 Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu
His Gln 1 5 10 15 ata gca gct tta ttc aca gtg aca gtc cct aag gaa
ctg tac ata ata 96 Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu
Leu Tyr Ile Ile 20 25 30 gag cat ggc agc aat gtg acc ctg gaa tgc
aac ttt gac act gga agt 144 Glu His Gly Ser Asn Val Thr Leu Glu Cys
Asn Phe Asp Thr Gly Per 35 40 45 cat gtg aac ctt gga gca ata aca
gcc agt ttg caa aag gtg gaa aat 192 His Val Asn Leu Gly Ala Ile Thr
Ala Ser Leu Gln Lys Val Glu Asn 50 55 60 gat aca tcc cca cac cgt
gaa aga gcc act ttg ctg gag gag cag ctg 240 Asp Thr Ser Pro His Arg
Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu 65 70 75 80 ccc cta ggg aag
gcc tcg ttc cac ata cct caa gtc caa gtg agg gac 200 Pro Leu Gly Lys
Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90 95 gaa gga
cag tac caa tgc ata atc atc tat ggg gtc gcc tgg gac tac 336 Glu Gly
Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105 110
aag tac ctg act ctg aaa gtc aaa gct tcc tac agg aaa ata aac act 384
Lys Tyr Leu Thr Lee Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr 115
120 125 cac atc cta aag gtt cca gaa aca gat gag gta gag ctc acc tgc
cag 432 His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys
Gln 130 135 140 gct aca ggt tat cct ctg gca gac gta tcc tgg cca aac
gtc agc gtt 480 Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn
Val Ser Val 145 150 155 160 cct gcc aac acc agc cac tcc agg acc cct
gaa ggc ctc tac cag gtc 528 Pro Ala Asn Thr Ser His Ser Arg Thr Pro
Glu Gly Leu Tyr Gln Val 165 170 175 acc agt gtt ctg cgc cta aag cca
ccc cct ggc aga aac ttc agc tgt 576 Thr Ser Val Leu Arg Leu Lys Pro
Pro Pro Gly Arg Asn Phe Ser Cys
180 185 190 gtg ttc tgg aat act cac gtg agg gaa ctt act ttg gcc agc
att gac 524 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser
Ile Asp 195 200 205 ctt caa agt cag atg gaa ccc agg acc cat cca act
tgg ctg ctt cac 672 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr
Trp Leu Leu His 210 215 220 att ttc atc ccc tcc tgc atc att gct ttc
att ttc ata gcc aca gtg 720 Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe
Ile Phe Ile Ala Thr Val 225 230 235 240 ata gcc cta aga aaa caa ctc
tgt caa aag ctg tat tct tca aaa gac 768 Ile Ala Leu Arg Lys Gln Leu
Cys Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255 aca aca aaa aga cct
gtc acc aca aca aag agg gaa gtg aac agt gct 816 Thr Thr Lys Arg Pro
Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265 270 atc 819 Ile
SEQ ID NO: 46 Human PD-L2 Amino Acid Sequence Met Ile Phe Leu Leu
Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu
His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40
45 His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn
50 55 60 Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu
Gln Leu 65 70 75 80 Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val
Gln Val Arg Asp 85 90 95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr
Gly Val Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys
Ala Ser Tyr Arg Lys Ile Asn Thr 115 120 125 His Ile Leu Lys Val Pro
Glu Thr Asp Glu Val Glu Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr
Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val 145 150 155 160 Pro
Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170
175 Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys
180 185 190 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser
Ile Asp 195 200 205 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr
Trp Leu Leu His 210 215 220 Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe
Ile Phe Ile Ala Thr Val 225 230 235 240 Ile Ala Leu Arg Lys Gln Leu
Cys Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro
Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 250 265 270 Ile SEQ ID
NO: 47 Human TIM-3 cDNA Sequence 1 atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 61 tcctcagaag tggaatacag
agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 121 accccagccg
ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 181
tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
241 agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 301 actctagcag acagtgggat ctactgctgc cggatccaaa
tcccaggcat aatgaatgat 361 gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactcgg 421 cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 481 gagacacaga
cactggggag cctccctgat ataaatctaa cacaaatatc cacattggcc 541
aatgagttac gggactctag attggccaat gacttacggg actctggagc aaccatcaga
601 ataggcatct acatcggagc agggatctgt gctgggctgg ctctggctct
tatcttcggc 661 gctttaattt tcaaatggta ttctcatagc aaagagaaga
tacagaattt aagcctcatc 721 tctttggcca acctccctcc ctcaggattg
gcaaatgcag tagcagaggg aattcgctca 781 gaagaaaaca tctataccat
tgaagagaac gtatatgaag tggaggagcc caatgagtat 841 tattgctatg
tcagcagcag gcagcaaccc tcacaacctt tgggttgtcg ctttgcaatg 901 ccatag
SEQ ID NO: 48 Human TIM-3 Amino Acid Sequence 1 mfshlpfdcv
lllllllltr sseveyraev gqnaylpcfy tpaapgnlvp vcwgkgacpv 61
fecgnvvlrt derdvnywts rywlngdfrk gdvsltienv tladsgiycc riqipgimnd
121 ekfnlklvik pakvtpaptr qrdftaafpr mlttrghgpa etqtlgslpd
inltqistla 181 nelrdsrlan dlrdsgatir igiyigagic aglalalifg
alifkwyshs kekiqnlsli 241 slanlppsgl anavaegirs eeniytieen
vyeveepney ycyvssrqqp sqplgcrfam 301 p SEQ ID NO: 49 Mouse TIM-3
cDNA Sequence 1 atgttttcag gtcttaccct caactgtgtc ctgctgctgc
tgcaactact acttgcaagg 61 tcattggaaa atgcttatgt gtttgaggtt
ggtaagaatg cctatctgcc ctgcagttac 121 actctatcta cacctgggac
acttgtgcct atgtgctggg gcaagggatt ctgtccttgg 181 tcacagtgta
ccaacgagtt gctcagaact gatgaaagaa atgtgacata tcagaaatcc 241
agcagatacc agctaaaggg cgatctcaac aaaggagacg tgtctctgat cataaagaat
301 gtgactctgg atgaccatag gacctactgc tgcaggatac agttccctgg
tcttatgaat 361 gataaaaaat tagaactgaa attagacatc aaagcagcca
aggtcactcc agctcagact 421 gcccatgggg actctactac agcttctcca
agaaccctaa ccacggagag aaatggttca 481 gagacacaga cactggtgac
cctccataat aacaatggaa caaaaatttc cacatgggct 541 gatgaaatta
aggactctgg agaaacgatc agaactgcta tccacattgg agtaggagtc 601
tctgctgggt tgaccctggc acttatcatt ggtgtcttaa tccttaaatg gtattcctgt
661 aagaaaaaga agttatcgag tttgagcctt attacactgg ccaacttgcc
tccaggaggg 721 ttggcaaatg caggagcagt caggattcgc tctgaggaaa
atatctacac catcgaggag 781 aacgtatatg aagtggagaa ttcaaatgag
tactactgct acgtcaacag ccagcagcca 841 tcctga SEQ ID NO: 50 Mouse
TIM-3 Amino Acid Sequence 1 mfsgltlncv llllqlllar slenayvfev
gknaylpcsy tlstpgalvp mcwgkgfcpw 61 sqctnellrt dernvtyqks
sryqlkgdln kgdvsliikn vtlddhgtyc criqfpglmn 121 dkklekldi
kaakvtpaqt ahgdsttasp rtltterngs etqtlvtlhn nngtkistwa 181
deikdsgeti rtaihigvgv sagltlalii gvlilkwysc kkkklsslsl itlanlppgg
241 lanagavrir seeniytiee nvyevensne yycyvnsqqp s SEQ ID NO: 51
Human LAG-3 cDNA Sequence 1 atgtgggagg ctcagttcct gggcttgctg
tttctgcagc cgctttgggt ggctccagtg 61 aagcctctcc agccagggac
tgaggtcccg gtggtgtgga cccaggaggg ggctcctgcc 121 cagctcccct
gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 181
gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg
241 gcccccggcc ctcacccgac ggcgccctcc tcctgggggc ccaggccccg
ccgctacacg 301 gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc
tgcccctgca gccccgcgtc 361 cagctggatg agcgcggccg gcagcgcggg
gacttctcgc tatggctgcg cccagcccgg 421 cgcgcggacg ccggcgagta
ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc 481 cgcctccgtc
tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga 541
gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg
601 cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc
ccatcaccac 661 ttagcggaaa gcttcctctt cctgccccaa gtcagcccca
tggactctgg gccctggggc 721 tgcatcctca cctacagaga tggcttcaac
gtctccatca tgtataacct cactgttctg 781 ggtctggagc ccccaactcc
cttgacagtg tacgctggag caggttccag ggtggggctg 841 ccctgccgcc
tgcctgctgg tgtggggacc cggtctttcc tcactgccaa gtgaactcct 901
cctgggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttcgacta
961 gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct
gcaggaacag 1021 cagctcaatg ccactgtcac attggcaatc atcacagtga
ctcccaaatc ctttgggtca 1081 cctggatccc tggggaagct gctttgtgag
gtgactccag tatctggaca agaacgcttt 1141 gtgtggagct ctctggacac
cccatcccag aggagtttct caggaccttg gctggaggca 1201 caggaggccc
agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggctt 1261
cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga
1321 gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcatccttgg
tgtcctttct 1381 ctgctccttt tggtgactgg agcctttggc tttcaccttt
ggagaagaca gtggcgacca 1441 agacgatttt ctgccttaca gcaagggatt
caccctccgc aggctcagag caagatagag 1501 gagctggagc aagaaccgga
gccggagccg gagccggaac cggagcccga gcccgagccc 1561 gagccggagc
agctctga SEQ ID NO: 52 Human LAG-3 Amino Acid Sequence 1 mweaqflgll
flqplwvapv kplqpgaevp vvwaqegapa qlpcsptipl qdlsllrrag 61
vtwqhqpdsg ppaaapghpl apgphpaaps swgprprryt vlsvgpgglr sgrlplqprv
121 qldergrqrg dfslwlrpar radageyraa vhlrdralsc rlrlrlgqas
mtasppgslr 181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh
laesflflpq vspmdsgpwg 241 ciltyrdgfn vsimynltvl glepptpltv
yagagsrvgl pcrlpagvqt rsfltakwtp 301 pgggpdllvt gdngdftlrl
edvsqaqagt ytchihlqeq glnatvtlai itvtpksfgs 361 pgslgkllce
vtpvsgqerf vwssldtpsq rsfsgpwlea qeaqllsqpw qcqlyggerl 421
lgaavyftel sspgaqrsgr apgalpaghl llflilgvls llllvtgafg fhlwrrqwrp
481 rrfsaleqqi hppqaqskie eleqepepep epepepepep epeql SEQ ID NO: 53
Mouse LAG-3 cDNA Sequence 1 atgagggagg acctgctcct tggctttttg
cttctgggac tgctttggga agctccagtt 61 gtgtcttcag ggcctgggaa
agagctcccc gtggtgtggg cccaggaggg agctcccgtc 121 catcttccct
gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg 181
gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc ccttgacctt
241 caccagggga tgccctcgcc tagacaaccc gcacccggtc gctacacggt
gctgagcgtg 301 gctccaggag gcctgcgcag cgggaggcag cccctgcatc
cccacgtgca gctggaggag 361 cgcggcctcc agcgcgggga cttctctctg
tggttgcgcc cagctctgcg caccgatgcg 421 ggcgagtacc acgccaccgt
gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg 481 cgcgtcggcc
aggcctcgat gattgctagt ccctcaggag tcctcaagct gtctgattgg 541
gtccttttga actgctcctt cagccgtcct gaccgcccag tctctgtgca ctggttccag
601 ggccagaacc gagtgcctgt ctacaactca ccgcgtcatt ttttagctga
aactttcctg 661 ttactgcccc aagtcagccc cctggactct gggacctggg
gctgtgtcct cacctacaga 721 gatggcttca atgtctccat cacgtacaac
ctcaaggttc tgggtctgga gcccgtagcc 781 cctctgacag tgtacgctgc
tgaaggttct agggtggagc tgccctgtca tttgccccca 841 ggagtgggga
ccccttcttt gctcattgcc aagtggactc ctcctggagg aggtcctgag 901
ctccccgtgg ctggaaagag tggcaatttt acccttcacc ttgaggctgt gggtctggca
961 caggctggga cctacacctg tagcatccat ctgcagggac agcagctcaa
tgccactgtc 1021 acgttggcgg tcatcacagt gactcccaaa tccttcgggt
tacctggctc ccgggggaag 1081 ctgttgtgtg aggtaacccc ggcatctgga
aaggaaagat ttgtgtggcg tcccctgaac 1141 aatctgtcca ggagttgccc
gggccctgtg ctggagattc aggaggccag gctccttgct 1201 gagcgatggc
agtgtcagct gtacgagggc cagaggcttc ttggagcgac agtgtacgcc 1261
gcagagtcta gctcaggcgc ccacagtgct aggagaatct caggtgacct taaaggaggc
1321 catctcgttc tcgttctcat ccttggtgcc ctctccctgt tccttttggt
ggccggggcc 1381 tttggctttc actggtggag aaaacagttg ctactgagaa
gattttctgc cttagaacat 1441 gggattcagc catttccggc tcagaggaag
atagaggagc tggagcgaga actggagacg 1501 gagatgggac aggagccgga
gcccgagccg gagccacagc tggagccaga gcccaggcag 1561 ctctga SEQ ID NO:
54 Mouse LAG-3 Amino Acid Sequence 1 mredlllgfl llgllweapv
vssgpgkelp vvwaqegapv hlpcslkspn ldpnflrrgg 61 viwqhqpdsg
qptpipaldl hqgmpsprqp apgrytvlsv apgglrsgrq plhphvqlee 121
rglqrqdfsl wlrpalrtda geyhatvrlp nralscslrl rvgqasmias psgvlklsdw
181 vllncsfsrp drpvsvhwfq gqnrvpvyns prnhflaetfl llpqvsplds
gtwgcvltyr 241 dgfnvsityn lkvlglepva pltvyaaegs rvelpchlpp
gvgtpsllia kwtppgggpe 301 lpvagksgnf tlhleavgla qagtytcsih
lqgqqlnatv tlavitvtpk sfglpqsrgk 361 llcevtpasg kerfvwrpln
nlsrscpgpv leiqearlla erwqcqlyeg qrllgatvya 421 aesssgahsa
rrisgdlkgg hlvlvlilga lslfllvaga fgfhwwrkgl llrrfsaleh 481
giqpfpaqrk ieelerelet emgqepepep epqlepeprq l * Included in Table 3
are RNA nucleic acid molecules (e.g., thymines replaced with
uredines), nucleic acid molecules encoding orthologs of the encoded
proteins, as well as DNA or RNA nucleic acid sequences comprising a
nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.5%, or more identity across their full length with the
nucleic acid sequence of any SEQ ID NO listed in Table 3, or a
portion thereof. Such nucleic acid molecules can have a function of
the full-length nucleic acid as described further herein. *
Included in Table 3 are orthologs of the proteins, as well as
polypeptide molecules comprising an amino acid sequence having at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity
across their full length with an amino acid sequence of any SEQ ID
NO listed in Table 3, or a portion thereof. Such polypeptides can
have a function of the full-length polypeptide as described further
herein.
II. Subjects
[0178] In one embodiment, the subject for whom combination PI3Kbeta
and immune checkpoint inhibitor therapy is administered, is a
mammal (e.g., mouse, rat, primate, non-human mammal, domestic
animal, such as a dog, cat, cow, horse, and the like), and is
preferably a human. In another embodiment, the subject is an animal
model of an epithelial cancer (e.g, a breast cancer (such as TNBC),
an ovarian cancer, or a prostate cancer). For example, the animal
model can be an orthotopic xenograft animal model of a
human-derived epithelial cancer (e.g., a breast cancer (such as
TNBC), an ovarian cancer, or a prostate cancer).
[0179] In another embodiment of the methods of the present
invention, the subject has not undergone treatment, such as
chemotherapy, radiation therapy, targeted therapy, and/or
anti-immune checkpoint therapy. In still another embodiment, the
subject has undergone treatment, such as chemotherapy, radiation
therapy, targeted therapy, and/or anti-immune checkpoint
therapy.
[0180] In certain embodiments, the subject has had surgery to
remove cancerous or pre-cancerous tissue. In other embodiments, the
cancerous tissue has not been removed, e.g., the cancerous tissue
may be located in an inoperable region of the body, such as in a
tissue that is essential for life, or in a region where a surgical
procedure would cause considerable risk of harm to the patient.
[0181] The methods of the present invention can be used to treat
and/or determine the responsiveness to combination PI3Kbeta and
immune checkpoint inhibitor therapy of many different cancers in
subjects such as those described herein.
III. Anti-Cancer Therapies
[0182] In one aspect, combination PI3Kbeta and immune checkpoint
inhibitor therapy or combinations of therapies (e.g., one or more
PI3Kbeta-selective inhibitors, such as KIN193, in combination with
one or more immune checkpoint inhibitors, such as an anti-PD-1
antibody, either alone or in combination with yet additional
anti-cancer therapies, such as targeted therapy) can be
administered, particularly if a subject has first been indicated as
being a likely responder to combination PI3Kbeta and immune
checkpoint inhibitor therapy, such as through selection for p53
and/or PTEN deficiency in their target cancer cells In another
embodiment, such combination PI3Kbeta and immune checkpoint
inhibitor therapy can be avoided once a subject is indicated as not
being a likely responder to such therapy and an alternative
treatment regimen, such as targeted and/or untargeted anti-cancer
therapies can be administered.
[0183] Combination therapies are also contemplated and can
comprise, for example, one or more chemotherapeutic agents and
radiation, one or more chemotherapeutic agents and immunotherapy,
or one or more chemotherapeutic agents, radiation and chemotherapy,
each combination of which can be with anti-immune checkpoint
therapy. As described below, agents can be administered in
combination therapy with, e.g., chemotherapeutic agents, hormones,
antiangiogens, radiolabelled, compounds, or with surgery,
cryotherapy, and/or radiotherapy. The preceding treatment methods
can be administered in conjunction with other forms of conventional
therapy (e.g., standard-of-care treatments for cancer well-known to
the skilled artisan), either consecutively with, pre- or
post-conventional therapy.
[0184] For example, these modulatory agents can be administered
with a therapeutically effective dose of chemotherapeutic agent. In
another embodiment, these modulatory agents are administered in
conjunction with chemotherapy to enhance the activity and efficacy
of the chemotherapeutic agent. The Physicians' Desk Reference (PDR)
discloses dosages of chemotherapeutic agents that have been used in
the treatment of various cancers. The dosing regimen and dosages of
these aforementioned chemotherapeutic drugs that are
therapeutically effective will depend on the particular melanoma,
being treated, the extent of the disease and other factors familiar
to the physician of skill in the art and can be determined by the
physician.
[0185] The term "targeted therapy" refers to administration of
agents that selectively interact with a chosen biomolecule to
thereby treat cancer. One example includes epithelial cancer
antigens, such as breast, ovarian, or prostate cancer antigens.
[0186] The terms "PI3Kbeta therapy" and "immune checkpoint
inhibitor" are described above. However, for additional
illustration of immune checkpoint inhibition, description of
anti-PD-1 pathway agents is illustrative of the class of targets
and their inhibitors. Anti-PD-1 pathway agents, such as therapeutic
monoclonal blocking antibodies, are well-known in the art and
described above, can be used to target tumor microenvironments and
cells expressing unwanted components of the PD-1 pathway, such as
the PD-1 receptor and its ligands, PD-L1 and PD-T2. "PD-1 pathway
inhibitors" block or otherwise reduce the interaction between PD-1
and one or both of its ligands such that the immunoinhibitory
signaling otherwise generated by the interaction is blocked or
otherwise reduced. Anti-immune checkpoint inhibitors can be direct
or indirect. Direct anti-immune checkpoint inhibitors block or
otherwise reduce the interaction between an immune checkpoint and
at least one of its ligands. For example, PD-1 inhibitors can block
PD-1 binding with one or both of its ligands. Direct PD-1
combination inhibitors are well-known in the art, especially since
the natural binding partners of PD-1 (e.g., PD-L1 and PD-L2), PD-L1
(e.g., PD-1 and B7-1), and PD-L2 (e.g., PD-1 and RGMb) are
known.
[0187] For example, agents which directly block the interaction
between PD-1 and PD-L1, PD-1 and PD-L2, PD-1 and both PD-L1 and
PD-L2, such as a bispecific antibody, can prevent inhibitory
signaling and upregulate an immune response (i.e., as a PD-1
pathway inhibitor). Alternatively, agents that indirectly block the
interaction between PD-1 and one or both of its ligands can prevent
inhibitory signaling and upregulate an immune response. For
example, B7-1 or a soluble form thereof, by binding to a PD-L1
polypeptide indirectly reduces the effective concentration of PD-L1
polypeptide available to bind to PD-1. Exemplary agents include
monospecific or bispecific blocking antibodies against PD-1, PD-L1,
and/or PD-L2 that block the interaction between the receptor and
ligand(s); a non-activating form of PD-1, PD-L1, and/or PD-L2
(e.g., a dominant negative or soluble polypeptide), small molecules
or peptides that block the interaction between PD-1. PD-L1, and/or
PD-L2; fusion proteins (e.g. the extracellular portion of PD-1,
PD-L1, and/or PD-L2, fused to the Fc portion of an antibody or
immunoglobulin) that bind to PD-1, PD-L1, and/or PD-L2 and inhibit
the interaction between the receptor and ligand(s); a
non-activating form of a natural PD-1, PD-L2, and/or PD-L2 ligand,
and a soluble form of a natural PD-1, PD-L2, and/or PD-L2
ligand.
[0188] Indirect anti-immune checkpoint inhibitors block or
otherwise reduce the immunoinhibitory signaling generated by the
interaction between the immune checkpoint and at least one of its
ligands. For example, an inhibitor can block the interaction
between PD-1 and one or both of its ligands without necessarily
directly blocking the interaction between PD-1 and one or both of
its ligands For example, indirect inhibitors include intrabodies
that bind the intracellular portion of PD-1 and/or PD-L1 required
to signal to block or otherwise reduce the immunoinhibitory
signaling. Similarly, nucleic acids that reduce the expression of
PD-1, PD-L1, and/or PD-L2 can indirectly inhibit the interaction
between PD-1 and one or both of its ligands by removing the
availability of components for interaction. Such nucleic acid
molecules can block PD-L1, PD-L2, and/or PD-L2 transcription or
translation.
[0189] Alternatively, immunotherapy is one form of targeted therapy
that may comprise, for example, the use of cancer vaccines and/or
sensitized antigen presenting cells. For example, an oncolytic
virus is a virus that is able to infect and lyse cancer cells,
while leaving normal cells unharmed, making them potentially useful
in cancer therapy. Replication of oncolytic viruses both
facilitates tumor cell destruction and also produces dose
amplification at the tumor site. They may also act as vectors for
anticancer genes, allowing them to be specifically delivered to the
tumor site. The immunotherapy can involve passive immunity for
short-term protection of a host, achieved by the administration of
pre-formed antibody directed against a cancer antigen or disease
antigen (e.g., administration of a monoclonal antibody, optionally
linked to a chemotherapeutic agent or toxin, to a tumor antigen).
Immunotherapy can also focus on using the cytotoxic
lymphocyte-recognized epitopes of cancer cell lines. Alternatively,
antisense polynucleotides, ribozymes, RNA interference molecules,
triple helix polynucleotides and the like, can be used to
selectively modulate biomolecules that are linked to the
initiation, progression, and/or pathology of a tumor or cancer.
[0190] The term "untargeted therapy" refers to administration of
agents that do not selectively interact with a chosen biomolecule
yet treat cancer. Representative examples of untargeted therapies
include, without limitation, chemotherapy, gene therapy, and
radiation therapy.
[0191] In one embodiment, chemotherapy is used. Chemotherapy
includes the administration of a chemotherapeutic agent. Such a
chemotherapeutic agent may be, but is not limited to, those
selected from among the following groups of compounds: platinum
compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic
agents, alkylating agents, arsenic compounds, DNA topoisomerase
inhibitors, taxanes, nucleoside analogues, plant alkaloids, and
toxins; and synthetic derivatives thereof. Exemplary compounds
include, but are not limited to, alkylating agents: cisplatin,
treosulfan, and trofosfamide; plant alkaloids: vinblastine,
paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide,
crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic
acid, and hydroxyurea; pyrimidine analogs 5-fluorouracil,
doxifluridine, and cytosine arabinoside; purine analogs:
mercaptopurine and thioguanine; DNA antimetabolites:
2'-deoxy-5-fluorouridine, aphidicolin glycinate, and
pyrazoloimidazole; and antimitotic agents: halichondrin,
colchicine, and rhizoxin. Compositions comprising one or more
chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG
comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP
comprises cyclophosphamide, vincristine, doxorubicin, and
prednisone. In another embodiments, PARP (e.g., PARP-1 and/or
PARP-2) inhibitors are used and such inhibitors are well-known in
the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research
Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34
(Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide
(Trevigen); 4-amino-1,8-naphthalimide; (Trevigen);
6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re.
36,397); and NU1025 (Bowman et al.). The mechanism of action is
generally related to the ability of PARP inhibitors to bind PARP
and decrease its activity. PARP catalyzes the conversion of
.beta.-nicotinamide adenine dinucleotide (NAD+) into nicotinamide
and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have
been linked to regulation of transcription, cell proliferation,
genomic stability, and carcinogenesis (Bouchard V. J. et. al.
Experimental Hematology, Volume 31, Number 6, June 2003, pp.
446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research/Fundamental and
Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun.
2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a
key molecule in the repair of DNA single-strand breaks (SSBs) (de
Murcia J. et al. 1997. Proc Nat Acad Sci USA 94:7303-7307;
Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol
Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev
11:2347-2358). Knockout of SSB repair by inhibition of PARP1
function induces DNA double-strand breaks (DSBs) that can trigger
synthetic lethality in cancer cells with defective
homology-directed DSB repair (Bryant H E, et al. (2005) Nature
434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The
foregoing examples of chemotherapeutic agents are illustrative, and
are not intended to be limiting.
[0192] In another embodiment, radiation therapy is used. The
radiation used in radiation therapy can be ionizing radiation.
Radiation therapy can also be gamma rays, X-rays, or proton beams.
Examples of radiation therapy include, but are not limited to,
external-beam radiation therapy, interstitial implantation of
radioisotopes (1-125, palladium, iridium), radioisotopes such as
strontium-89, thoracic radiation therapy, intraperitoneal P-32
radiation therapy, and/or total abdominal and pelvic radiation
therapy. For a general overview of radiation therapy, see Hellman,
Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th
edition, 2001, DeVita et al., eds., J. B. Lippencott Company,
Philadelphia. The radiation therapy can be administered as external
beam radiation or teletherapy wherein the radiation is directed
from a remote source. The radiation treatment can also be
administered as internal therapy or brachytherapy wherein a
radioactive source is placed inside the body close to cancer cells
or a tumor mass. Also encompassed is the use of photodynamic
therapy comprising the administration of photosensitizers, such as
hematoporphyrin and its derivatives, Vertoporfin (BPD-MA),
phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and
2BA-2-DMHA.
[0193] In another embodiment, surgical intervention can occur to
physically remove cancerous cells and/or tissues.
[0194] In still another embodiment, hormone therapy is used.
Hormonal therapeutic treatments can comprise, for example, hormonal
agonists, hormonal antagonists (e.g., flutamide, bicalutamide,
tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH
antagonists), inhibitors of hormone biosynthesis and processing,
and steroids (e.g., dexamethasone, retinoids, deltoids,
betamethasone, cortisol, cortisone, prednisone,
dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen,
testosterone, progestins), vitamin A derivatives (e.g., all-trans
retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g.,
mifepristone, onapristone), or antiandrogens (e.g., cyproterone
acetate).
[0195] In yet another embodiment, hyperthermia, a procedure in
which body tissue is exposed to high temperatures (up to
106.degree. F.) is used. Heat may help shrink tumors by damaging
cells or depriving them of substances they need to live.
Hyperthermia therapy can be local, regional, and whole-body
hyperthermia, using external and internal heating devices.
Hyperthermia is almost always used with other forms of therapy
(e.g., radiation therapy, chemotherapy, and biological therapy) to
try to increase their effectiveness. Local hyperthermia refers to
heat that is applied to a very small area, such as a tumor. The
area may be heated externally with high-frequency waves aimed at a
tumor from a device outside the body. To achieve internal heating,
one of several types of sterile probes may be used, including thin,
heated wires or hollow tubes filled with warm water; implanted
microwave antennae; and radiofrequency electrodes. In regional
hyperthermia, an organ or a limb is heated. Magnets and devices
that produce high energy are placed over the region to be heated.
In another approach, called perfusion, some of the patient's blood
is removed, heated, and then pumped (perfused) into the region that
is to be heated internally. Whole-body heating is used to treat
metastatic cancer that has spread throughout the body. It can be
accomplished using warm-water blankets, hot wax, inductive coils
(like those in electric blankets), or thermal chambers (similar to
large incubators). Hyperthermia does not cause any marked increase
in radiation side effects or complications. Heat applied directly
to the skin, however, can cause discomfort or even significant
local pain in about half the patients treated. It can also cause
blisters, which generally heal rapidly.
[0196] In still another embodiment, photodynamic therapy (also
called PDT, photoradiation therapy, phototherapy, or
photochemotherapy) is used for the treatment of some types of
cancer. It is based on the discovery that certain chemicals known
as photosensitizing agents can kill one-celled organisms when the
organisms are exposed to a particular type of light. PDT destroys
cancer cells through the use of a fixed-frequency laser light in
combination with a photosensitizing agent. In PDT, the
photosensitizing agent is injected into the bloodstream and
absorbed by cells all over the body. The agent remains in cancer
cells for a longer time than it does in normal cells. When the
treated cancer cells are exposed to laser light, the
photosensitizing agent absorbs the light and produces an active
form of oxygen that destroys the treated cancer cells. Light
exposure must be timed carefully so that it occurs when most of the
photosensitizing agent has left healthy cells but is still present
in the cancer cells. The laser light used in PDT can be directed
through a fiber-optic (a very thin glass strand). The fiber-optic
is placed close to the cancer to deliver the proper amount of
light. The fiber-optic can be directed through a bronchoscope into
the lungs for the treatment of lung cancer or through an endoscope
into the esophagus for the treatment of esophageal cancer. An
advantage of PDT is that it causes minimal damage to healthy
tissue. However, because the laser light currently in use cannot
pass through more than about 3 centimeters of tissue (a little more
than one and an eighth inch), PDT is mainly used to treat tumors on
or just under the skin or on the lining of internal organs.
Photodynamic therapy makes the skin and eyes sensitive to light for
6 weeks or more after treatment. Patients are advised to avoid
direct sunlight and bright indoor light for at least 6 weeks. If
patients must go outdoors, they need to wear protective clothing,
including sunglasses. Other temporary side effects of PDT are
related to the treatment of specific areas and can include
coughing, trouble swallowing, abdominal pain, and painful breathing
or shortness of breath. In December 1995, the U.S. Food and Drug
Administration (FDA) approved a photosensitizing agent called
porfimer sodium, or Photofrin.RTM., to relieve symptoms of
esophageal cancer that is causing an obstruction and for esophageal
cancer that cannot be satisfactorily treated with lasers alone. In
January 1998, the FDA approved porfimer sodium for the treatment of
early non-small cell lung cancer in patients for whom the usual
treatments for lung cancer are not appropriate. The National Cancer
Institute and other institutions are supporting clinical trials
(research studies) to evaluate the use of photodynamic therapy for
several types of cancer, including cancers of the bladder, brain,
larynx, and oral cavity.
[0197] In yet another embodiment, laser therapy is used to harness
high-intensity light to destroy cancer cells. This technique is
often used to relieve symptoms of cancer such as bleeding or
obstruction, especially when the cancer cannot be cured by other
treatments. It may also be used to treat cancer by shrinking or
destroying tumors. The term "laser" stands for light amplification
by stimulated emission of radiation. Ordinary light, such as that
from a light bulb, has many wavelengths and spreads in all
directions. Laser light, on the other hand, has a specific
wavelength and is focused in a narrow beam. This type of
high-intensity light contains a lot of energy. Lasers are very
powerful and may be used to cut through steel or to shape diamonds.
Lasers also can be used for very precise surgical work, such as
repairing a damaged retina in the eye or cutting through tissue (in
place of a scalpel). Although there are several different kinds of
lasers, only three kinds have gained wide use in medicine: Carbon
dioxide (CO.sub.2) laser--This type of laser can remove thin layers
from the skin's surface without penetrating the deeper layers. This
technique is particularly useful in treating tumors that have not
spread deep into the skin and certain precancerous conditions. As
an alternative to traditional scalpel surgery, the CO.sub.2 laser
is also able to cut the skin. The laser is used in this way to
remove skin cancers. Neodymium:yttrium-aluminum-garnet (Nd:YAG)
laser--Light from this laser can penetrate deeper into tissue than
light from the other types of lasers, and it can cause blood to
clot quickly. It can be carried through optical fibers to less
accessible parts of the body. This type of laser is sometimes used
to treat throat cancers. Argon laser--This laser can pass through
only superficial layers of tissue and is therefore useful in
dermatology and in eye surgery. It also is used with
light-sensitive dyes to treat tumors in a procedure known as
photodynamic therapy (PDT). Lasers have several advantages over
standard surgical tools, including Lasers are more precise than
scalpels Tissue near an incision is protected, since there is
little contact with surrounding skin or other tissue. The heat
produced by lasers sterilizes the surgery site, thus reducing the
risk of infection. Less operating time may be needed because the
precision of the laser allows for a smaller incision. Healing time
is often shortened; since laser heat seals blood vessels, there is
less bleeding, swelling, or scarring. Laser surgery may be less
complicated. For example, with fiber optics, laser light can be
directed to parts of the body without making a large incision. More
procedures may be done on an outpatient basis. Lasers can be used
in two ways to treat cancer: by shrinking or destroying a tumor
with heat, or by activating a chemical--known as a photosensitizing
agent--that destroys cancer cells. In PDT, a photosensitizing agent
is retained in cancer cells and can be stimulated by light to cause
a reaction that kills cancer cells. CO.sub.2 and Nd:YAG lasers are
used to shrink or destroy tumors. They may be used with endoscopes,
tubes that allow physicians to see into certain areas of the body,
such as the bladder. The light from some lasers can be transmitted
through a flexible endoscope fitted with fiber optics. This allows
physicians to see and work in parts of the body that could not
otherwise be reached except by surgery and therefore allows very
precise aiming of the laser beam. Lasers also may be used with
low-power microscopes, giving the doctor a clear view of the site
being treated. Used with other instruments, laser systems can
produce a cutting area as small as 200 microns in diameter--less
than the width of a very fine thread. Lasers are used to treat many
types of cancer. Laser surgery is a standard treatment for certain
stages of glottis (vocal cord), cervical, skin, lung, vaginal,
vulvar, and penile cancers. In addition to its use to destroy the
cancer, laser surgery is also used to help relieve symptoms caused
by cancer (palliative care). For example, lasers may be used to
shrink or destroy a tumor that is blocking a patient's trachea
(windpipe), making it easier to breathe. It is also sometimes used
for palliation in colorectal and anal cancer. Laser-induced
interstitial thermotherapy (LITT) is one of the most recent
developments in laser therapy. LITT uses the same idea as a cancer
treatment called hyperthermia; that heat may help shrink tumors by
damaging cells or depriving them of substances they need to live.
In this treatment, lasers are directed to interstitial areas (areas
between organs) in the body. The laser light then raises the
temperature of the tumor, which damages or destroys cancer
cells.
[0198] The duration and/or dose of treatment with therapies may
vary according to the particular therapeutic agent or combination
thereof. An appropriate treatment time for a particular cancer
therapeutic agent will be appreciated by the skilled artisan The
present invention contemplates the continued assessment of optimal
treatment schedules for each cancer therapeutic agent, where the
phenotype of the cancer of the subject as determined by the methods
of the present invention is a factor in determining optimal
treatment doses and schedules.
[0199] Any means for the introduction of a polynucleotide into
mammals, human or non-human, or cells thereof may be adapted to the
practice of this invention for the delivery of the various
constructs of the present invention into the intended recipient. In
one embodiment of the present invention, the DNA constructs are
delivered to cells by transfection, i.e., by delivery of "naked"
DNA or in a complex with a colloidal dispersion system. A colloidal
system includes macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a lipid-complexed or
liposome-formulated DNA. In the former approach, prior to
formulation of DNA, e.g., with lipid, a plasmid containing a
transgene bearing the desired DNA constructs may first be
experimentally optimized for expression (e.g., inclusion of an
intron in the 5' untranslated region and elimination of unnecessary
sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995).
Formulation of DNA, e.g. with various lipid or liposome materials,
may then be effected using known methods and materials and
delivered to the recipient mammal. See, e.g., Canonico et al, Am J
Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268;
Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.
5,679,647 by Carson et al.
[0200] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs, which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0201] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand. Naked
DNA or DNA associated with a delivery vehicle, e.g., liposomes, can
be administered to several sites in a subject (see below).
[0202] Nucleic acids can be delivered in any desired vector. These
include viral or non-viral vectors, including adenovirus vectors,
adeno-associated virus vectors, retrovirus vectors, lentivirus
vectors, and plasmid vectors. Exemplary types of viruses include
HSV (herpes simplex virus), AAV (adeno associated virus), HIV
(human immunodeficiency virus), BIV (bovine immunodeficiency
virus), and MLV (murine leukemia virus). Nucleic acids can be
administered in any desired format that provides sufficiently
efficient delivery levels, including in virus particles, in
liposomes, in nanoparticles, and complexed to polymers.
[0203] The nucleic acids encoding a protein or nucleic acid of
interest may be in a plasmid or viral vector, or other vector as is
known in the art. Such vectors are well-known and any can be
selected for a particular application. In one embodiment of the
present invention, the gene delivery vehicle comprises a promoter
and a demethylase coding sequence. Preferred promoters are
tissue-specific promoters and promoters which are activated by
cellular proliferation, such as the thymidine kinase and
thymidylate synthase promoters. Other preferred promoters include
promoters which are activatable by infection with a virus, such as
the .alpha.- and .beta.-interferon promoters, and promoters which
are activatable by a hormone, such as estrogen. Other promoters
which can be used include the Moloney virus LTR, the CMV promoter,
and the mouse albumin promoter. A promoter may be constitutive or
inducible.
[0204] In another embodiment, naked polynucleotide molecules are
used as gene delivery vehicles, as described in WO 90/11092 and
U.S. Pat. No. 5,580,859. Such gene delivery vehicles can be either
growth factor DNA or RNA and, in certain embodiments, are linked to
killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992.
Other vehicles which can optionally be used include DNA-ligand (Wu
et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA
combinations (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413
7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci.
84:7851-7855, 1987) and microprojectiles (Williams et al., Proc.
Natl. Acad. Sci. 88:2726-2730, 1991).
[0205] A gene delivery vehicle can optionally comprise viral
sequences such as a viral origin of replication or packaging signal
These viral sequences can be selected from viruses such as
astrovirus, coronavirus, orthomyxovirus, papovavirus,
paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus,
togavirus or adenovirus. In a preferred embodiment, the growth
factor gene delivery vehicle is a recombinant retroviral vector.
Recombinant retroviruses and various uses thereof have been
described in numerous references including, for example, Mann et
al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.
USA 81.6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990,
U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT
Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806.
Numerous retroviral gene delivery vehicles can be utilized in the
present invention, including for example those described in EP
0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S.
Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer
Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967,
1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J.
Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg.
79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP
0,345,242 and WO91/02805).
[0206] Other viral vector systems that can be used to deliver a
polynucleotide of the present invention have been derived from
herpes virus, e.g., Herpes Simplex Virus (U.S. Pat. No. 5,631,236
by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex),
vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression
vectors," In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of
molecular cloning vectors and their uses. Stoneham: Butterworth;
Baichwal and Sugden (1986) "Vectors for gene transfer derived from
animal DNA viruses: Transient and stable expression of transferred
genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum
Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA
viruses. Preferred viruses include an alphavirus, a poxivirus, an
arena virus, a vaccinia virus, a polio virus, and the like. They
offer several attractive features for various mammalian cells
(Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988, supra;
Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et
al. (1990) J. Virol., 64:642-650).
[0207] In other embodiments, target DNA in the genome can be
manipulated using well-known methods in the art. For example, the
target DNA in the genome can be manipulated by deletion, insertion,
and/or mutation are retroviral insertion, artificial chromosome
techniques, gene insertion, random insertion with tissue specific
promoters, gene targeting, transposable elements and/or any other
method for introducing foreign DNA or producing modified
DNA/modified nuclear DNA. Other modification techniques include
deleting DNA sequences from a genome and/or altering nuclear DNA
sequences. Nuclear DNA sequences, for example, may be altered by
site-directed mutagenesis.
[0208] In other embodiments, recombinant biomarker polypeptides,
and fragments thereof, can be administered to subjects. In some
embodiments, fusion proteins can be constructed and administered
which have enhanced biological properties. In addition, the
biomarker polypeptides, and fragment thereof, can be modified
according to well-known pharmacological methods in the art (e.g.,
pegylation, glycosylation, oligomerization, etc.) in order to
further enhance desirable biological activities, such as increased
bioavailability and decreased proteolytic degradation.
[0209] Clinical efficacy can be measured by any method known in the
art. For example, the response to a therapy, such as combination
PI3Kbeta and immune checkpoint inhibitor therapy, relates to any
response of the cancer, e.g., a tumor, to the therapy, preferably
to a change in tumor mass and/or volume after initiation of
neoadjuvant or adjuvant chemotherapy. Tumor response may be
assessed in a neoadjuvant or adjuvant situation where the size of a
tumor after systemic intervention can be compared to the initial
size and dimensions as measured by CT, PET, mammogram, ultrasound
or palpation and the cellularity of a tumor can be estimated
histologically and compared to the cellularity of a tumor biopsy
taken before initiation of treatment. Response may also be assessed
by caliper measurement or pathological examination of the tumor
after biopsy or surgical resection. Response may be recorded in a
quantitative fashion like percentage change in tumor volume or
cellularity or using a semi-quantitative scoring system such as
residual cancer burden (Symmans et al., J. Clin. Oncol. (2007)
25:4414-4422) or Miller-Payne score (Ogston et al., (2003) Breast
(Edinburgh, Scotland) 12:320-327) in a qualitative fashion like
"pathological complete response" (pCR), "clinical complete
remission" (cCR), "clinical partial remission" (cPR), "clinical
stable disease" (cSD), "clinical progressive disease" (cPD) or
other qualitative criteria. Assessment of tumor response may be
performed early after the onset of neoadjuvant or adjuvant therapy,
e.g., after a few hours, days, weeks or preferably after a few
months. A typical endpoint for response assessment is upon
termination of neoadjuvant chemotherapy or upon surgical removal of
residual tumor cells and/or the tumor bed.
[0210] In some embodiments, clinical efficacy of the therapeutic
treatments described herein may be determined by measuring the
clinical benefit rate (CBR). The clinical benefit rate is measured
by determining the sum of the percentage of patients who are in
complete remission (CR), the number of patients who are in partial
remission (PR) and the number of patients having stable disease
(SD) at a time point at least 6 months out from the end of therapy.
The shorthand for this formula is CBR=CR+PR+SD over 6 months. In
some embodiments, the CBR for a particular anti-immune checkpoint
therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, or more.
[0211] Additional criteria for evaluating the response to
anti-immune checkpoint therapies are related to "survival," which
includes all of the following: survival until mortality, also known
as overall survival (wherein said mortality may be either
irrespective of cause or tumor related); "recurrence-free survival"
(wherein the term recurrence shall include both localized and
distant recurrence); metastasis free survival; disease free
survival (wherein the term disease shall include cancer and
diseases associated therewith). The length of said survival may be
calculated by reference to a defined start point (e.g., time of
diagnosis or start of treatment) and end point (e.g., death,
recurrence or metastasis). In addition, criteria for efficacy of
treatment can be expanded to include response to chemotherapy,
probability of survival, probability of metastasis within a given
time period, and probability of tumor recurrence.
[0212] For example, in order to determine appropriate threshold
values, a particular anti-cancer therapeutic regimen can be
administered to a population of subjects and the outcome can be
correlated to biomarker measurements that were determined prior to
administration of any combination PI3Kbeta and immune checkpoint
inhibitor therapy. The outcome measurement may be pathologic
response to therapy given in the neoadjuvant setting.
Alternatively, outcome measures, such as overall survival and
disease-free survival can be monitored over a period of time for
subjects following anti-immune checkpoint therapy for whom
biomarker measurement values are known. In certain embodiments, the
same doses of anti-immune checkpoint agents are administered to
each subject. In related embodiments, the doses administered are
standard doses known in the art for anti-immune checkpoint agents.
The period of time for which subjects are monitored can vary. For
example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker
measurement threshold values that correlate to outcome of an
anti-immune checkpoint therapy can be determined using methods such
as those described in the Examples section.
3. Pharmaceutical Compositions
[0213] The present invention provides pharmaceutically acceptable
compositions which comprise a therapeutically-effective amount of
an agent that modulates (e.g., decreases) biomarker expression
and/or activity, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes, (2) parenteral administration,
for example, by subcutaneous, intramuscular or intravenous
injection as, for example, a sterile solution or suspension; (3)
topical application, for example, as a cream, ointment or spray
applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; or (5) aerosol, for example,
as an aqueous aerosol, liposomal preparation or solid particles
containing the compound.
[0214] The phrase "therapeutically-effective amount" as used herein
means that amount of an agent that modulates (e.g., inhibits)
biomarker expression and/or activity, or expression and/or activity
of the complex, or composition comprising an agent that modulates
(e.g., inhibits) biomarker expression and/or activity, or
expression and/or activity of the complex, which is effective for
producing some desired therapeutic effect, e.g., cancer treatment,
at a reasonable benefit/risk ratio.
[0215] The phrase "pharmaceutically acceptable" is employed herein
to refer to those agents, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0216] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the subject.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol, (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution,
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0217] The term "pharmaceutically-acceptable salts" refers to the
relatively non-toxic, inorganic and organic acid addition salts of
the agents that modulates (e.g., inhibits) biomarker expression
and/or activity, or expression and/or activity of the complex
encompassed by the present invention. These salts can be prepared
in situ during the final isolation and purification of the
therapeutic agents, or by separately reacting a purified
therapeutic agent in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like (See, for example. Berge et al. (1977) "Pharmaceutical Salts".
J. Pharm. Sci. 66:1-19).
[0218] In other cases, the agents useful in the methods of the
present invention may contain one or more acidic functional groups
and, thus, are capable of forming pharmaceutically-acceptable salts
with pharmaceutically-acceptable bases. The term
"pharmaceutically-acceptable salts" in these instances refers to
the relatively non-toxic, inorganic and organic base addition salts
of agents that modulates (e.g., inhibits) biomarker expression
and/or activity, or expression and/or activity of the complex.
These salts can likewise be prepared in situ during the final
isolation and purification of the therapeutic agents, or by
separately reacting the purified therapeutic agent in its free acid
form with a suitable base, such as the hydroxide, carbonate or
bicarbonate of a pharmaceutically-acceptable metal cation, with
ammonia, or with a pharmaceutically-acceptable organic primary,
secondary or tertiary amine. Representative alkali or alkaline
earth salts include the lithium, sodium, potassium, calcium,
magnesium, and aluminum salts and the like. Representative organic
amines useful for the formation of base addition salts include
ethylamine, diethylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine and the like (see, for example, Berge et
al., supra).
[0219] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0220] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0221] Formulations useful in the methods of the present invention
include those suitable for oral, nasal, topical (including buccal
and sublingual), rectal, vaginal, aerosol and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well-known in
the art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the host being treated, the particular
mode of administration. The amount of active ingredient, which can
be combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 1 percent to about ninety-nine percent
of active ingredient, preferably from about 5 percent to about 70
percent, most preferably from about 10 percent to about 30
percent.
[0222] Methods of preparing these formulations or compositions
include the step of bringing into association an agent that
modulates (e.g., inhibits) biomarker expression and/or activity,
with the carrier and, optionally, one or more accessory
ingredients. In general, the formulations are prepared by uniformly
and intimately bringing into association a therapeutic agent with
liquid carriers, or finely divided solid carriers, or both, and
then, if necessary, shaping the product.
[0223] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a therapeutic agent as an active ingredient. A compound
may also be administered as a bolus, electuary or paste.
[0224] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more
pharmaceutically-acceptable carriers, such as sodium citrate or
dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0225] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered peptide or peptidomimetic moistened with an
inert liquid diluent.
[0226] Tablets, and other solid dosage forms, such as dragees,
capsules, pills and granules, may optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well-known in the pharmaceutical-formulating art. They may
also be formulated so as to provide slow or controlled release of
the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions, which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions, which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0227] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0228] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0229] Suspensions, in addition to the active agent may contain
suspending agents as, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and mixtures thereof.
[0230] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more therapeutic agents with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active agent.
[0231] Formulations which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing such carriers as are known in the art
to be appropriate.
[0232] Dosage forms for the topical or transdermal administration
of an agent that modulates (e.g., inhibits) biomarker expression
and/or activity include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
component may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0233] The ointments, pastes, creams and gels may contain, in
addition to a therapeutic agent, excipients, such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0234] Powders and sprays can contain, in addition to an agent that
modulates (e.g., inhibits) biomarker expression and/or activity,
excipients such as lactose, talc, silicic acid, aluminum hydroxide,
calcium silicates and polyamide powder, or mixtures of these
substances. Sprays can additionally contain customary propellants,
such as chlorofluorohydrocarbons and volatile unsubstituted
hydrocarbons, such as butane and propane.
[0235] The agent that modulates (e.g., inhibits) biomarker
expression and/or activity, can be alternatively administered by
aerosol. This is accomplished by preparing an aqueous aerosol,
liposomal preparation or solid particles containing the compound. A
nonaqueous (e.g., fluorocarbon propellant) suspension could be
used. Sonic nebulizers are preferred because they minimize exposing
the agent to shear, which can result in degradation of the
compound.
[0236] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0237] Transdermal patches have the added advantage of providing
controlled delivery of a therapeutic agent to the body. Such dosage
forms can be made by dissolving or dispersing the agent in the
proper medium. Absorption enhancers can also be used to increase
the flux of the peptidomimetic across the skin. The rate of such
flux can be controlled by either providing a rate controlling
membrane or dispersing the peptidomimetic in a polymer matrix or
gel.
[0238] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0239] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more therapeutic agents
in combination with one or more pharmaceutically-acceptable sterile
isotonic aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0240] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants.
[0241] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0242] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0243] Injectable depot forms are made by forming microencapsule
matrices of an agent that modulates (e.g., inhibits) biomarker
expression and/or activity, in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions, which are
compatible with body tissue.
[0244] When the therapeutic agents of the present invention are
administered as pharmaceuticals, to humans and animals, they can be
given per se or as a pharmaceutical composition containing, for
example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0245] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be determined by
the methods of the present invention so as to obtain an amount of
the active ingredient, which is effective to achieve the desired
therapeutic response for a particular subject, composition, and
mode of administration, without being toxic to the subject.
[0246] The nucleic acid molecules of the present invention can be
inserted into vectors and used as gene therapy vectors. Gene
therapy vectors can be delivered to a subject by, for example,
intravenous injection, local administration (see U.S. Pat. No.
5,328,470) or by stereotactic injection (see e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054 3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
[0247] The present invention also encompasses kits for detecting
and/or modulating biomarkers described herein. A kit of the present
invention may also include instructional materials disclosing or
describing the use of the kit or an antibody of the disclosed
invention in a method of the disclosed invention as provided
herein. A kit may also include additional components to facilitate
the particular application for which the kit is designed For
example, a kit may additionally contain means of detecting the
label (e.g., enzyme substrates for enzymatic labels, filter sets to
detect fluorescent labels, appropriate secondary labels such as a
sheep anti-mouse-HRP, etc.) and reagents necessary for controls
(e.g., control biological samples or standards). A kit may
additionally include buffers and other reagents recognized for use
in a method of the disclosed invention. Non-limiting examples
include agents to reduce non-specific binding, such as a carrier
protein or a detergent.
4. Further Uses and Methods of the Present Invention
[0248] The compositions described herein can be used in a variety
of diagnostic, prognostic, and therapeutic applications. In any
method described herein, such as a diagnostic method, prognostic
method, therapeutic method, or combination thereof, all steps of
the method can be performed by a single actor or, alternatively, by
more than one actor. For example, diagnosis can be performed
directly by the actor providing therapeutic treatment.
Alternatively, a person providing a therapeutic agent can request
that a diagnostic assay be performed. The diagnostician and/or the
therapeutic interventionist can interpret the diagnostic assay
results to determine a therapeutic strategy Similarly, such
alternative processes can apply to other assays, such as prognostic
assays.
1. Predictive Medicine
[0249] The present invention can pertain to the field of predictive
medicine in which diagnostic assays, prognostic assays, and
monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining the amount and/or activity level
of a biomarker described herein, such as p53 and/or PTEN deficiency
status, in the context of a biological sample (e.g., blood, serum,
cells, or tissue) to thereby determine whether an individual
afflicted with a cancer is likely to respond to combination
PI3Kbeta and immune checkpoint inhibitor therapy, such as in
epithelial cancers (e.g., breast cancers, ovarian cancers, or
prostate cancers). Such assays can be used for prognostic or
predictive purpose alone, or can be coupled with a therapeutic
intervention to thereby prophylactically treat an individual prior
to the onset or after recurrence of a disorder characterized by or
associated with biomarker polypeptide, nucleic acid expression or
activity. The skilled artisan will appreciate that any method can
use one or more (e.g., combinations) of biomarkers described
herein, such as those in the tables, figures, examples, and
otherwise described in the specification.
[0250] Another aspect of the present invention pertains to
monitoring the influence of agents (e.g., drugs, compounds, and
small nucleic acid-based molecules) on the expression or activity
of a biomarker or cancer state described herein. These and other
agents are described in further detail in the following
sections.
[0251] The skilled artisan will also appreciated that, in certain
embodiments, the methods of the present invention implement a
computer program and computer system. For example, a computer
program can be used to perform algorithms described herein. A
computer system can also store and manipulate data generated by the
methods of the present invention which comprises a plurality of
biomarker signal changes/profiles which can be used by a computer
system in implementing the methods of this invention. In certain
embodiments, a computer system receives biomarker expression data;
(ii) stores the data; and (iii) compares the data in any number of
ways described herein (e.g., analysis relative to appropriate
controls) to determine the state of informative biomarkers from
cancerous or pre-cancerous tissue. In other embodiments, a computer
system (i) compares the determined expression biomarker level to a
threshold value; and (ii) outputs an indication of whether said
biomarker level is significantly modulated (e.g., above or below)
the threshold value, or a phenotype based on said indication.
[0252] In certain embodiments, such computer systems are also
considered part of the present invention. Numerous types of
computer systems can be used to implement the analytic methods of
this invention according to knowledge possessed by a skilled
artisan in the bioinformatics and/or computer arts. Several
software components can be loaded into memory during operation of
such a computer system. The software components can comprise both
software components that are standard in the art and components
that are special to the present invention (e.g., dCHIP software
described in Lin et al. (2004) Bioinformatics 20, 1233-1240; radial
basis machine learning algorithms (RBM) known in the art).
[0253] The methods of the present invention can also be programmed
or modeled in mathematical software packages that allow symbolic
entry of equations and high-level specification of processing,
including specific algorithms to be used, thereby freeing a user of
the need to procedurally program individual equations and
algorithms. Such packages include, e.g., Matlab from Mathworks
(Natick, Mass.), Mathematica from Wolfram Research (Champaign,
Ill.) or S-Plus from MathSoft (Seattle, Wash.).
[0254] In certain embodiments, the computer comprises a database
for storage of biomarker data. Such stored profiles can be accessed
and used to perform comparisons of interest at a later point in
time. For example, biomarker expression profiles of a sample
derived from the non-cancerous tissue of a subject and/or profiles
generated from population-based distributions of informative loci
of interest in relevant populations of the same species can be
stored and later compared to that of a sample derived from the
cancerous tissue of the subject or tissue suspected of being
cancerous of the subject.
[0255] In addition to the exemplary program structures and computer
systems described herein, other, alternative program structures and
computer systems will be readily apparent to the skilled artisan.
Such alternative systems, which do not depart from the above
described computer system and programs structures either in spirit
or in scope, are therefore intended to be comprehended within the
accompanying claims.
2. Diagnostic Assays
[0256] The present invention provides, in part, methods, systems,
and code for accurately classifying whether a biological sample is
associated with a cancer that is likely to respond to combination
PI3Kbeta and immune checkpoint inhibitor therapy In some
embodiments, the present invention is useful for classifying a
sample (e.g., from a subject) as associated with or at risk for
responding to or not responding to combination PI3Kbeta and immune
checkpoint inhibitor therapy using a statistical algorithm and/or
empirical data (e.g., the amount or activity of a biomarker
described herein, such as in the tables, figures, examples, and
otherwise described in the specification).
[0257] An exemplary method for detecting the amount or activity of
a biomarker described herein, and thus useful for classifying
whether a sample is likely or unlikely to respond to combination
PI3Kbeta and immune checkpoint inhibitor therapy involves obtaining
a biological sample from a test subject and contacting the
biological sample with an agent, such as a protein-binding agent
like an antibody or antigen-binding fragment thereof, or a nucleic
acid-binding agent like an oligonucleotide, capable of detecting
the amount or activity of the biomarker, such as a p53 and/or PTEN
nucleic acid or protein of interest, in the biological sample. In
some embodiments, at least one antibody or antigen-binding fragment
thereof is used, wherein two, three, four, five, six, seven, eight,
nine, ten, or more such antibodies or antibody fragments can be
used in combination (e.g., in sandwich ELISAs) or in serial. In
certain instances, the statistical algorithm is a single learning
statistical classifier system. For example, a single learning
statistical classifier system can be used to classify a sample as a
based upon a prediction or probability value and the presence or
level of the biomarker. The use of a single learning statistical
classifier system typically classifies the sample as, for example,
a likely anti-immune checkpoint therapy responder or progressor
sample with a sensitivity, specificity, positive predictive value,
negative predictive value, and/or overall accuracy of at least
about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%.
[0258] Other suitable statistical algorithms are well-known to
those of skill in the art. For example, learning statistical
classifier systems include a machine learning algorithmic technique
capable of adapting to complex data sets (e.g., panel of markers of
interest) and making decisions based upon such data sets. In some
embodiments, a single learning statistical classifier system such
as a classification tree (e.g., random forest) is used. In other
embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
learning statistical classifier systems are used, preferably in
tandem. Examples of learning statistical classifier systems
include, but are not limited to, those using inductive learning
(e.g., decision/classification trees such as random forests,
classification and regression trees (C&RT), boosted trees,
etc.), Probably Approximately Correct (PAC) learning, connectionist
learning (e.g., neural networks (NN), artificial neural networks
(ANN), neuro fuzzy networks (NFN), network structures, perceptrons
such as multi-layer perceptrons, multi-layer feed-forward networks,
applications of neural networks, Bayesian learning in belief
networks, etc.), reinforcement learning (e.g., passive learning in
a known environment such as naive learning, adaptive dynamic
learning, and temporal difference learning, passive learning in an
unknown environment, active learning in an unknown environment,
learning action-value functions, applications of reinforcement
learning, etc.), and genetic algorithms and evolutionary
programming. Other learning statistical classifier systems include
support vector machines (e.g., Kernel methods), multivariate
adaptive regression splines (MARS), Levenberg-Marquardt algorithms,
Gauss-Newton algorithms, mixtures of Gaussians, gradient descent
algorithms, and learning vector quantization (LVQ). In certain
embodiments, the method of the present invention further comprises
sending the sample classification results to a clinician, e.g., an
oncologist.
[0259] In another embodiment, the diagnosis of a subject is
followed by administering to the individual a therapeutically
effective amount of a defined treatment based upon the
diagnosis.
[0260] In one embodiment, the methods further involve obtaining a
control biological sample (e.g., biological sample from a subject
who does not have a cancer or whose cancer is susceptible to
combination PI3Kbeta and immune checkpoint inhibitor therapy), a
biological sample from the subject during remission, or a
biological sample from the subject during treatment for developing
a cancer progressing despite combination PI3Kbeta and immune
checkpoint inhibitor therapy.
3. Prognostic Assays
[0261] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
cancer that is likely or unlikely to be responsive to combination
PI3Kbeta and immune checkpoint inhibitor therapy. The assays
described herein, such as the preceding diagnostic assays or the
following assays, can be utilized to identify a subject having or
at risk of developing a disorder associated with a misregulation of
the amount or activity of at least one biomarker described herein,
such as in cancer. Alternatively, the prognostic assays can be
utilized to identify a subject having or at risk for developing a
disorder associated with a misregulation of the at least one
biomarker described herein, such as in cancer. Furthermore, the
prognostic assays described herein can be used to determine whether
a subject can be administered an agent (e.g., an agonist,
antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,
small molecule, or other drug candidate) to treat a disease or
disorder associated with the aberrant biomarker expression or
activity.
4. Biomarker Nucleic Acids and Polypeptides
[0262] The therapeutic and other methods of the present invention
uses biomarkers of interest. In some embodiments, the biomarkers of
interest are isolated nucleic acid molecules that correspond to
biomarker nucleic acids that encode a biomarker polypeptide or a
portion of such a polypeptide. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0263] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Preferably, an
"isolated" nucleic acid molecule is free of sequences (preferably
protein-encoding sequences) which naturally flank the nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1
kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0264] A biomarker nucleic acid molecule of the present invention
can be isolated using standard molecular biology techniques and the
sequence information in the database records described herein.
Using all or a portion of such nucleic acid sequences, nucleic acid
molecules of the present invention can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0265] A nucleic acid molecule of the present invention can be
amplified using cDNA, mRNA, or genomic DNA as a template and
appropriate oligonucleotide primers according to standard PCR
amplification techniques. The nucleic acid molecules so amplified
can be cloned into an appropriate vector and characterized by DNA
sequence analysis. Furthermore, oligonucleotides corresponding to
all or a portion of a nucleic acid molecule of the present
invention can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0266] Moreover, a nucleic acid molecule of the present invention
can comprise only a portion of a nucleic acid sequence, wherein the
full length nucleic acid sequence comprises a marker of the present
invention or which encodes a polypeptide corresponding to a marker
of the present invention. Such nucleic acid molecules can be used,
for example, as a probe or primer. The probe/primer typically is
used as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7,
preferably about 15, more preferably about 25, 50, 75, 100, 125,
150, 175, 200, 250, 300, 350, or 400 or more consecutive
nucleotides of a biomarker nucleic acid sequence. Probes based on
the sequence of a biomarker nucleic acid molecule can be used to
detect transcripts or genomic sequences corresponding to one or
more markers of the present invention. The probe comprises a label
group attached thereto, e.g., a radioisotope, a fluorescent
compound, an enzyme, or an enzyme co-factor.
[0267] A biomarker nucleic acid molecules that differ, due to
degeneracy of the genetic code, from the nucleotide sequence of
nucleic acid molecules encoding a protein which corresponds to the
biomarker, and thus encode the same protein, are also
contemplated.
[0268] In addition, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequence can exist within a population (e.g., the human
population). Such genetic polymorphisms can exist among individuals
within a population due to natural allelic variation. An allele is
one of a group of genes which occur alternatively at a given
genetic locus. In addition, it will be appreciated that DNA
polymorphisms that affect RNA expression levels can also exist that
may affect the overall expression level of that gene (e.g., by
affecting regulation or degradation).
[0269] The term "allele," which is used interchangeably herein with
"allelic variant," refers to alternative forms of a gene or
portions thereof. Alleles occupy the same locus or position on
homologous chromosomes. When a subject has two identical alleles of
a gene, the subject is said to be homozygous for the gene or
allele. When a subject has two different alleles of a gene, the
subject is said to be heterozygous for the gene or allele. For
example, biomarker alleles can differ from each other in a single
nucleotide, or several nucleotides, and can include substitutions,
deletions, and insertions of nucleotides. An allele of a gene can
also be a form of a gene containing one or more mutations.
[0270] The term "allelic variant of a polymorphic region of gene"
or "allelic variant", used interchangeably herein, refers to an
alternative form of a gene having one of several possible
nucleotide sequences found in that region of the gene in the
population. As used herein, allelic variant is meant to encompass
functional allelic variants, non-functional allelic variants, SNPs,
mutations and polymorphisms.
[0271] The term "single nucleotide polymorphism" (SNP) refers to a
polymorphic site occupied by a single nucleotide, which is the site
of variation between allelic sequences The site is usually preceded
by and followed by highly conserved sequences of the allele (e.g.,
sequences that vary in less than 1/100 or 1/1000 members of a
population). A SNP usually arises due to substitution of one
nucleotide for another at the polymorphic site. SNPs can also arise
from a deletion of a nucleotide or an insertion of a nucleotide
relative to a reference allele. Typically the polymorphic site is
occupied by a base other than the reference base. For example,
where the reference allele contains the base "T" (thymidine) at the
polymorphic site, the altered allele can contain a "C" (cytidine),
"G" (guanine), or "A" (adenine) at the polymorphic site. SNP's may
occur in protein-coding nucleic acid sequences, in which case they
may give rise to a defective or otherwise variant protein, or
genetic disease. Such a SNP may alter the coding sequence of the
gene and therefore specify another amino acid (a "missense" SNP) or
a SNP may introduce a stop codon (a "nonsense" SNP). When a SNP
does not alter the amino acid sequence of a protein, the SNP is
called "silent." SNP's may also occur in noncoding regions of the
nucleotide sequence. This may result in defective protein
expression, e.g., as a result of alternative spicing, or it may
have no effect on the function of the protein.
[0272] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide corresponding to a marker of the present
invention. Such natural allelic variations can typically result in
1-5% variance in the nucleotide sequence of a given gene.
Alternative alleles can be identified by sequencing the gene of
interest in a number of different individuals. This can be readily
carried out by using hybridization probes to identify the same
genetic locus in a variety of individuals. Any and all such
nucleotide variations and resulting amino acid polymorphisms or
variations that are the result of natural allelic variation and
that do not alter the functional activity are intended to be within
the scope of the present invention.
[0273] In another embodiment, a biomarker nucleic acid molecule is
at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300,
350, 400, 450, 550, 650, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800,
3000, 3500, 4000, 4500, or more nucleotides in length and
hybridizes under stringent conditions to a nucleic acid molecule
corresponding to a marker of the present invention or to a nucleic
acid molecule encoding a protein corresponding to a marker of the
present invention. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% (65%, 70%, 75%, 80%, preferably 85%) identical to each other
typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989). A preferred, non-limiting
example of stringent hybridization conditions are hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C.
[0274] In addition to naturally-occurring allelic variants of a
nucleic acid molecule of the present invention that can exist in
the population, the skilled artisan will further appreciate that
sequence changes can be introduced by mutation thereby leading to
changes in the amino acid sequence of the encoded protein, without
altering the biological activity of the protein encoded thereby.
For example, one can make nucleotide substitutions leading to amino
acid substitutions at "non-essential" amino acid residues. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequence without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologs of various species
may be non-essential for activity and thus would be likely targets
for alteration. Alternatively, amino acid residues that are
conserved among the homologs of various species (e.g., murine and
human) may be essential for activity and thus would not be likely
targets for alteration.
[0275] Accordingly, another aspect of the present invention
pertains to nucleic acid molecules encoding a polypeptide of the
present invention that contain changes in amino acid residues that
are not essential for activity. Such polypeptides differ in amino
acid sequence from the naturally-occurring proteins which
correspond to the markers of the present invention, yet retain
biological activity. In one embodiment, a biomarker protein has an
amino acid sequence that is at least about 40% identical, 50%, 60%,
70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or identical to the amino acid sequence of a
biomarker protein described herein.
[0276] An isolated nucleic acid molecule encoding a variant protein
can be created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of nucleic
acids of the present invention, such that one or more amino acid
residue substitutions, additions, or deletions are introduced into
the encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0277] In some embodiments, the present invention further
contemplates the use of anti-biomarker antisense nucleic acid
molecules, i.e., molecules which are complementary to a sense
nucleic acid of the present invention, e.g., complementary to the
coding strand of a double-stranded cDNA molecule corresponding to a
marker of the present invention or complementary to an mRNA
sequence corresponding to a marker of the present invention.
Accordingly, an antisense nucleic acid molecule of the present
invention can hydrogen bond to (i.e. anneal with) a sense nucleic
acid of the present invention. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can also be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a polypeptide of the
present invention. The non-coding regions ("5' and 3' untranslated
regions") are the 5' and 3' sequences which flank the coding region
and are not translated into amino acids.
[0278] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in
length. An antisense nucleic acid can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been sub-cloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0279] The antisense nucleic acid molecules of the present
invention are typically administered to a subject or generated in
situ such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a polypeptide corresponding to a selected
marker of the present invention to thereby inhibit expression of
the marker, e.g., by inhibiting transcription and/or translation.
The hybridization can be by conventional nucleotide complementarity
to form a stable duplex, or, for example, in the case of an
antisense nucleic acid molecule which binds to DNA duplexes,
through specific interactions in the major groove of the double
helix. Examples of a route of administration of antisense nucleic
acid molecules of the present invention includes direct injection
at a tissue site or infusion of the antisense nucleic acid into a
blood- or bone marrow-associated body fluid. Alternatively,
antisense nucleic acid molecules can be modified to target selected
cells and then administered systemically. For example, for systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies which bind to cell surface receptors or
antigens. The antisense nucleic acid molecules can also be
delivered to cells using the vectors described herein. To achieve
sufficient intracellular concentrations of the antisense molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0280] An antisense nucleic acid molecule of the present invention
can be an a-anomeric nucleic acid molecule. An a-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual a-units, the
strands run parallel to each other (Gaultier et al., 1987, Nucleic
Acids Res. 15:6625-6641). The antisense nucleic acid molecule can
also comprise a 2'-o-methylribonucleotide (Inoue et al., 1987,
Nucleic Acids Res. 15.6131-6148) or a chimeric RNA-DNA analogue
(Inoue et al., 1987, FEBS Lett. 215:327-330).
[0281] The present invention also encompasses ribozymes. Ribozymes
are catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes as described in Haselhoff and Gerlach,
1988, Nature 334:585-591) can be used to catalytically cleave mRNA
transcripts to thereby inhibit translation of the protein encoded
by the mRNA. A ribozyme having specificity for a nucleic acid
molecule encoding a polypeptide corresponding to a marker of the
present invention can be designed based upon the nucleotide
sequence of a cDNA corresponding to the marker. For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved (see Cech et al. U.S. Pat.
No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
Alternatively, an mRNA encoding a polypeptide of the present
invention can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules (see, e.g.,
Bartel and Szostak, 1993, Science 261:1411-1418).
[0282] The present invention also encompasses nucleic acid
molecules which form triple helical structures. For example,
expression of a biomarker protein can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
gene encoding the polypeptide (e.g., the promoter and/or enhancer)
to form triple helical structures that prevent transcription of the
gene in target cells. See generally Helene (1991) Anticancer Drug
Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher (1992) Bioassays 14(12):807-15.
[0283] In various embodiments, the nucleic acid molecules of the
present invention can be modified at the base moiety, sugar moiety
or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of the molecule. For example, the
deoxyribose phosphate backbone of the nucleic acid molecules can be
modified to generate peptide nucleic acid molecules (see Hyrup et
al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As
used herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup et al. (1996), supra;
Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA
93:14670-675.
[0284] PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, e.g., inducing
transcription or translation arrest or inhibiting replication. PNAs
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or
primers for DNA sequence and hybridization (Hyrup, 1996, supra;
Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA
93:14670-675).
[0285] In another embodiment, PNAs can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic
or other helper groups to PNA, by the formation of PNA-DNA
chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras can be
generated which can combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes, e.g., RNASE H and
DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996).
supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
For example, a DNA chain can be synthesized on a solid support
using standard phosphoramidite coupling chemistry and modified
nucleoside analogs. Compounds such as
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be
used as a link between the PNA and the 5' end of DNA (Mag et al.,
1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled
in a step-wise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids Res.
24(17):3357-63). Alternatively, chimeric molecules can be
synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et
al., 1975, Bioorganic Med. (hem. Lett. 5:1119-11124).
[0286] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide
can be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.
[0287] Another aspect of the present invention pertains to the use
of biomarker proteins and biologically active portions thereof. In
one embodiment, the native polypeptide corresponding to a marker
can be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, polypeptides corresponding to a marker of
the present invention are produced by recombinant DNA techniques.
Alternative to recombinant expression, a polypeptide corresponding
to a marker of the present invention can be synthesized chemically
using standard peptide synthesis techniques.
[0288] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
[0289] Biologically active portions of a biomarker polypeptide
include polypeptides comprising amino acid sequences sufficiently
identical to or derived from a biomarker protein amino acid
sequence described herein, but which includes fewer amino acids
than the full length protein, and exhibit at least one activity of
the corresponding full-length protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the corresponding protein. A biologically active
portion of a protein of the present invention can be a polypeptide
which is, for example, 10, 25, 50, 100 or more amino acids in
length. Moreover, other biologically active portions, in which
other regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of the native form of a polypeptide of the
present invention.
[0290] Preferred polypeptides have an amino acid sequence of a
biomarker protein encoded by a nucleic acid molecule described
herein. Other useful proteins are substantially identical (e.g., at
least about 40%, preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to one of
these sequences and retain the functional activity of the protein
of the corresponding naturally-occurring protein yet differ in
amino acid sequence due to natural allelic variation or
mutagenesis.
[0291] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100) In one embodiment the two sequences are the
same length.
[0292] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, e a. (1990) J. Mol. Biol. 215:403-410. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the present invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the present invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules. When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) Compul Appl Biosci, 4:11-7. Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Yet another useful algorithm for identifying regions
of local sequence similarity and alignment is the FASTA algorithm
as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
USA 85:2444-2448. When using the FASTA algorithm for comparing
nucleotide or amino acid sequences, a PAM120 weight residue table
can, for example, be used with a k-tuple value of 2.
[0293] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0294] The present invention also provides chimeric or fusion
proteins corresponding to a biomarker protein As used herein, a
"chimeric protein" or "fusion protein" comprises all or part
(preferably a biologically active part) of a polypeptide
corresponding to a marker of the present invention operably linked
to a heterologous polypeptide (i.e., a polypeptide other than the
polypeptide corresponding to the marker) Within the fusion protein,
the term "operably linked" is intended to indicate that the
polypeptide of the present invention and the heterologous
polypeptide are fused in-frame to each other. The heterologous
polypeptide can be fused to the amino-terminus or the
carboxyl-terminus of the polypeptide of the present invention.
[0295] One useful fusion protein is a GST fusion protein in which a
polypeptide corresponding to a marker of the present invention is
fused to the carboxyl terminus of GST sequences. Such fusion
proteins can facilitate the purification of a recombinant
polypeptide of the present invention.
[0296] In another embodiment, the fusion protein contains a
heterologous signal sequence, immunoglobulin fusion protein, toxin,
or other useful protein sequence. Chimeric and fusion proteins of
the present invention can be produced by standard recombinant DNA
techniques. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed and re-amplified to generate a
chimeric gene sequence (see, e.g., Ausubel et al., supra).
Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g., a GST polypeptide). A nucleic
acid encoding a polypeptide of the present invention can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the polypeptide of the present invention.
[0297] A signal sequence can be used to facilitate secretion and
isolation of the secreted protein or other proteins of interest.
Signal sequences are typically characterized by a core of
hydrophobic amino acids which are generally cleaved from the mature
protein during secretion in one or more cleavage events. Such
signal peptides contain processing sites that allow cleavage of the
signal sequence from the mature proteins as they pass through the
secretory pathway. Thus, the present invention pertains to the
described polypeptides having a signal sequence, as well as to
polypeptides from which the signal sequence has been
proteolytically cleaved (i.e., the cleavage products). In one
embodiment, a nucleic acid sequence encoding a signal sequence can
be operably linked in an expression vector to a protein of
interest, such as a protein which is ordinarily not secreted or is
otherwise difficult to isolate. The signal sequence directs
secretion of the protein, such as from a eukaryotic host into which
the expression vector is transformed, and the signal sequence is
subsequently or concurrently cleaved. The protein can then be
readily purified from the extracellular medium by art recognized
methods. Alternatively, the signal sequence can be linked to the
protein of interest using a sequence which facilitates
purification, such as with a GST domain.
[0298] The present invention also pertains to variants of the
biomarker polypeptides described herein. Such variants have an
altered amino acid sequence which can function as either agonists
(mimetics) or as antagonists. Variants can be generated by
mutagenesis, e.g., discrete point mutation or truncation. An
agonist can retain substantially the same, or a subset, of the
biological activities of the naturally occurring form of the
protein. An antagonist of a protein can inhibit one or more of the
activities of the naturally occurring form of the protein by, for
example, competitively binding to a downstream or upstream member
of a cellular signaling cascade which includes the protein of
interest. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein can have fewer side
effects in a subject relative to treatment with the naturally
occurring form of the protein.
[0299] Variants of a biomarker protein which function as either
agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the present invention for agonist or
antagonist activity. In one embodiment, a variegated library of
variants is generated by combinatorial mutagenesis at the nucleic
acid level and is encoded by a variegated gene library. A
variegated library of variants can be produced by, for example,
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential protein
sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display). There are a variety of methods which can be used to
produce libraries of potential variants of the polypeptides of the
present invention from a degenerate oligonucleotide sequence.
Methods for synthesizing degenerate oligonucleotides are known in
the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al.,
1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science
198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).
[0300] In addition, libraries of fragments of the coding sequence
of a polypeptide corresponding to a marker of the present invention
can be used to generate a variegated population of polypeptides for
screening and subsequent selection of variants. For example, a
library of coding sequence fragments can be generated by treating a
double stranded PCR fragment of the coding sequence of interest
with a nuclease under conditions wherein nicking occurs only about
once per molecule, denaturing the double stranded DNA, renaturing
the DNA to form double stranded DNA which can include
sense/antisense pairs from different nicked products, removing
single stranded portions from reformed duplexes by treatment with
S1 nuclease, and ligating the resulting fragment library into an
expression vector. By this method, an expression library can be
derived which encodes amino terminal and internal fragments of
various sizes of the protein of interest.
[0301] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques, which
are amenable to high throughput analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the present invention (Arkin and
Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et
al., 1993, Protein Engineering 6(3):327-331).
[0302] The production and use of biomarker nucleic acid and/or
biomarker polypeptide molecules described herein can be facilitated
by using standard recombinant techniques. In some embodiments, such
techniques use vectors, preferably expression vectors, containing a
nucleic acid encoding a biomarker polypeptide or a portion of such
a polypeptide. As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors, namely
expression vectors, are capable of directing the expression of
genes to which they are operably linked. In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids (vectors). However, the present invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0303] The recombinant expression vectors of the present invention
comprise a nucleic acid of the present invention in a form suitable
for expression of the nucleic acid in a host cell. This means that
the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operably linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel,
Methods in Enzymology: Gene Expression Technology vol. 185,
Academic Press, San Diego, Calif. (1991). Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cell and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the present
invention can be introduced into host cells to thereby produce
proteins or peptides, including fusion proteins or peptides,
encoded by nucleic acids as described herein.
[0304] The recombinant expression vectors for use in the present
invention can be designed for expression of a polypeptide
corresponding to a marker of the present invention in prokaryotic
(e.g., E. coli) or eukaryotic cells (e.g., insect cells {using
baculovirus expression vectors}, yeast cells or mammalian cells).
Suitable host cells are discussed further in Goeddel, supra.
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
[0305] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0306] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET
11d (Studier et al., p. 60-89, In Gene Expression Technology:
Methods in Enzymology vol. 185, Academic Press, San Diego, Calif.,
1991). Target biomarker nucleic acid expression from the pTrc
vector relies on host RNA polymerase transcription from a hybrid
trp-lac fusion promoter. Target biomarker nucleic acid expression
from the pET 11d vector relies on transcription from a T7 gn10-lac
fusion promoter mediated by a co-expressed viral RNA polymerase (T7
gn1). This viral polymerase is supplied by host strains BL21 (DE3)
or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene
under the transcriptional control of the lacUV 5 promoter.
[0307] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacterium with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, p. 119-128. In Gene Expression Technology: Methods in
Enzymology vol. 185, Academic Press, San Diego, Calif., 1990.
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., 1992, Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
present invention can be carried out by standard DNA synthesis
techniques
[0308] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al., 1987, EMBOJ.
6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30.933-943),
pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0309] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
[0310] In yet another embodiment, a nucleic acid of the present
invention is expressed in mammalian cells using a mammalian
expression vector. Examples of mammalian expression vectors include
pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al.,
1987, EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral
regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook
et al., supra.
[0311] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., 1987, Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton,
1988, Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and
Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al., 1985, Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss, 1990, Science
249:374-379) and the a-fetoprotein promoter (Camper and Tilghman,
1989, Genes Dev. 3:537-546).
[0312] The present invention further provides a recombinant
expression vector comprising a DNA molecule cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to the mRNA encoding a
polypeptide of the present invention. Regulatory sequences operably
linked to a nucleic acid cloned in the antisense orientation can be
chosen which direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters
and/or enhancers, or regulatory sequences can be chosen which
direct constitutive, tissue-specific or cell type specific
expression of antisense RNA. The antisense expression vector can be
in the form of a recombinant plasmid, phagemid, or attenuated virus
in which antisense nucleic acids are produced under the control of
a high efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes (see Weintraub et al., 1986, Trends in Genetics,
Vol. 1(1)).
[0313] Another aspect of the present invention pertains to host
cells into which a recombinant expression vector of the present
invention has been introduced. The terms "host cell" and
"recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject
cell but to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[0314] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0315] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0316] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker gene will
survive, while the other cells die).
5. Analyzing Biomarker Nucleic Acids and Polypeptides
[0317] For any method described herein, biomarker nucleic acids
and/or biomarker polypeptides can be analyzed according to the
methods described herein and techniques known to the skilled
artisan to identify such genetic or expression alterations useful
for the present invention including, but not limited to, 1) an
alteration in the level of a biomarker transcript or polypeptide,
2) a deletion or addition of one or more nucleotides from a
biomarker gene, 4) a substitution of one or more nucleotides of a
biomarker gene, 5) aberrant modification of a biomarker gene, such
as an expression regulatory region, and the like.
[0318] a. Methods for Detection of Biomarker Copy Number
[0319] Methods of evaluating the copy number of a biomarker nucleic
acid are well-known to those of skill in the art. The presence or
absence of chromosomal gain or loss can be evaluated simply by a
determination of copy number of the regions or markers identified
herein.
[0320] In one embodiment, a biological sample is tested for the
presence of copy number changes in genomic loci containing the
genomic marker. A copy number of at least 3, 4, 5, 6, 7, 8, 9, or
10 is predictive of poorer outcome of PI3K/mTOR combination
inhibitor treatment.
[0321] Methods of evaluating the copy number of a biomarker locus
include, but are not limited to, hybridization-based assays.
Hybridization-based assays include, but are not limited to,
traditional "direct probe" methods, such as Southern blots, in situ
hybridization (e.g., FISH and FISH plus SKY) methods, and
"comparative probe" methods, such as comparative genomic
hybridization (CGIT), e.g., cDNA-based or oligonucleotide-based
CGH. The methods can be used in a wide variety of formats
including, but not limited to, substrate (e.g. membrane or glass)
bound methods or array-based approaches.
[0322] In one embodiment, evaluating the biomarker gene copy number
in a sample involves a Southern Blot. In a Southern Blot, the
genomic DNA (typically fragmented and separated on an
electrophoretic gel) is hybridized to a probe specific for the
target region. Comparison of the intensity of the hybridization
signal from the probe for the target region with control probe
signal from analysis of normal genomic DNA (e.g., a non-amplified
portion of the same or related cell, tissue, organ, etc.) provides
an estimate of the relative copy number of the target nucleic acid.
Alternatively, a Northern blot may be utilized for evaluating the
copy number of encoding nucleic acid in a sample. In a Northern
blot, mRNA is hybridized to a probe specific for the target region.
Comparison of the intensity of the hybridization signal from the
probe for the target region with control probe signal from analysis
of normal RNA (e.g., a non-amplified portion of the same or related
cell, tissue, organ, etc.) provides an estimate of the relative
copy number of the target nucleic acid. Alternatively, other
methods well-known in the art to detect RNA can be used, such that
higher or lower expression relative to an appropriate control
(e.g., a non-amplified portion of the same or related cell tissue,
organ, etc.) provides an estimate of the relative copy number of
the target nucleic acid.
[0323] An alternative means for determining genomic copy number is
in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152:
649). Generally, in situ hybridization comprises the following
steps: (1) fixation of tissue or biological structure to be
analyzed; (2) prehybridization treatment of the biological
structure to increase accessibility of target DNA, and to reduce
nonspecific binding; (3) hybridization of the mixture of nucleic
acids to the nucleic acid in the biological structure or tissue;
(4) post-hybridization washes to remove nucleic acid fragments not
bound in the hybridization and (5) detection of the hybridized
nucleic acid fragments. The reagent used in each of these steps and
the conditions for use vary depending on the particular
application. In a typical in situ hybridization assay, cells are
fixed to a solid support, typically a glass slide. If a nucleic
acid is to be probed, the cells are typically denatured with heat
or alkali. The cells are then contacted with a hybridization
solution at a moderate temperature to permit annealing of labeled
probes specific to the nucleic acid sequence encoding the protein.
The targets (e.g., cells) are then typically washed at a
predetermined stringency or at an increasing stringency until an
appropriate signal to noise ratio is obtained The probes are
typically labeled, e.g., with radioisotopes or fluorescent
reporters. In one embodiment, probes are sufficiently long so as to
specifically hybridize with the target nucleic acid(s) under
stringent conditions Probes generally range in length from about
200 bases to about 1000 bases. In some applications it is necessary
to block the hybridization capacity of repetitive sequences. Thus,
in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used
to block non-specific hybridization.
[0324] An alternative means for determining genomic copy number is
comparative genomic hybridization. In general, genomic DNA is
isolated from normal reference cells, as well as from test cells
(e.g., tumor cells) and amplified, if necessary. The two nucleic
acids are differentially labeled and then hybridized in situ to
metaphase chromosomes of a reference cell. The repetitive sequences
in both the reference and test DNAs are either removed or their
hybridization capacity is reduced by some means, for example by
prehybridization with appropriate blocking nucleic acids and/or
including such blocking nucleic acid sequences for said repetitive
sequences during said hybridization. The bound, labeled DNA
sequences are then rendered in a visualizable form, if necessary.
Chromosomal regions in the test cells which are at increased or
decreased copy number can be identified by detecting regions where
the ratio of signal from the two DNAs is altered. For example,
those regions that have decreased in copy number in the test cells
will show relatively lower signal from the test DNA than the
reference compared to other regions of the genome. Regions that
have been increased in copy number in the test cells will show
relatively higher signal from the test DNA. Where there are
chromosomal deletions or multiplications, differences in the ratio
of the signals from the two labels will be detected and the ratio
will provide a measure of the copy number. In another embodiment of
CGH, array CGH (aCGH), the immobilized chromosome element is
replaced with a collection of solid support bound target nucleic
acids on an array, allowing for a large or complete percentage of
the genome to be represented in the collection of solid support
bound targets. Target nucleic acids may comprise cDNAs, genomic
DNAs, oligonucleotides (e.g., to detect single nucleotide
polymorphisms) and the like. Array-based CGH may also be performed
with single-color labeling (as opposed to labeling the control and
the possible tumor sample with two different dyes and mixing them
prior to hybridization, which will yield a ratio due to competitive
hybridization of probes on the arrays). In single color CGH, the
control is labeled and hybridized to one array and absolute signals
are read, and the possible tumor sample is labeled and hybridized
to a second array (with identical content) and absolute signals are
read. Copy number difference is calculated based on absolute
signals from the two arrays. Methods of preparing immobilized
chromosomes or arrays and performing comparative genomic
hybridization are well-known in the art (see, e.g., U.S. Pat. Nos.
6,335,167; 6,197,501; 5,830,645; and 5,665,549 and Albertson (1984)
EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85:
9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol.
33: In situ Hybridization Protocols, Choo, ed., Humana Press,
Totowa, N.J. (1994), etc.) In another embodiment, the hybridization
protocol of Pinkel, et al. (1998) Nature Genetics 20: 207-211, or
of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992)
is used.
[0325] In still another embodiment, amplification-based assays can
be used to measure copy number. In such amplification-based assays,
the nucleic acid sequences act as a template in an amplification
reaction (e.g., Polymerase Chain Reaction (PCR). In a quantitative
amplification, the amount of amplification product will be
proportional to the amount of template in the original sample.
Comparison to appropriate controls, e.g. healthy tissue, provides a
measure of the copy number.
[0326] Methods of "quantitative" amplification are well-known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided in Innis, et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.). Measurement of DNA copy number at microsatellite loci
using quantitative PCR analysis is described in Ginzonger, et al.
(2000) Cancer Research 60:5405-5409. The known nucleic acid
sequence for the genes is sufficient to enable one of skill in the
art to routinely select primers to amplify any portion of the gene.
Fluorogenic quantitative PCR may also be used in the methods of the
present invention. In fluorogenic quantitative PCR, quantitation is
based on amount of fluorescence signals, e.g., TaqMan and SYBR
green.
[0327] Other suitable amplification methods include, but are not
limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4: 560, Landegren, et al. (1988) Science 241:1077, and
Barringer et al. (1990) Gene 89: 117), transcription amplification
(Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173),
self-sustained sequence replication (Guatelli, et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR,
etc.
[0328] Loss of heterozygosity (LOH) and major copy proportion (MCP)
mapping (Wang, Z. C., et al. (2004) Cancer Res 64(1):64-71;
Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4; Hahn, S. A.,
et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. (1996)
Genes Chromosomes Cancer 17, 88-93; Li et al., (2008) MBC
Bioinform. 9, 204-219) may also be used to identify regions of
amplification or deletion.
[0329] b. Methods for Detection of Biomarker Nucleic Acid
Expression
[0330] Biomarker expression may be assessed by any of a wide
variety of well-known methods for detecting expression of a
transcribed molecule or protein. Non-limiting examples of such
methods include immunological methods for detection of secreted,
cell-surface, cytoplasmic, or nuclear proteins, protein
purification methods, protein function or activity assays, nucleic
acid hybridization methods, nucleic acid reverse transcription
methods, and nucleic acid amplification methods.
[0331] In preferred embodiments, activity of a particular gene is
characterized by a measure of gene transcript (e.g. mRNA), by a
measure of the quantity of translated protein, or by a measure of
gene product activity. Marker expression can be monitored in a
variety of ways, including by detecting mRNA levels, protein
levels, or protein activity, any of which can be measured using
standard techniques. Detection can involve quantification of the
level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein,
or enzyme activity), or, alternatively, can be a qualitative
assessment of the level of gene expression, in particular in
comparison with a control level. The type of level being detected
will be clear from the context.
[0332] In another embodiment, detecting or determining expression
levels of a biomarker and functionally similar homologs thereof,
including a fragment or genetic alteration thereof (e.g., in
regulatory or promoter regions thereof) comprises detecting or
determining RNA levels for the marker of interest. In one
embodiment, one or more cells from the subject to be tested are
obtained and RNA is isolated from the cells. In a preferred
embodiment, a sample of breast tissue cells is obtained from the
subject.
[0333] In one embodiment, RNA is obtained from a single cell. For
example, a cell can be isolated from a tissue sample by laser
capture microdissection (LCM). Using this technique, a cell can be
isolated from a tissue section, including a stained tissue section,
thereby assuring that the desired cell is isolated (see, e.g.,
Bonner et al. (1997) Science 278: 1481; Emmert-Buck et al (1996)
Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61 and
Murakami et al. (2000) Kidney Int. 58:1346). For example, Murakami
et al., supra, describe isolation of a cell from a previously
immunostained tissue section
[0334] It is also be possible to obtain cells from a subject and
culture the cells in vitro, such as to obtain a larger population
of cells from which RNA can be extracted. Methods for establishing
cultures of non-transformed cells, i.e., primary cell cultures, are
known in the art.
[0335] When isolating RNA from tissue samples or cells from
individuals, it may be important to prevent any further changes in
gene expression after the tissue or cells has been removed from the
subject. Changes in expression levels are known to change rapidly
following perturbations, e.g., heat shock or activation with
lipopolysaccharide (LPS) or other reagents. In addition, the RNA in
the tissue and cells may quickly become degraded. Accordingly, in a
preferred embodiment, the tissue or cells obtained from a subject
is snap frozen as soon as possible.
[0336] RNA can be extracted from the tissue sample by a variety of
methods, e.g., the guanidium thiocyanate lysis followed by CsCl
centrifugation (Chirgwin et al., 1979, Biochemistry 18:5294-5299).
RNA from single cells can be obtained as described in methods for
preparing cDNA libraries from single cells, such as those described
in Dulac, C. (1998) Curr. Top. Dev. Biol. 36, 245 and Jena et al.
(1996) J. Immunol. Methods 190:199. Care to avoid RNA degradation
must be taken, e.g., by inclusion of RNAsin.
[0337] The RNA sample can then be enriched in particular species.
In one embodiment, poly(A)+ RNA is isolated from the RNA sample. In
general, such purification takes advantage of the poly-A tails on
mRNA. In particular and as noted above, poly-T oligonucleotides may
be immobilized within on a solid support to serve as affinity
ligands for mRNA. Kits for this purpose are commercially available,
e.g., the MessageMaker kit (Life Technologies, Grand Island,
N.Y.).
[0338] In a preferred embodiment, the RNA population is enriched in
marker sequences. Enrichment can be undertaken, e.g., by
primer-specific cDNA synthesis, or multiple rounds of linear
amplification based on cDNA synthesis and template-directed in
vitro transcription (see, e.g., Wang et al. (1989) PNAS 86, 9717;
Dulac et al., supra, and Jena et al., supra).
[0339] The population of RNA, enriched or not in particular species
or sequences, can further be amplified. As defined herein, an
"amplification process" is designed to strengthen, increase, or
augment a molecule within the RNA. For example, where RNA is mRNA,
an amplification process such as RT-PCR can be utilized to amplify
the mRNA, such that a signal is detectable or detection is
enhanced. Such an amplification process is beneficial particularly
when the biological, tissue, or tumor sample is of a small size or
volume.
[0340] Various amplification and detection methods can be used For
example, it is within the scope of the present invention to reverse
transcribe mRNA into cDNA followed by polymerase chain reaction
(RT-PCR); or, to use a single enzyme for both steps as described in
U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA
followed by symmetric gap ligase chain reaction (RT-AGLCR) as
described by R. L. Marshall, et al., PCR Methods and Applications
4: 80-84 (1994). Real time PCR may also be used.
[0341] Other known amplification methods which can be utilized
herein include but are not limited to the so-called "NASBA" or
"3SR" technique described in PNAS USA 87: 1874-1878 (1990) and also
described in Nature 350 (No. 6313): 91-92 (1991); Q-beta
amplification as described in published European Patent Application
(EPA) No. 4544610; strand displacement amplification (as described
in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European
Patent Application No. 684315; target mediated amplification, as
described by PCT Publication WO9322461; PCR; ligase chain reaction
(LCR) (see, e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren
et al., Science 241, 1077 (1988)); self-sustained sequence
replication (SSR) (see, e.g., Guatelli et al., Proc. Nat. Acad.
Sci. USA, 87, 1874 (1990)); and transcription amplification (see,
e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)).
[0342] Many techniques are known in the state of the art for
determining absolute and relative levels of gene expression,
commonly used techniques suitable for use in the present invention
include Northern analysis, RNase protection assays (RPA),
microarrays and PCR-based techniques, such as quantitative PCR and
differential display PCR. For example, Northern blotting involves
running a preparation of RNA on a denaturing agarose gel, and
transferring it to a suitable support, such as activated cellulose,
nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or
RNA is then hybridized to the preparation, washed and analyzed by
autoradiography.
[0343] In situ hybridization visualization may also be employed,
wherein a radioactively labeled antisense RNA probe is hybridized
with a thin section of a biopsy sample, washed, cleaved with RNase
and exposed to a sensitive emulsion for autoradiography. The
samples may be stained with hematoxylin to demonstrate the
histological composition of the sample, and dark field imaging with
a suitable light filter shows the developed emulsion.
Non-radioactive labels such as digoxigenin may also be used.
[0344] Alternatively, mRNA expression can be detected on a DNA
array, chip or a microarray. Labeled nucleic acids of a test sample
obtained from a subject may be hybridized to a solid surface
comprising biomarker DNA. Positive hybridization signal is obtained
with the sample containing biomarker transcripts. Methods of
preparing DNA arrays and their use are well-known in the art (see,
e.g., U.S. Pat. Nos: 6,618,6796; 6,379,897, 6,664,377, 6,451,536;
548,257; U.S. 20030157485 and Schena et al. (1995) Science 20,
467-470; Gerhold et al. (1999) Trends In Biochem. Sci. 24, 168-173;
and Lennon et al. (2000) Drug Discovery Today 5, 59-65, which are
herein incorporated by reference in their entirety). Serial
Analysis of Gene Expression (SAGE) can also be performed (See for
example U.S. Patent Application 20030215858).
[0345] To monitor mRNA levels, for example, mRNA is extracted from
the biological sample to be tested, reverse transcribed, and
fluorescently-labeled cDNA probes are generated. The microarrays
capable of hybridizing to marker cDNA are then probed with the
labeled cDNA probes, the slides scanned and fluorescence intensity
measured. This intensity correlates with the hybridization
intensity and expression levels.
[0346] Types of probes that can be used in the methods described
herein include cDNA, riboprobes, synthetic oligonucleotides and
genomic probes. The type of probe used will generally be dictated
by the particular situation, such as riboprobes for in situ
hybridization, and cDNA for Northern blotting, for example. In one
embodiment, the probe is directed to nucleotide regions unique to
the RNA. The probes may be as short as is required to
differentially recognize marker mRNA transcripts, and may be as
short as, for example, 15 bases; however, probes of at least 17,
18, 19 or 20 or more bases can be used. In one embodiment, the
primers and probes hybridize specifically under stringent
conditions to a DNA fragment having the nucleotide sequence
corresponding to the marker. As herein used, the term "stringent
conditions" means hybridization will occur only if there is at
least 95% sa identity in nucleotide sequences. In another
embodiment, hybridization under "stringent conditions" occurs when
there is at least 97% identity between the sequences.
[0347] The form of labeling of the probes may be any that is
appropriate, such as the use of radioisotopes, for example,
.sup.32P and .sup.35S. Labeling with radioisotopes may be achieved,
whether the probe is synthesized chemically or biologically, by the
use of suitably labeled bases.
[0348] In one embodiment, the biological sample contains
polypeptide molecules from the test subject. Alternatively, the
biological sample can contain mRNA molecules from the test subject
or genomic DNA molecules from the test subject.
[0349] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting marker
polypeptide, mRNA, genomic DNA, or fragments thereof, such that the
presence of the marker polypeptide, mRNA, genomic DNA, or fragments
thereof, is detected in the biological sample, and comparing the
presence of the marker polypeptide, mRNA, genomic DNA, or fragments
thereof, in the control sample with the presence of the marker
polypeptide, mRNA, genomic DNA, or fragments thereof in the test
sample.
[0350] c. Methods for Detection of Biomarker Protein Expression
[0351] The activity or level of a biomarker protein can be detected
and/or quantified by detecting or quantifying the expressed
polypeptide. The polypeptide can be detected and quantified by any
of a number of means well-known to those of skill in the art.
Aberrant levels of polypeptide expression of the polypeptides
encoded by a biomarker nucleic acid and functionally similar
homologs thereof, including a fragment or genetic alteration
thereof (e.g., in regulatory or promoter regions thereof) are
associated with the likelihood of response of a cancer to PI3K/mTOR
combination inhibitor therapy. Any method known in the art for
detecting polypeptides can be used. Such methods include, but are
not limited to, immunodiffusion, immunoelectrophoresis,
radioimmunoassay (RIA), enzyme-linked immunosorbent assays
(ELISAs), immunofluorescent assays, Western blotting, binder-ligand
assays, immunohistochemical techniques, agglutination, complement
assays, high performance liquid chromatography (HPLC), thin layer
chromatography (TLC), hyperdiffusion chromatography, and the like
(e.g., Basic and Clinical Immunology, Sites and Terr, eds.,
Appleton and Lange, Norwalk, Conn. pp 217-262, 1991 which is
incorporated by reference). Preferred are binder-ligand immunoassay
methods including reacting antibodies with an epitope or epitopes
and competitively displacing a labeled polypeptide or derivative
thereof.
[0352] For example, ELISA and RIA procedures may be conducted such
that a desired biomarker protein standard is labeled (with a
radioisotope such as .sup.125I or .sup.35S, or an assayable enzyme,
such as horseradish peroxidase or alkaline phosphatase), and,
together with the unlabeled sample, brought into contact with the
corresponding antibody, whereon a second antibody is used to bind
the first, and radioactivity or the immobilized enzyme assayed
(competitive assay). Alternatively, the biomarker protein in the
sample is allowed to react with the corresponding immobilized
antibody, radioisotope- or enzyme-labeled anti-biomarker protein
antibody is allowed to react with the system, and radioactivity or
the enzyme assayed (ELISA-sandwich assay). Other conventional
methods may also be employed as suitable
[0353] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. A "one-step" assay involves
contacting antigen with immobilized antibody and, without washing,
contacting the mixture with labeled antibody. A "two-step" assay
involves washing before contacting, the mixture with labeled
antibody. Other conventional methods may also be employed as
suitable.
[0354] In one embodiment, a method for measuring biomarker protein
levels comprises the steps of: contacting a biological specimen
with an antibody or variant (e.g., fragment) thereof which
selectively binds the biomarker protein, and detecting whether said
antibody or variant thereof is bound to said sample and thereby
measuring the levels of the biomarker protein.
[0355] Enzymatic and radiolabeling of biomarker protein and/or the
antibodies may be effected by conventional means. Such means will
generally include covalent linking of the enzyme to the antigen or
the antibody in question, such as by glutaraldehyde, specifically
so as not to adversely affect the activity of the enzyme, by which
is meant that the enzyme must still be capable of interacting with
its substrate, although it is not necessary for all of the enzyme
to be active, provided that enough remains active to permit the
assay to be effected. Indeed, some techniques for binding enzyme
are non-specific (such as using formaldehyde), and will only yield
a proportion of active enzyme.
[0356] It is usually desirable to immobilize one component of the
assay system on a support, thereby allowing other components of the
system to be brought into contact with the component and readily
removed without laborious and time-consuming labor. It is possible
for a second phase to be immobilized away from the first, but one
phase is usually sufficient.
[0357] It is possible to immobilize the enzyme itself on a support,
but if solid-phase enzyme is required, then this is generally best
achieved by binding to antibody and affixing the antibody to a
support, models and systems for which are well-known in the art.
Simple polyethylene may provide a suitable support.
[0358] Enzymes employable for labeling are not particularly
limited, but may be selected from the members of the oxidase group,
for example. These catalyze production of hydrogen peroxide by
reaction with their substrates, and glucose oxidase is often used
for its good stability, ease of availability and cheapness, as well
as the ready availability of its substrate (glucose). Activity of
the oxidase may be assayed by measuring the concentration of
hydrogen peroxide formed after reaction of the enzyme-labeled
antibody with the substrate under controlled conditions well-known
in the art.
[0359] Other techniques may be used to detect biomarker protein
according to a practitioner's preference based upon the present
disclosure. One such technique is Western blotting (Towbin et al.,
Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated
sample is run on an SDS-PAGE gel before being transferred to a
solid support, such as a nitrocellulose filter. Anti-biomarker
protein antibodies (unlabeled) are then brought into contact with
the support and assayed by a secondary immunological reagent, such
as labeled protein A or anti-immunoglobulin (suitable labels
including .sup.125I, horseradish peroxidase and alkaline
phosphatase). Chromatographic detection may also be used.
[0360] Immunohistochemistry may be used to detect expression of
biomarker protein, e.g., in a biopsy sample. A suitable antibody is
brought into contact with, for example, a thin layer of cells,
washed, and then contacted with a second, labeled antibody.
Labeling may be by fluorescent markers, enzymes, such as
peroxidase, avidin, or radiolabeling. The assay is scored visually,
using microscopy.
[0361] Anti-biomarker protein antibodies, such as intrabodies, may
also be used for imaging purposes, for example, to detect the
presence of biomarker protein in cells and tissues of a subject.
Suitable labels include radioisotopes, iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulphur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc),
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0362] For in vivo imaging purposes, antibodies are not detectable,
as such, from outside the body, and so must be labeled, or
otherwise modified, to permit detection. Markers for this purpose
may be any that do not substantially interfere with the antibody
binding, but which allow external detection. Suitable markers may
include those that may be detected by X-radiography, NMR or MRI.
For X-radiographic techniques, suitable markers include any
radioisotope that emits detectable radiation but that is not
overtly harmful to the subject, such as barium or cesium, for
example. Suitable markers for NMR and MRI generally include those
with a detectable characteristic spin, such as deuterium, which may
be incorporated into the antibody by suitable labeling of nutrients
for the relevant hybridoma, for example.
[0363] The size of the subject, and the imaging system used, will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of technetium-99 The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain biomarker protein. The
labeled antibody or antibody fragment can then be detected using
known techniques.
[0364] Antibodies that may be used to detect biomarker protein
include any antibody, whether natural or synthetic, full length or
a fragment thereof, monoclonal or polyclonal, that binds
sufficiently strongly and specifically to the biomarker protein to
be detected. An antibody may have a K.sub.d of at most about
10.sup.-6M, 10.sup.-7M, 10.sup.-8M, 10.sup.-9M, 10.sup.-10M,
10.sup.-11M, 10.sup.-12M. The phrase "specifically binds" refers to
binding of, for example, an antibody to an epitope or antigen or
antigenic determinant in such a manner that binding can be
displaced or competed with a second preparation of identical or
similar epitope, antigen or antigenic determinant. An antibody may
bind preferentially to the biomarker protein relative to other
proteins, such as related proteins.
[0365] Antibodies are commercially available or may be prepared
according to methods known in the art.
[0366] Antibodies and derivatives thereof that may be used
encompass polyclonal or monoclonal antibodies, chimeric, human,
humanized, primatized (CDR-grafted), veneered or single-chain
antibodies as well as functional fragments, i.e., biomarker protein
binding fragments, of antibodies. For example, antibody fragments
capable of binding to a biomarker protein or portions thereof,
including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments
can be used. Such fragments can be produced by enzymatic cleavage
or by recombinant techniques. For example, papain or pepsin
cleavage can generate Fab or F(ab') 2 fragments, respectively.
Other proteases with the requisite substrate specificity can also
be used to generate Fab or F(ab') 2 fragments. Antibodies can also
be produced in a variety of truncated forms using antibody genes in
which one or more stop codons have been introduced upstream of the
natural stop site. For example, a chimeric gene encoding a F(ab') 2
heavy chain portion can be designed to include DNA sequences
encoding the CH, domain and hinge region of the heavy chain.
[0367] Synthetic and engineered antibodies are described in, e.g.,
Cabilly et al., U.S. Pat. No. 4,816,567 Cabilly et al., European
Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss
et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,
WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.
0,239,400 B1; Queen et al., European Patent No. 0451216 B1; and
Padlan, E. A. et al., EP 0519596 A1. See also, Newman, R. et al.,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody,
and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al.,
Science, 242: 423-426 (1988)) regarding single-chain antibodies.
Antibodies produced from a library, e.g., phage display library,
may also be used.
[0368] In some embodiments, agents that specifically bind to a
biomarker protein other than antibodies are used, such as peptides.
Peptides that specifically bind to a biomarker protein can be
identified by any means known in the art. For example, specific
peptide binders of a biomarker protein can be screened for using
peptide phage display libraries.
[0369] d. Methods for Detection of Biomarker Structural
Alterations
[0370] The following illustrative methods can be used to identify
the presence of a structural alteration in a biomarker nucleic acid
and/or biomarker polypeptide molecule in order to, for example,
identify PI3K/mTOR pathway proteins that are overexpressed,
overfunctional, and the like.
[0371] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in a biomarker nucleic acid such as a biomarker gene (see
Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method
can include the steps of collecting a sample of cells from a
subject, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a biomarker
gene under conditions such that hybridization and amplification of
the biomarker gene (if present) occurs, and detecting the presence
or absence of an amplification product, or detecting the size of
the amplification product and comparing the length to a control
sample. It is anticipated that PCR and/or LCR may be desirable to
use as a preliminary amplification step in conjunction with any of
the techniques used for detecting mutations described herein.
[0372] Alternative amplification methods include: self-sustained
sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. et al. (1989) Proc Natl. Acad Sci. USA 86:1173-1177),
Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology
6:1197), or any other nucleic acid amplification method, followed
by the detection of the amplified molecules using techniques
well-known to those of skill in the art. These detection schemes
are especially useful for the detection of nucleic acid molecules
if such molecules are present in very low numbers.
[0373] In an alternative embodiment, mutations in a biomarker
nucleic acid from a sample cell can be identified by alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined by gel electrophoresis and compared. Differences in
fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0374] In other embodiments, genetic mutations in biomarker nucleic
acid can be identified by hybridizing a sample and control nucleic
acids, e.g., DNA or RNA, to high density arrays containing hundreds
or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996)
Hum. Mutat. 7:244-255; Kozal, M. J. et al. (1996) Nat. Med.
2:753-759). For example, biomarker genetic mutations can be
identified in two dimensional arrays containing light-generated DNA
probes as described in Cronin et al. (1996) supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential,
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene. Such biomarker genetic mutations
can be identified in a variety of contexts, including, for example,
germline and somatic mutations.
[0375] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence a
biomarker gene and detect mutations by comparing the sequence of
the sample biomarker with the corresponding wild-type (control)
sequence. Examples of sequencing reactions include those based on
techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad.
Sci. (ISA 74:560 or Sanger (1977) Proc. Natl. Acad Sci. (USA
74:5463. It is also contemplated that any of a variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve (1995) Biotechniques 19:448-53), including
sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.
36:127-162, and Griffin et al. (1993) Appl. Biochem. Biotechnol.
38:147-159).
[0376] Other methods for detecting mutations in a biomarker gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes formed by
hybridizing (labeled) RNA or DNA containing the wild-type biomarker
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to base pair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digest the mismatched regions. In other embodiments, either DNA/DNA
or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al.
(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[0377] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in
biomarker cDNAs obtained from samples of cells. For example, the
mutY enzyme of E. coli cleaves A at G/A mismatches and the
thymidine DNA glycosylase from HeLa cells cleaves T at G/T
mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
According to an exemplary embodiment, a probe based on a biomarker
sequence, e.g., a wild-type biomarker treated with a DNA mismatch
repair enzyme, and the cleavage products, if any, can be detected
from electrophoresis protocols or the like (e.g., U.S. Pat. No.
5,459,039.)
[0378] In other embodiments, alterations in electrophoretic
mobility can be used to identify mutations in biomarker genes For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA 86:2766; see also Cotton (1993) Mutat. Res. 285125-144 and
Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded
DNA fragments of sample and control biomarker nucleic acids will be
denatured and allowed to renature. The secondary structure of
single-stranded nucleic acids varies according to sequence, the
resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments may be
labeled or detected with labeled probes. The sensitivity of the
assay may be enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method utilizes heteroduplex
analysis to separate double stranded heteroduplex molecules on the
basis of changes in electrophoretic mobility (Keen et al. (1991)
Trends Genet. 7:5).
[0379] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to ensure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys.
Chem. 265:12753).
[0380] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki
et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0381] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0382] e. Sampling Methods
[0383] In some embodiments, biomarker amount and/or activity
measurement(s) in a sample from a subject is compared to a
predetermined control (standard) sample. The sample from the
subject is typically from a diseased tissue, such as cancer cells
or tissues. The control sample can be from the same subject or from
a different subject. The control sample is typically a normal,
non-diseased sample. However, in some embodiments, such as for
staging of disease or for evaluating the efficacy of treatment, the
control sample can be from a diseased tissue. The control sample
can be a combination of samples from several different subjects. In
some embodiments, the biomarker amount and/or activity
measurement(s) from a subject is compared to a pre-determined
level. This pre-determined level is typically obtained from normal
samples. As described herein, a "pre-determined" biomarker amount
and/or activity measurement(s) may be a biomarker amount and/or
activity measurement(s) used to, by way of example only, evaluate a
subject that may be selected for treatment (e.g., based on the
number of genomic mutations and/or the number of genomic mutations
causing non-functional proteins for DNA repair genes), evaluate a
response to a PI3K/mTOR combination inhibitor therapy, and/or
evaluate a response to a PI3K/mTOR combination inhibitor therapy
with one or more additional anti-cancer therapies. A pre-determined
biomarker amount and/or activity measurement(s) may be determined
in populations of patients with or without cancer. The
pre-determined biomarker amount and/or activity measurement(s) can
be a single number, equally applicable to every patient, or the
pre-determined biomarker amount and/or activity measurement(s) can
vary according to specific subpopulations of patients. Age, weight,
height, and other factors of a subject may affect the
pre-determined biomarker amount and/or activity measurement(s) of
the individual. Furthermore, the pre-determined biomarker amount
and/or activity can be determined for each subject individually. In
one embodiment, the amounts determined and/or compared in a method
described herein are based on absolute measurements.
[0384] In another embodiment, the amounts determined and/or
compared in a method described herein are based on relative
measurements, such as ratios (e.g., biomarker copy numbers, level,
and/or activity before a treatment vs. after a treatment, such
biomarker measurements relative to a spiked or man-made control,
such biomarker measurements relative to the expression of a
housekeeping gene, and the like). For example, the relative
analysis can be based on the ratio of pre-treatment biomarker
measurement as compared to post-treatment biomarker measurement.
Pre-treatment biomarker measurement can be made at any time prior
to initiation of anti-cancer therapy. Post-treatment biomarker
measurement can be made at any time after initiation of anti-cancer
therapy. In some embodiments, post-treatment biomarker measurements
are made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 weeks or more after initiation of anti-cancer therapy,
and even longer toward indefinitely for continued monitoring.
Treatment can comprise anti-cancer therapy, such as a therapeutic
regimen comprising one or more PI3K/mTOR combination inhibitors
alone or in combination with other anti-cancer agents, such as with
immune checkpoint inhibitors.
[0385] The pre-determined biomarker amount and/or activity
measurement(s) can be any suitable standard. For example, the
pre-determined biomarker amount and/or activity measurement(s) can
be obtained from the same or a different human for whom a patient
selection is being assessed. In one embodiment, the pre-determined
biomarker amount and/or activity measurement(s) can be obtained
from a previous assessment of the same patient. In such a manner,
the progress of the selection of the patient can be monitored over
time. In addition, the control can be obtained from an assessment
of another human or multiple humans, e.g., selected groups of
humans, if the subject is a human. In such a manner, the extent of
the selection of the human for whom selection is being assessed can
be compared to suitable other humans, e.g., other humans who are in
a similar situation to the human of interest, such as those
suffering from similar or the same condition(s) and/or of the same
ethnic group.
[0386] In some embodiments of the present invention the change of
biomarker amount and/or activity measurement(s) from the
pre-determined level is about 0, 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or
greater, or any range in between, inclusive. Such cutoff values
apply equally when the measurement is based on relative changes,
such as based on the ratio of pre-treatment biomarker measurement
as compared to post-treatment biomarker measurement
[0387] Biological samples can be collected from a variety of
sources from a patient including a body fluid sample, cell sample,
or a tissue sample comprising nucleic acids and/or proteins. "Body
fluids" refer to fluids that are excreted or secreted from the body
as well as fluids that are normally not (e.g., amniotic fluid,
aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,
cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,
chyme, stool, female ejaculate, interstitial fluid, intracellular
fluid, lymph, menses, breast milk, mucus, pleural fluid, pus,
saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine,
vaginal lubrication, vitreous humor, vomit). In a preferred
embodiment, the subject and/or control sample is selected from the
group consisting of cells, cell lines, histological slides,
paraffin embedded tissues, biopsies, whole blood, nipple aspirate,
serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine,
stool, and bone marrow. In one embodiment, the sample is serum,
plasma, or urine. In another embodiment, the sample is serum.
[0388] The samples can be collected from individuals repeatedly
over a longitudinal period of time (e.g., once or more on the order
of days, weeks, months, annually, biannually, etc.). Obtaining
numerous samples from an individual over a period of time can be
used to verify results from earlier detections and/or to identify
an alteration in biological pattern as a result of, for example,
disease progression, drug treatment, etc. For example, subject
samples can be taken and monitored every month, every two months,
or combinations of one, two, or three month intervals according to
the present invention. In addition, the biomarker amount and/or
activity measurements of the subject obtained over time can be
conveniently compared with each other, as well as with those of
normal controls during the monitoring period, thereby providing the
subject's own values, as an internal, or personal, control for
long-term monitoring.
[0389] Sample preparation and separation can involve any of the
procedures, depending on the type of sample collected and/or
analysis of biomarker measurement(s). Such procedures include, by
way of example only, concentration, dilution, adjustment of pH,
removal of high abundance polypeptides (e.g., albumin, gamma
globulin, and transferrin, etc), addition of preservatives and
calibrants, addition of protease inhibitors, addition of
denaturants, desalting of samples, concentration of sample
proteins, extraction and purification of lipids.
[0390] The sample preparation can also isolate molecules that are
bound in non-covalent complexes to other protein (e.g., carrier
proteins). This process may isolate those molecules bound to a
specific carrier protein (e.g., albumin), or use a more general
process, such as the release of bound molecules from all carrier
proteins via protein denaturation, for example using an acid,
followed by removal of the carrier proteins.
[0391] Removal of undesired proteins (e.g., high abundance,
uninformative, or undetectable proteins) from a sample can be
achieved using high affinity reagents, high molecular weight
filters, ultracentrifugation and/or electrodialysis. High affinity
reagents include antibodies or other reagents (e.g., aptamers) that
selectively bind to high abundance proteins. Sample preparation
could also include ion exchange chromatography, metal ion affinity
chromatography, gel filtration, hydrophobic chromatography,
chromatofocusing, adsorption chromatography, isoelectric focusing
and related techniques. Molecular weight filters include membranes
that separate molecules on the basis of size and molecular weight.
Such filters may further employ reverse osmosis, nanofiltration,
ultrafiltration and microfiltration.
[0392] Ultracentrifugation is a method for removing undesired
polypeptides from a sample. Ultracentrifugation is the
centrifugation of a sample at about 15,000-60,000 rpm while
monitoring with an optical system the sedimentation (or lack
thereof) of particles. Electrodialysis is a procedure which uses an
electromembrane or semipermable membrane in a process in which ions
are transported through semi-permeable membranes from one solution
to another under the influence of a potential gradient. Since the
membranes used in electrodialysis may have the ability to
selectively transport ions having positive or negative charge,
reject ions of the opposite charge, or to allow species to migrate
through a semipermable membrane based on size and charge, it
renders electrodialysis useful for concentration, removal, or
separation of electrolytes.
[0393] Separation and purification in the present invention may
include any procedure known in the art, such as capillary
electrophoresis (e.g., in capillary or on-chip) or chromatography
(e.g., in capillary, column or on a chip). Electrophoresis is a
method which can be used to separate ionic molecules under the
influence of an electric field. Electrophoresis can be conducted in
a gel, capillary, or in a microchannel on a chip. Examples of gels
used for electrophoresis include starch, acrylamide, polyethylene
oxides, agarose, or combinations thereof. A gel can be modified by
its cross-linking, addition of detergents, or denaturants,
immobilization of enzymes or antibodies (affinity electrophoresis)
or substrates (zymography) and incorporation of a pH gradient
Examples of capillaries used for electrophoresis include
capillaries that interface with an electrospray.
[0394] Capillary electrophoresis (CE) is preferred for separating
complex hydrophilic molecules and highly charged solutes. CE
technology can also be implemented on microfluidic chips. Depending
on the types of capillary and buffers used, CE can be further
segmented into separation techniques such as capillary zone
electrophoresis (CZE), capillary isoelectric focusing (CIEF),
capillary isotachophoresis (cITP) and capillary
electrochromatography (CEC). An embodiment to couple CE techniques
to electrospray ionization involves the use of volatile solutions,
for example, aqueous mixtures containing a volatile acid and/or
base and an organic such as an alcohol or acetonitrile.
[0395] Capillary isotachophoresis (cITP) is a technique in which
the analytes move through the capillary at a constant speed but are
nevertheless separated by their respective mobilities. Capillary
zone electrophoresis (CZE), also known as free-solution CE (FSCE),
is based on differences in the electrophoretic mobility of the
species, determined by the charge on the molecule, and the
frictional resistance the molecule encounters during migration
which is often directly proportional to the size of the molecule.
Capillary isoelectric focusing (CIEF) allows weakly-ionizable
amphoteric molecules, to be separated by electrophoresis in a pH
gradient. CEC is a hybrid technique between traditional high
performance liquid chromatography (HPLC) and CE.
[0396] Separation and purification techniques used in the present
invention include any chromatography procedures known in the art.
Chromatography can be based on the differential adsorption and
elution of certain analytes or partitioning of analytes between
mobile and stationary phases. Different examples of chromatography
include, but not limited to, liquid chromatography (LC), gas
chromatography (GC), high performance liquid chromatography (HPLC),
etc.
EXAMPLES
Example 1: Materials and Methods for Examples 2-5
[0397] a. Mice
[0398] K14Cre, Pten.sup.L/L, Pik3ca.sup.L/L, Pik3cb.sup.L/L,
Trp3.sup.L/L, and MMTV-Neu-IRES-Cre (NIC) mice of FVB/N background
were intercrossed to generate mice for this study. All animals were
housed and treated in accordance with protocols approved by the
Institutional Animal Care and Use Committees at Dana-Farber Cancer
Institute and Harvard Medical School.
[0399] b. Breast tumors
[0400] In order to obtain K14Cre;Pten.sup.L/L; Trp53.sup.L/L with
or without additional Pik3ca.sup.L/L or Pik3cb.sup.L/L mammary
tumors while avoiding epidermal complications arising from Pten
deletion in K14-positive basal cells (Wang et al. (2013) Genes and
Development 27:1568-1580), mammary glands from
genetically-engineered K14Cre;Pen.sup.L/L; Trp53.sup.L/L with or
without additional Pik3ca.sup.L/L or Pik3cb.sup.L/L mice were
excised, and fragments transplanted orthotopically into cleared
mammary fat pads of four week-old female nude mice
(CrTac:NCr-Foxn1.sup.nu). Mice were monitored until mammary tumors
formed. Tumors were excised and transplanted into additional nude
mice for expansion. To obtain NIC-Pten.sup.L/L tumors for
experimentation, tumors formed de novo in GEM models were excised
and transplanted into the 3.sup.rd and 4.sup.th fat pad of
additional six week-old FVB/NJ (Taconic) mice. Orthotopic tumors
were obtained by injecting 1.0.times.10.sup.5 primary cells in 40%
matrigel into the 3.sup.rd mammary fat pad of six week-old FVB/NJ
(Taconic). Tumors growth was assessed by measuring the long and
short axes with digital calipers, and tumor volume was calculated
by use of the modified ellipsoid formula
(long.times.short.sup.2.times.0.52) For all experiments involving
tumor transplantations, all mice were transplanted from single cell
pools on the same day. Mice were tagged two to three days later and
randomly assigned to treatment or control groups. All tumor
measurements were performed by the same researcher in order to
avoid variation due to methodology. All analyses were conducted by
a second researcher, who was blinded to tumor measurements.
[0401] c. Derivation of Primary Breast Cancer Cells
[0402] Primary K14Cre;Pten.sup.L/L; Trp53.sup.L/L with or without
additional Pik3cd.sup.L/L or Pik3cb.sup.L/L breast tumors or
NIC-Pten.sup.L/L tumors were excised, minced and dissociated in
collagenase/hyaluronidase buffer (10 mM HEPES, 5% FBS, 20 ug/mL
DNase I [Stem Cell Technologies], collagenase/hyaluronidase [Stem
Cell Technologies] to 1.times.) for 45 minutes at 37.degree. C.,
followed by red blood cell lysis (4 volumes of 0.8% NH.sub.4Cl 0.1
mM EDTA [Stem Cell Technologies] plus 1 volume PBS), and strained
through a 40 um strainer into a cell culture dish. Primary cells
from K14Cre;Pten.sup.L/L; Trp53.sup.L/L with or without additional
Pik3ca.sup.L/L or Pik3cb.sup.L/L breast tumors were grown at
37.degree. C. in a humidified incubator under 5% CO.sub.2 in
F12/DMEM (1:3) media supplemented with 25 ng/mL hydrocortisone, 5
ug/mL insulin, 8.5 ng/mL cholera toxin, 0.125 ng/mL EGF, 10 ug/mL
gentamicin and 5 uM Y-27632. Primary cells derived from
NIC-Pten.sup.C1L tumors were grown in F12/DMEM (1:1) media
supplemented with 0.6% FBS, 0.5 ug/mL hydrocortisone, 2.5 ug/mL
insulin, 1.0 ng/mL cholera toxin, 20 ng/mL EGF, 10 ug/mL gentamicin
and 5 uM Y-27632.
[0403] d. Ovarian Tumors
[0404] To obtain serous ovarian cancer (SOC) tumors, we isolated
ovarian surface epithelial (OSE) cells from Pten.sup.L/L;
Trp53.sup.L/L mouse ovaries and introduced Cre and Myc expression
via adenoviral and lentiviral transduction in vitro, respectively.
Transduced OSE cells were subsequently injected into the ovarian
bursa of recipient nude mice. Resulting tumors were excised, minced
and dissociated as outlined above. In order to evaluate tumor
growth in vivo, Firefly luciferase expression was introduced by
lentiviral transduction of pLenti-Blasticidin-Luciferase in vitro,
prior to injecting the cells back into the ovarian bursa of
recipient FVB/NJ mice. The resulting Luciferase-expressing SOC
tumors were used for orthotopic transplants into experimental
cohorts of FVB/NJ mice.
[0405] e. In Vivo Drug Treatment
[0406] BKM120, BYL719 and KIN193 were reconstituted in one volume
of NMP (1-methyl-2-pyrrolidone; Sigma) and 9 volumes of PEG-300
(polyethylene glycol 300; Fluka Analytical). Mice were dosed at 30
mg/kg once daily via oral gavage. Monoclonal antibody specific to
mouse PD-1 (clone 332.8H3) was administered at 250 ug per mouse
once every three days via intraperitoneal injection.
[0407] f. Flow Cytometry
[0408] Single-cell tumor suspensions in PBS containing 0.2% BSA and
5 mM EDTA were incubated with .alpha.CD16/32 antibody for 10
minutes at 4.degree. C. to block Fc receptors, and subsequently
incubated with fluorophore-labeled primary antibodies as
appropriate for 20 minutes at 4.degree. C. Cell sorting was
performed on LSR II and analyzed with FloJo software.
[0409] g. Histology and Immunohistochemistry
[0410] For histology analyses, formalin-fixed tissue sections were
embedded in paraffin, sectioned, and stained with hematoxylin and
eosin by the Dana Farber/Harvard Cancer Center Rodent
Histopathology Core. For immunohistochemistry, sections were
de-paraffinized, incubated in primary antibody overnight, followed
by secondary antibody and DAB incubation, and counterstained with
hematoxylin.
[0411] h. Cell Viability and Cell Invasion Assays
[0412] For cell viability assays, one thousand primary cells were
seeded in ultra-low attachment 96-well plates and incubated with
the appropriate drug concentration for five days. Viability was
determined by incubating with CellTiter-Glo.RTM. (Promega).
Briefly, cells were lysed by adding CellTiter-Glo.RTM., and then
transferred to opaque plates to measure luminescence. Cell invasion
assays were carried out using 6.5 mm, 8.0 .mu.m-pore polycarbonate
membrane transwell inserts (BD Biosciences) by seeding
1.0.times.10.sup.5 cells in serum-free media into the inner
chamber, and adding normal growth media to the outer chamber. Cells
were incubated overnight. Non-migrating cells were carefully
removed with a cotton swab. Migrating cells were stained with 0.5%
crystal violet in 70% ethanol for ten minutes at room temperature,
and photographed under a Zeiss light microscope. At least five
random fields were counted and averaged.
[0413] i. Western Blotting
[0414] Cells were lysed in EBC.sub.250 lysis buffer (250 mM NaCl,
50 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40, 0.2 mM PMSF, 2 .mu.g/mL
aprotinin, and 2 .mu.g/mL leupeptin, 5 mM NaF, and 0.5 mM
NaVO.sub.4) Equal amounts of protein were resolved by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
polyvinylidene difluoride membranes (PVDF), and hybridized to an
appropriate primary antibody and fluorophore-conjugated secondary
antibody for subsequent detection by Odyssey.RTM. Scanner
(Li-Cor).
[0415] j. Analysis of Gene Expression and Quantitative-Polymerase
Chain Reaction (Q-PCR)
[0416] Total RNA was isolated using RNeasy.RTM. Mini Kit (Qiagen)
and reverse transcribed using SuperScript.RTM. III First-Strand
Synthesis System (ThermoFisher). Q-PCR was performed in a 7300
Real-Time PCR System (Applied Biosystems, CA) using QuantiTect.RTM.
SYBR.RTM. Green PCR Kit (Qiagen). Reactions were carried out in
96-well plates at 95.degree. C. for 15 minutes, followed by 40
cycles of 94.degree. C. for 15 seconds, 51.degree. C. for 30
seconds and 72.degree. C. for 30 seconds. Gene expression was
normalized to GAPDH expression by the .DELTA..DELTA.Ct method. For
RNA sequencing (RNA Seq), RNA was isolated from bulk tumor
fragments using TRizol reagent according to manufacturer's
specifications. RNA Seq was carried out by Ion Torrent (Thermo
Fisher) using a panel of 3,826 mouse-specific genes Gene set
expression analysis (GSEA) was carried out by GSEA software (The
Broad Institute of MIT and Harvard).
Example 2: Experimental TNBC Animal Model
[0417] In order to investigate the role of p110.beta. in
PTEN-deficient TNBC and to develop novel therapeutic strategies, a
genetically-engineered mouse (GEM) model of Pten-null TNBC was
generated (FIG. 1A). Consistent with previous results in genetic
models of prostate and ovarian cancer (Jia et al. (2008) Nature
454:776-9, Schmit et al (2014) Proc. Natl. Acad. Sc. USA
111:6395-400), Pten deletion led to tumor formation, which was
prevented by additional loss of Pik3cb, but not by Pik3ca deletion
(FIG. 1B). These initial findings confirmed the hypothesis that
PTEN-deficient TNBC depends on p110.beta. for survival, and
supports the hypothesis that PI3K.beta.-based combination therapies
could lead to more effective treatments against PTEN-deficient
TNBC.
[0418] In order to obtain a more robust phenotype suitable for
studying TNBC, the tumor suppressor p53 (encoded by TP53 in humans
and Trp53 in mice) was concomitantly deleted by crossing K14-Cre;
Pten.sup.L/L mice with Trp53.sup.L/L mice to obtain K14-Cre;
Pten.sup.L/L;Trp53.sup.L/L mice. Of note, approximately 84% of TNBC
exhibit deleted or mutant p53. From the resulting tumors,
early-passage primary Pten;Trp53-null (PP) TNBC cells were obtained
that maintained the biochemical and phenotypic characteristics of
the parental tumors, including expression of diagnostic markers
(FIG. 2A) and sensitivity to isoform-specific PI3K inhibition, as
shown by treatment with a pan-PI3K inhibitor (BKM120), a p110a
inhibitor (BYL719) or a p110.beta. inhibitor (KIN-193) (FIGS.
2B-2C). This system constitutes a durable research tool allowing
the analysis of PI3K isoform-specific signaling networks within a
Pten-null TNBC context, and further allowing the analysis of the
impact of targeting these pathways on tumor growth and cancer
progression.
[0419] Clear differences between p110.alpha.- and
p110.beta.-specific signaling pathways have been observed. For
example, primary Pten; Trp53;Pik3ca-null (PPA) cells exhibit clear
signs of epithelial to mesenchymal transition (EMT), including
increased expression of mesenchymal markers (N-Cadherin and
Vimentin) and undetectable levels of the epithelial marker,
E-Cadherin. Conversely, primary Pten;Trp53;Pik3ch-null (PPB) cells
express higher levels of E-Cadherin and undetectable levels of
Vimentin (FIG. 3A). It has previously been shown that p110.alpha.
and p110.beta. compete for binding to the p85 PI3K regulatory
subunit, so that deletion of one PI3K catalytic isoform results in
increased signaling output through the other isoform (Utermark et
a/(2012). Genes Dev 26:1573-86). In other words, PPA cells contain
upregulated p110.beta. signaling, while PPB cells contain
upregulated p110.alpha. activity. In addition, primary PPA cells
display increased ability to invade through matrigel, compared to
primary PP and PPB cells (FIG. 3B), which can be blocked by
specific p110.beta.inhibition (KIN-193; FIG. 3C). Likewise, primary
PP cell invasion could be prevented by inhibiting p110P, but not by
p110.alpha. inhibition (BYL719; FIG. 3D). These results indicate
that p110.beta. signaling may regulate a cell invasion and motility
program that could potentially impact on a susceptibility axis to
combine with p110.beta. inhibition.
[0420] The experimental model indicates that PTEN loss could
promote metastatic spread via sustained p110.beta. signaling. In
support of this hypothesis, loss of PTEN expression in the majority
of samples of distal metastases analyzed from patients with
advanced TNBC was confirmed (n=20) (FIG. 4). In addition, a case
study was recently reported in which an initially responsive
patient eventually developed resistance to p110a inhibition and
presented numerous metastatic lesions that had all lost PTEN
expression (Juric et al (2014) Nature 518:240-44). Taken together,
these results and clinical observations suggest that a
PI3K.beta.-based treatment regimen could prevent TNBC from
progressing into metastatic disease, or could be effective for
targeting metastatic TNBC lesions even if the primary tumor were
not PTEN-deficient. Interestingly, we observed that primary PP
cells grow at a faster rate in immunocompromised mice (athymic
nude) than in syngeneic immunocompetent mice (FVB strain; FIG. 5A).
Furthermore, when injected intravenously into nude mice, primary PP
cells formed numerous metastatic nodules in the lungs, while no
metastatic nodules could be observed in the immunocompetent FVB
mice (FIGS. 5B-5C). These results indicate that the growth of these
tumors could be subject to initial rejection by the immune system,
but that eventual resistance develops. Moreover, primary PPA tumor
cells grow at an aggressively fast pace in immunocompetent FVB
mice, while primary PPB tumor cells fail to form tumors in
immunocompetent mice (FIG. 6) but do form tumors in immunodeficient
nude mice. Together, these results indicate that PI3K.beta.
activity is required by Pten/p53-deficient breast cancer cells to
inhibit anti-tumor immune activity.
Example 3: Combined PI3Kbeta and Immune Checkpoint Blockade Delays
Breast Cancer Tumor Growth
[0421] The combination of isoform-selective PI3K inhibition and ICB
immunotherapy in the model of PTEN-deficient breast cancer
described in Example 2 was evaluated. Specifically, immunocompetent
FVB mice bearing orthotopic Pten;Trp53-null tumors were treated
with the immune checkpoint blocker (ICB) anti-mouse PD-1 monoclonal
antibody (.alpha.-mPD-1) either alone or in combination with
BKM120, a pan-PI3K inhibitor. A robust response to the combination
treatment that significantly delayed tumor growth was observed
(FIG. 7A). Moreover, fluorescence-activated cell sorting (FACS)
analysis revealed increased CD8+ T-cell infiltration into the
tumors in the combination treatment (FIGS. 7B-7C).
[0422] Next, isoform-specific combination treatments using either
BYL719, a PI3Kalpha-specific inhibitor, and KIN193, a
PI3Kbeta-specific inhibitor, (Ni et al. (2012) Cancer Discov.
2:425-433; Nylander et al (2012) J. Thromb. Haemost. 10:2127-36) in
combination with the anti-mouse PD-1 monoclonal antibody
(.alpha.-mPD-1) were tested as described above. It was determined
that BYL719 treatment, either alone or in combination with
anti-mouse PD-1, did not affect tumor growth. Similarly, KIN-193
treatment alone only marginally affected tumor growth. By contrast,
KIN-193 in combination with ICB surprisingly inhibited tumor growth
in a strong fashion (FIG. 8A). Notably, three of the six tumors
treated with this combination therapy regressed to undetectable
levels (FIG. 8B). This was confirmed by histological analysis,
which demonstrated a complete loss of tumor cellularity (FIG. 8C).
Increased CD8+ T-cell infiltration in tumors from mice treated with
combined PI3Kbeta inhibition and ICB, as assessed by FACS, was also
consistently observed (FIG. 8D).
[0423] These results were confirmed on a second cohort of mice,
which yielded similar results. Combined PI3Kbeta inhibition and ICB
with an anti-mouse PD-1 monoclonal antibody strongly inhibited
tumor growth, while neither agent alone led to a significant
decrease in tumor growth compared to the vehicle control (FIG. 9A).
In this second cohort, complete responses were observed in two out
of six cases treated with the combined treatment, and an additional
two cases showed almost complete responses (FIG. 9B).
[0424] The analysis can be furthered by characterizing the tumors
by IHC for expression of markers of interest, including staining
for CD8+ cells, Foxp3+ regulatory T cells that can dampen immune
response, and PD-1 Ligand 1 (PD-L1), which is often over-expressed
by tumor cells to inhibit anti-tumor immune response. It is
believed that the results allow inference of whether immune
suppression is due to increased Treg infiltration or an adaptive
tumor response.
[0425] In addition, ablation of PD-L1 expression in PP primary TNBC
cells using lentiviral-based GIPZ shRNA constructs obtained from
Dharmacon, which allow selection by puromycin resistance and GFP
expression, is performed. Without being bound by theory, it is
believed that PP cells with silenced PD-L1 expression display
slower and reduced tumor growth upon orthotopic transplantation
into FVB mice.
[0426] Since a complete remission in 40-50% of the tumors treated
with combined p110D inhibition and ICB immunotherapy was observed,
robustness of this response is determined by continuing treatment
for an additional two months, and then discontinuing treatment.
Tumor growth and recurrence is monitored during this time and for
an additional ten months. In the event of tumor recurrence,
treatment is reinstated and responsiveness and/or resistance is
evaluated.
[0427] In order to investigate changes in genes expression induced
by combined PI3Kbeta and ICB, gene expression levels were analyzed
in tumors from mice treated with each agent alone or in combination
for 96 hours. The results showed that combined treatment results in
enriched gene signatures related to anti-tumor immune response to a
much greater and significant extent than either agent alone (FIG.
10).
[0428] In addition, a clear correlation between response and CD8+
T-cell infiltration has been observed (FIG. 11) with different
responses between tumors from the same mouse indicating that
treatment efficacy is not necessarily due to systemic factors.
[0429] In order to extend these results to other types of breast
cancer with PTEN loss, combined PI3Kbeta inhibition and ICB was
evaluated in a Her2-positive model of breast cancer driven by
Her2/Neu over-expression and harboring wild type p53 expression. In
this model, treatment with lapatinib alone, a small molecule
EGFR/Her2 inhibitor used in clinical settings, did not
significantly affect tumor growth. Similarly, treatment with
lapatinib and anti-mouse PD-1 ICB did not affect tumor growth.
However, treatment with lapatinib plus anti-mouse PD-1 and KIN-193
(a PI3Kbeta inhibitor) significantly inhibited tumor growth (FIG.
12). These results indicate that combined PI3Kbeta inhibition and
ICB inhibits PTEN-null tumor growth in multiple breast cancer
models.
[0430] In order to determine whether CD8+ T-cell infiltration prior
to treatment correlates with response to therapy, PP tumors are
biopsied prior to, during, and after treatment, and CD8+
infiltration is determined by FACS. Furthermore, to investigate if
transcriptional differences between T-cell populations account for
differences in response, CD8+ T-cells are isolated from the tumors
and spleen (which represent systemic lymphocyte levels) from mice
treated with or without combined p110.beta. targeted therapy and
ICB and gene expression is analyzed by transcriptome analysis. Gene
expression is also analyzed in purified tumor and stromal cells.
Tumor and T cells are isolated using magnetic cell isolation and
cell separation kits (Miltenyi Biotec). Without being bound by
theory, it is believed that these analyses indicate how the tumor
microenvironment affects response to immunotherapy. These results
inform the rational design of adjuvant therapies to potentiate
anti-tumor immune response.
[0431] For statistical analyses involving only two groups, a
Student's t-test is used to assess statistical significance. The
majority of analyses, however, involve at least three groups (e.g.,
differences in gene expression between PP, PPA and PPB primary TNBC
cells). For statistical analyses involving three or more groups,
one-way ANOVA followed by Tukey's HSD post-hoc analysis is used.
Based on preliminary results, orthotopically transplanted PP tumors
in FVB mice reach a mean volume of 1,365 mm.sup.3 at endpoint (n=8;
SD=702 mm.sup.3). In order to achieve a reduction of 60% by the
combination PI3Kbeta selective inhibitor and ICB treatment,
assuming similar variance, the following power calculation is
believed to apply:
TABLE-US-00005 TABLE 4 Anticipated values Mean (mm.sup.3) St. Dev.
(mm.sup.3) St. Dev. (% of mean) Control 1365 702 51.4 Combination
546 280 51.3 Difference in means: 60%
TABLE-US-00006 TABLE 5 Sample size needed in each group Power Alpha
level 95% 90% 80% 50% 0.1 9 7 5 2 0.05 11 9 7 3 0.02 13 11 9 5 0.01
15 13 10 6
[0432] Thus, to observe a statistically significant difference with
a P-value of 0.05 and power level of 95%, at least 11 tumors per
condition are analyzed. Since a mean reduction of 72% between
control mice and mice treated with KIN-193 and anti-mouse PD-1
described above, it is believed that a 60% difference in means for
our power calculation is a conservative suitable estimate. For all
experiments involving tumor transplantations, all mice are
transplanted from single cell pools on the same day. Mice are
tagged two to three days later and randomly assigned to treatment
or control groups. All tumor measurements are performed by the same
research technician in order to avoid variation due to methodology.
All analyses are conducted by the same investigator, who is blinded
to tumor measurements. In the unlikely case of death before
endpoint unrelated to tumor burden, data from the mouse are
discarded, as post-mortem analysis of the tumors is
compromised.
Example 4: Patient-Derived Xenograft (PD) TNBC Models
[0433] The combination of isoform-selective PI3K inhibition and ICB
immunotherapy in the model of PTEN-deficient breast cancer
described in Examples 2 and 3 are extended to analysis of
pre-clinical cancer models using a panel of orthotopic
patient-derived xenografts from advanced TNBC. One of the major
advantages of PDX models is that they provide a clinically-relevant
system, as genetic and epigenetic alterations found within these
models correspond to alterations actually found in the clinic.
Likewise, PDX models recapitulate the heterogeneity found within a
single tumor and between patients. Since PDX models are amenable to
molecular and pharmacological manipulations, this system can be
used to evaluate response to novel cancer therapies, examine
genetic and epigenetic differences between responder and
non-responder tumors, investigate molecular mechanisms of
resistance or recurrence, and even help in the design of
personalized therapeutic options for patients. Therefore, PDX
models provide a uniquely strong tool to evaluate novel cancer
therapies, and an excellent system to validate results obtained
from genetically engineered mouse (GEM) models.
[0434] Over twenty PDX models of TNBC from primary tumors and
distal metastases (i.e., lungs, liver and brain) that faithfully
recapitulate ER, PR and HER2 status, as shown by
immunohistochemistry (IHC), have been established (FIG. 13A). This
collection is under continuous expansion via tissue collection from
newly diagnosed cases. Initial analyses of two PDX models by
whole-exome sequencing demonstrate that these models retain the
genetic features of patient-matched tumors (FIG. 13B). In addition,
a luciferase gene can be introduced into the tumor cells to allow
for live imaging of metastatic lesions (FIG. 13C)
[0435] IHC staining for PTEN and phosphorylated AKT, a major
downstream target and effector of PI3K, are performed. IHC slides
are scored blindly by two independent pathologists and potential
mutations and copy number alterations in PTEN, as well as in other
relevant genes, are identified such as by whole-exome sequencing.
RNA-Seq is also used to examine gene expression profiles.
PI3Kbeta-selective inhibitors, such as KIN-193, are tested on the
PDX metastatic TNBC models. For example, early-passage,
luciferase-tagged metastatic PDX tumors are transplanted into the
third mammary fat pad of NODSCIDgamma (NSG) mice and treated as
described above. Tumor growth is assessed as described above and
metastatic dissemination is examined using luciferase imaging.
Responses to PI3Kbeta inhibition are monitored and combination
treatments using inhibitors to putative targets, such as immune
checkpoints, are evaluated.
[0436] Without being bound by theory, it is believed that the
majority of metastatic TNBC PDX models exhibit loss of PTEN
expression, keeping in line with results described above. Based on
the described genetically engineered mouse (GEM) models and in vivo
studies on tumors derived from human PTEN-null cell lines, it is
believed that PTEN-deficient PDX models are sensitive to PI3Kbeta
inhibition. It is further believed that the combination of two
pre-clinical approaches (i.e., a pure genetic model in
immunocompetent mice and patient-derived samples in immunodeficient
mice), effectively complement each other to assay therapeutic
effects of the combination of isoform-selective PI3K inhibition and
ICB immunotherapy.
Example 5: Combined PI3Kbeta and Immune Checkpoint Blockade Delays
Ovarian Cancer Tumor Growth
[0437] A previously generated GEM model of ovarian cancer (OvCa)
driven by co-loss of p53 and PTEN (PP OvCa) demonstrated that PP
OvCa tumors depend on PI3Kbeta for survival, as PP OvCa tumors with
additional p110.beta. deletion (PPB OvCa) exhibit significantly
reduced tumor growth in vivo, while additional p110a deletion (PPA
OvCa) does not affect tumor growth (Schmit et al (2014) Proc. Natl.
Acad. Sci. USA 111:6395-400). Consistently, pharmacological
p110.beta.-specific inhibition (KIN193), but not
p110.alpha.-specific (BYL719) inhibition, significantly inhibited
tumor growth (Schmit et al (2014) Proc. Natl. Acad. Sci. USA
111:6395-400).
[0438] Hence, a serous ovarian cancer model suitable for
experimental evaluation of combined PI3Kbeta and ICB was developed
by deleting PTEN and p53, and over-expressing Myc. The combination
of isoform-selective PI3Kbeta inhibition and ICB immunotherapy in
this model of PTEN-deficient serous ovarian cancer was evaluated
PD-1 blockade by anti-mouse PD-1 monoclonal antibody did not affect
tumor growth. Although PI3Kbeta inhibition by KIN193 delayed tumor
growth compared to the vehicle control, combined PI3Kbeta
inhibition and ICB significantly inhibited ovarian cancer tumor
growth (FIG. 14). These results indicate that combined PI3Kbeta
inhibition and ICB inhibits PTEN-null tumor growth in ovarian
cancer with PTEN deficiency.
Example 6: Treating PTEN-Deficient Prostate Cancers
[0439] Since PTEN lesions are so common in primary prostate cancer
(PCA), and become even more common (and focal) in advanced PCA, the
PTEN pathway makes an attractive treatment target in this disease.
However, the early clinical trials were not promising at all. One
likely reason for this failure was due to a lack of understanding
of the key catalytic isoforms of PI3K (see, for example, Lu et al.
(2017) Nature 728-735). Using GEM models, it was demonstrated that,
while tumors driven by oncogenes or receptor tyrosine kinases are
dependent on p110.alpha., those driven by PTEN loss are uniquely
dependent on the p110.beta. isoform. Notably, the prostate trials
used so-called pan-PI3K inhibitors, which actually fail to
sufficiently dampen the action of p110.beta.. It is believed that
PTEN loss, in addition to driving p1103 activation, also activates
pathways that render cells at least partially resistant to PI3K
therapies. The mechanistic findings suggest novel targets that
should be complementary to p110.beta. inhibition. The main thrust
is to initiate a clinical trial using p110.beta. inhibitors to
confirm the hypothesis and gain valuable information from the
resulting patient derived data about markers for tumor sensitivity
and both initial and acquired resistance. In addition, the
mechanisms linking PTEN loss to p110.beta. activation to obtain
sufficient additional pre-clinical data has been investigated to
prepare for trials attacking the novel mechanistic targets in
combination with p110.beta.-directed therapies and
immunotherapies.
[0440] Specifically, the molecular details that uniquely couple
PTEN loss to p110.beta. activation are believed to be illustrated
below, which not only explains how p110.beta. is activated in
response to PTEN loss, but also suggests why the same tumors might
quickly become partially or even totally resistant to PI3K
inhibition. The same mechanisms clearly suggest other therapeutic
targets, under the same treatment or in combination with
PI3K/p110.beta. in PTEN-null tumors.
[0441] Step 1: Localizing p110.beta. to Lipid Rafts
[0442] The first question to be addressed is how p110.beta. is
activated under physiological conditions by GPCRs. Since the plasma
membrane is the major site of PI3K activation and integration of
divergent growth factor signals, it was initially aimed to
understand how spatial compartmentalization in the plasma membrane
might contribute to the functions of the ubiquitous class IA
phosphoinositide 3-kinase (PI3K) isoforms, p110.alpha. and
p110.beta.. Notably, GPCRs are known to be localized in lipid
rafts. It was found that p110.beta. also localizes to membrane
rafts in a Rac1-dependent manner. This localization is required for
Akt activation by G-protein-coupled receptors (GPCRs). Thus,
genetic targeting of a Rac1 binding-deficient allele of p110.beta.
to rafts alleviated the requirement for p110.beta.-Rac1 association
for GPCR signaling, cell growth and migration. In contrast, p1 l0a,
which does not play a role in physiological GPCR signaling, is
found to reside exclusively in nonraft regions of the plasma
membrane. However it was shown that raft targeting of p110.alpha.
by fusing a raft targeting motif from c-Lyn allows GPCR mediated
activation of Akt by p110.alpha.. The discovery that Rac plays a
key role in p110.beta. activation via localization in lipid rafts
led to a question about Rac's role in PTEN-null tumors. Notably,
Rac interacts exclusively with p110.beta. but not with p110a
(Fritsch et al. (2013) Cell 153(5):1050-63). p110a is known to
interact instead with Ras (Rodriguez-Viciana et al. (1994) Nature
370: 527-32; Rodriguez-Viciana et al. (1996) EMBO J. 15:2442-51).
Thus, Rac activation is a necessary prelude to p110.beta.
activation, making Rac an upstream activator of p110.beta..
However, Rac family members are unique in that their activators,
the Rac GEFs, are known to be activated by the phosphoinositide
products of PI3Ks. Thus, Rac is also a downstream effector of
PI3Ks. Hence, the interactions of Rac and p110.beta. constitute the
very definition of a positive feedback loop. In the absence of
PTEN, there is nothing to keep this positive feedback loop in
check--a classic vicious cycle. This means that Rac inhibitors
might be very useful in PTEN null tumors (see below) and might be
ideal combination partners with p110.beta. inhibitors. This has
been proven and recently published (Yuzugullu et al. (2015) Nat
Commun. 6:8501), diagrammed in FIG. 15.
[0443] Notably, p110.beta.-dependent, PTEN null tumor cells
critically also rely upon the integrity of their lipid rafts. As
discussed below, lowering cholesterol can block both PI3K signaling
and cell growth both in tissue culture and in tumors in vivo.
Collectively, these findings provide a mechanistic account of how
membrane raft localization regulates differential activation of
distinct PI3K isoforms and offer insight into therapeutic
approaches to complement p110b inhibition in PTEN null tumors.
[0444] Step 2 Activation of p110.beta.
[0445] The data on the Rac/p110.beta. feedback loop described above
(see Yuzugullu et al. (2015) Nat Commun. 6.8501 for details) leaves
open a big question: how the feedback loop is activated in the
first place. While, in theory, p100.beta. could be activated via
its interactions with G.sub..beta..gamma. subunits of GPCRs, the
current data suggest this is generally not the case. There must be
something more to the activation process in the absence of PTEN.
Novel p110.beta. binding proteins is believed to solve the
mechanism. Using taptag technology, CRKL was identified as a novel
PI3K.beta. interacting protein. Silencing endogenous CRKL
expression in PTEN null human cancer cells led to a decrease in
p110.beta.-dependent PI3K signaling and cell proliferation. In
contrast, CRKL depletion did not impair p110.alpha.-mediated
signaling or growth. In addition, CRKL in turn binds to
tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. FAK/SRC
family kinases have already been shown both to be negatively
regulated by PTEN and to phosphorylate and activate p130Cas (Zhang
et al. (2017) Cell Reports 20:1-9). Notably, both p130 CAS and CRKL
also bind the very GEFs which activate Rac (Zhang et al. (2017),
supra). These GEFs are in turn activated by the 3'phosphorylated
phosphoinositides produced by p110.beta.. This leads to the model
shown in FIG. 11.
[0446] This model makes testable predictions. In this model, the
PTEN loss leads to the activation of a number of signaling
molecules in addition to PI3K. Two of these molecules, SRC and the
MEK/ERK, are known to render cells resistant to PI3K inhibition
(Cheng et al. (2016) Oncogene. 35:2961-2970). Further, inhibiting
these pathways could synergize with PI3K inhibitors. As shown
below, both predictions are believed to be true.
[0447] Pre-Clinical Testing of p110.beta. Inhibitors in Combination
with Compounds Targeting Candidates Discovered in Mechanistic
Studies
[0448] As targeted therapies are rarely effective as single agents,
novel combinations of targeted therapies were investigated.
Although inhibitor combinations that are successful in mice are
often too toxic in clinical trials, p110.beta. inhibitors are
proving to have much lower on-target toxicity than pan-PI3K or
p110a inhibitors. This is, in part, because p110.beta. plays a
lesser role in insulin action than p110a (Hill et al. (2009)
Endocrinology 150:4874-4882, Sopasakis et al. (2010) Cell
Metabolism 11:220-230; Xu et al. (2010) Cell Metabolism 12:88-95).
Indeed, it is possible to have a healthy adult mouse with no
p110.beta. activity in any tissue in its body. For example, the
tissue-specific p110.beta. deletion in the bone marrow (Gritsman et
al. (2014) J Clin Invest. 124:1794-1809; Yuzugullu et al. (2015)
Nat Commun. 6:8501), liver (Sopasakis et al. (2010) Cell
Metabolism. 11:220-230), mammary gland (Utermark et al. (2012)
Genes & Development. 26:1573-1586), ovary (Schmit et al. (2014)
Proc Natl Acad Sci USA. 111:6395-6400) or prostate (Jia et al.
(2008) Nature 454:776-779) had little effect on the targeted tissue
or the whole animal. Thus, efficacious combinations comprising
p110.beta. inhibitors are believed to be possible treatment
options. The following examples illustrate identification of
inhibitors targeting p110.beta. localization, as well as its
activation mode and/or novel downstream targets. In addition,
clinical samples are used to test involvement of this pathway in
human tumors (as outlined below). Established protocols are used,
including animal studies and organoid tests, using various organoid
models.
[0449] Targeting p110.beta. Localization
[0450] There are a large number of possible inhibitor combinations
targeting p110.beta. localization. Preliminary data indicated that
inhibitors of the interaction between Rac and its GEFs effectively
block the growth of PTEN-null human tumor cell lines and
genetically engineered mouse (GEM) tumor models (FIG. 16; Yuzugullu
et al. (2015) Nat Commun. 6:8501). However, lipid raft integrity
was also required for the growth of PTEN null tumors (FIG. 17). It
has been already found that statins are effective in vitro in a
number of models (FIG. 17 and Cizmecioglu et al. (2016) eLIFE
5:e17635). Furthermore, the preliminary data showed that statins
are effective in PTEN-null tumor models, in agreement with existing
literature (Platz et al. (2006) J Natl Cancer Inst.
98(24):1819-25). Effective in vitro genetic controls are used to
demonstrate that the effects of statins are actually occurring via
p110.beta. (FIG. 17 and Cizmecioglu et al. (2016) eLIFE 5:e17635).
Since statins can only achieve relatively small reductions in
cholesterol levels, other agents are also tested, including LXR
agonists as well as antibodies targeting PCSK9, which can lower
cholesterol to a much greater extent. In addition, prevention
models will be examined, particularly in prostate cancer
models.
[0451] Targeting p110.beta. Activation and Downstream Signaling
[0452] It has been shown that in some cases the mechanism of
activation of p110.beta. via PTEN loss involves Src family members.
Notably, published data showed that Src family kinases are
activated by PTEN loss (Dey et al. (2008) Cancer Res.
68(6):1862-71). Src inhibitors, such as dasatinib, synergize with
p110.beta. inhibition in vitro. The same is true for inhibitors of
PAK and MEK kinases that are activated downstream from Rac. In vivo
validation of such inhibitors will be done accordingly.
Combinations of PI3K or Akt inhibitors with MEK inhibitors have
been poorly tolerated in clinical studies. However, since neither
p110.beta. nor Src inhibition nor cholesterol lowering is
intrinsically toxic, further combinations may also be tolerated and
be more effective. Optimal dosing schedules will be tested and
found with similar protocols.
[0453] Testing of Immunotherapies Plus p110.beta. Inhibitors
[0454] Immuno-oncology represents a huge opportunity in cancer
therapy. At this point in time several so-called "checkpoint
inhibitors" have been approved for melanoma and lung cancers. They
also showing promise in prostate breast and brain cancers (e.g.,
Klein et al. (2015) J. Immunol. 194:213.10). The beauty of this
inhibitor class is that it offers much longer remissions than
targeted inhibitors have usually been able to achieve. However,
only a fraction of a given type of tumors respond. This is the
opposite of targeted therapies that give broad responses in tumors
of the correct genotype, but for which responses are usually of
limited duration. Thus, current research is aimed at combining the
best of both therapies to achieve broad and lasting tumor responses
in susceptible tumor types. Notably, PTEN loss has been suggested
to be a mode of resistance to checkpoint inhibitors, which may
explain why prostate tumors have responded to ICB less well than
melanomas. One worry with targeted therapies is that they might
adversely alter immune function. However, it was shown that
knocking out p110.beta. in hematopoietic stem cells had no effect
on the immune system. Moreover, since PTEN loss appears to
frequently function via p110.beta. inhibiting, p110.beta. is
expected to increase sensitivity to checkpoint inhibition. As
described above, promising data have been built up on the effects
of combining p110b knockout and inhibition with anti-PD1 in a
PTEN/P53 (PP) knockout GEMN model of triple negative beast cancer.
As shown in FIG. 5, PP TNBC tumors grow at a faster rate in
immunocompromised mice (athymic nude) than in syngeneic
immunocompetent mice (FVB strain). Signaling through
PI3K-p110.beta. is critical to mediate immune evasion, as PP TNBC
tumors with additional deletion of p110.beta.(PPB TNBC) fail to
grow in immunocompetent mice, but do form tumors in immunodeficient
mice (FIG. 18) A PTEN null/Erg allograft model of prostate cancer
is generated to test these results in prostate cancer models.
[0455] The optimal timing of the administration of the two
therapies (current theory suggests treating tumors with targeted
therapy first to elicit tumor cell killing, which generates innate
immune responses and primes later adaptive immune therapy) is
tested. For example, different sequences of, and/or timings
between, combinational therapies are to be tested. The most
effective sequence(s)/timing(s) of combination therapies are
arrived at by comparing the therapeutic results of each tested
therapy. Similarly, the effectiveness of p110.beta. inhibitors with
other immunotherapies, such as anti-PDL1/2, is also tested. Other
drugs targeting p110.beta. localization, activation, and signaling
discussed above, in combination with immunotherapies, are tested
similarly. Moreover, the effect of increasing the burden of
"neo-antigens" is also tested in the same models. For example, the
mutator alleles of DNA polymerase s, Pole (P286R) are exogenously
expressed (Kane et al. (2014) Cancer Res. 74(7):1895-901; Shinbrot
er al. (2014) Genome Res. 24(11):1740-50) to generate matched
tumors in which the numbers of neoantigens ranges from quite low to
very high. As described herein, multiple approaches are to be used
to maximize the therapeutic efficacy of combining immunotherapies
with PI3Kbeta-directed directed therapies in the very large class
of PTEN deficient human tumor.
[0456] Clinical Trial of a p110.beta. Inhibitor in Combination with
AR Targeted Therapy
[0457] An approved clinical trial for the SPORE project is ongoing.
Organoid and slice cultures are to be established from the trial.
Samples from the trial and existing samples are used to test
mechanistic hypotheses and to validate preclinical discovery of
combinational therapies. To measure pathway activation in pathology
samples, multiplexed methods for pathway analysis are used in both
frest tissue and fixed samples from previous trial. For example,
validation of mechanisms underlying any effects of PI3K pathway
activation on responses to checkpoint blockade, seen in preclinical
studies, is to be performed with trial samples. This work forms the
basis for a second trial combining p110.beta. inhibition with
checkpoint blockade. In addition to testing the effects of
p110.beta. inhibition on the tumors visibility to the immune
system, the effects of inhibitors on the immune system itself are
also to be tested. Pre-clinical studies showed that knockout of
p110.beta. has little effect on immune system function. However,
after testing clinical candidate p110.beta. inhibitors, the
inhibition may be effective in vivo.
INCORPORATION BY REFERENCE
[0458] All publications, patents, and patent applications mentioned
herein are hereby incorporated by reference in their entirety as if
each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference. In case of conflict, the present application, including
any definitions herein, will control.
[0459] Also incorporated by reference in their entirety are any
polynucleotide and polypeptide sequences which reference an
accession number correlating to an entry in a public database, such
as those maintained by The Institute for Genomic Research (TIGR) on
the world wide web at tigr.org and/or the National Center for
Biotechnology Information (NCBI) on the World Wide Web at
ncbi.nlm.nih.gov.
EQUIVALENTS
[0460] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
5413207DNAHomo sapiens 1atgcctccac gaccatcatc aggtgaactg tggggcatcc
acttgatgcc cccaagaatc 60ctagtagaat gtttactacc aaatggaatg atagtgactt
tagaatgcct ccgtgaggct 120acattaataa ccataaagca tgaactattt
aaagaagcaa gaaaataccc cctccatcaa 180cttcttcaag atgaatcttc
ttacattttc gtaagtgtta ctcaagaagc agaaagggaa 240gaattttttg
atgaaacaag acgactttgt gaccttcggc tttttcaacc ctttttaaaa
300gtaattgaac cagtaggcaa ccgtgaagaa aagatcctca atcgagaaat
tggttttgct 360atcggcatgc cagtgtgtga atttgatatg gttaaagatc
cagaagtaca ggacttccga 420agaaatattc tgaacgtttg taaagaagct
gtggatctta gggacctcaa ttcacctcat 480agtagagcaa tgtatgtcta
tcctccaaat gtagaatctt caccagaatt gccaaagcac 540atatataata
aattagataa agggcaaata atagtggtga tctgggtaat agtttctcca
600aataatgaca agcagaagta tactctgaaa atcaaccatg actgtgtacc
agaacaagta 660attgctgaag caatcaggaa aaaaactcga agtatgttgc
tatcctctga acaactaaaa 720ctctgtgttt tagaatatca gggcaagtat
attttaaaag tgtgtggatg tgatgaatac 780ttcctagaaa aatatcctct
gagtcagtat aagtatataa gaagctgtat aatgcttggg 840aggatgccca
atttgatgtt gatggctaaa gaaagccttt attctcaact gccaatggac
900tgttttacaa tgccatctta ttccagacgc atttccacag ctacaccata
tatgaatgga 960gaaacatcta caaaatccct ttgggttata aatagtgcac
tcagaataaa aattctttgt 1020gcaacctacg tgaatgtaaa tattcgagac
attgataaga tctatgttcg aacaggtatc 1080taccatggag gagaaccctt
atgtgacaat gtgaacactc aaagagtacc ttgttccaat 1140cccaggtgga
atgaatggct gaattatgat atatacattc ctgatcttcc tcgtgctgct
1200cgactttgcc tttccatttg ctctgttaaa ggccgaaagg gtgctaaaga
ggaacactgt 1260ccattggcat ggggaaatat aaacttgttt gattacacag
acactctagt atctggaaaa 1320atggctttga atctttggcc agtacctcat
ggattagaag atttgctgaa ccctattggt 1380gttactggat caaatccaaa
taaagaaact ccatgcttag agttggagtt tgactggttc 1440agcagtgtgg
taaagttccc agatatgtca gtgattgaag agcatgccaa ttggtctgta
1500tcccgagaag caggatttag ctattcccac gcaggactga gtaacagact
agctagagac 1560aatgaattaa gggaaaatga caaagaacag ctcaaagcaa
tttctacacg agatcctctc 1620tctgaaatca ctgagcagga gaaagatttt
ctatggagtc acagacacta ttgtgtaact 1680atccccgaaa ttctacccaa
attgcttctg tctgttaaat ggaattctag agatgaagta 1740gcccagatgt
attgcttggt aaaagattgg cctccaatca aacctgaaca ggctatggaa
1800cttctggact gtaattaccc agatcctatg gttcgaggtt ttgctgttcg
gtgcttggaa 1860aaatatttaa cagatgacaa actttctcag tatttaattc
agctagtaca ggtcctaaaa 1920tatgaacaat atttggataa cttgcttgtg
agatttttac tgaagaaagc attgactaat 1980caaaggattg ggcacttttt
cttttggcat ttaaaatctg agatgcacaa taaaacagtt 2040agccagaggt
ttggcctgct tttggagtcc tattgtcgtg catgtgggat gtatttgaag
2100cacctgaata ggcaagtcga ggcaatggaa aagctcatta acttaactga
cattctcaaa 2160caggagaaga aggatgaaac acaaaaggta cagatgaagt
ttttagttga gcaaatgagg 2220cgaccagatt tcatggatgc tctacagggc
tttctgtctc ctctaaaccc tgctcatcaa 2280ctaggaaacc tcaggcttga
agagtgtcga attatgtcct ctgcaaaaag gccactgtgg 2340ttgaattggg
agaacccaga catcatgtca gagttactgt ttcagaacaa tgagatcatc
2400tttaaaaatg gggatgattt acggcaagat atgctaacac ttcaaattat
tcgtattatg 2460gaaaatatct ggcaaaatca aggtcttgat cttcgaatgt
taccttatgg ttgtctgtca 2520atcggtgact gtgtgggact tattgaggtg
gtgcgaaatt ctcacactat tatgcaaatt 2580cagtgcaaag gcggcttgaa
aggtgcactg cagttcaaca gccacacact acatcagtgg 2640ctcaaagaca
agaacaaagg agaaatatat gatgcagcca ttgacctgtt tacacgttca
2700tgtgctggat actgtgtagc taccttcatt ttgggaattg gagatcgtca
caatagtaac 2760atcatggtga aagacgatgg acaactgttt catatagatt
ttggacactt tttggatcac 2820aagaagaaaa aatttggtta taaacgagaa
cgtgtgccat ttgttttgac acaggatttc 2880ttaatagtga ttagtaaagg
agcccaagaa tgcacaaaga caagagaatt tgagaggttt 2940caggagatgt
gttacaaggc ttatctagct attcgacagc atgccaatct cttcataaat
3000cttttctcaa tgatgcttgg ctctggaatg ccagaactac aatcttttga
tgacattgca 3060tacattcgaa agaccctagc cttagataaa actgagcaag
aggctttgga gtatttcatg 3120aaacaaatga atgatgcaca tcatggtggc
tggacaacaa aaatggattg gatcttccac 3180acaattaaac agcatgcatt gaactga
320721068PRTHomo sapiens 2Met Pro Pro Arg Pro Ser Ser Gly Glu Leu
Trp Gly Ile His Leu Met1 5 10 15Pro Pro Arg Ile Leu Val Glu Cys Leu
Leu Pro Asn Gly Met Ile Val 20 25 30Thr Leu Glu Cys Leu Arg Glu Ala
Thr Leu Ile Thr Ile Lys His Glu 35 40 45Leu Phe Lys Glu Ala Arg Lys
Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60Glu Ser Ser Tyr Ile Phe
Val Ser Val Thr Gln Glu Ala Glu Arg Glu65 70 75 80Glu Phe Phe Asp
Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95Pro Phe Leu
Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110Leu
Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120
125Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu
130 135 140Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser
Pro His145 150 155 160Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val
Glu Ser Ser Pro Glu 165 170 175Leu Pro Lys His Ile Tyr Asn Lys Leu
Asp Lys Gly Gln Ile Ile Val 180 185 190Val Ile Trp Val Ile Val Ser
Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205Leu Lys Ile Asn His
Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220Ile Arg Lys
Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys225 230 235
240Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly
245 250 255Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr
Lys Tyr 260 265 270Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn
Leu Met Leu Met 275 280 285Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro
Met Asp Cys Phe Thr Met 290 295 300Pro Ser Tyr Ser Arg Arg Ile Ser
Thr Ala Thr Pro Tyr Met Asn Gly305 310 315 320Glu Thr Ser Thr Lys
Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335Lys Ile Leu
Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350Lys
Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360
365Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn
370 375 380Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg
Ala Ala385 390 395 400Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly
Arg Lys Gly Ala Lys 405 410 415Glu Glu His Cys Pro Leu Ala Trp Gly
Asn Ile Asn Leu Phe Asp Tyr 420 425 430Thr Asp Thr Leu Val Ser Gly
Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445Pro His Gly Leu Glu
Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460Asn Pro Asn
Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe465 470 475
480Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala
485 490 495Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His
Ala Gly 500 505 510Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg
Glu Asn Asp Lys 515 520 525Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp
Pro Leu Ser Glu Ile Thr 530 535 540Glu Gln Glu Lys Asp Phe Leu Trp
Ser His Arg His Tyr Cys Val Thr545 550 555 560Ile Pro Glu Ile Leu
Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575Arg Asp Glu
Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590Ile
Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600
605Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr
610 615 620Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val
Leu Lys625 630 635 640Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg
Phe Leu Leu Lys Lys 645 650 655Ala Leu Thr Asn Gln Arg Ile Gly His
Phe Phe Phe Trp His Leu Lys 660 665 670Ser Glu Met His Asn Lys Thr
Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685Glu Ser Tyr Cys Arg
Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700Gln Val Glu
Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys705 710 715
720Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val
725 730 735Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly
Phe Leu 740 745 750Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu
Arg Leu Glu Glu 755 760 765Cys Arg Ile Met Ser Ser Ala Lys Arg Pro
Leu Trp Leu Asn Trp Glu 770 775 780Asn Pro Asp Ile Met Ser Glu Leu
Leu Phe Gln Asn Asn Glu Ile Ile785 790 795 800Phe Lys Asn Gly Asp
Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815Ile Arg Ile
Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830Met
Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840
845Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly
850 855 860Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His
Gln Trp865 870 875 880Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp
Ala Ala Ile Asp Leu 885 890 895Phe Thr Arg Ser Cys Ala Gly Tyr Cys
Val Ala Thr Phe Ile Leu Gly 900 905 910Ile Gly Asp Arg His Asn Ser
Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925Leu Phe His Ile Asp
Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940Phe Gly Tyr
Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe945 950 955
960Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu
965 970 975Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala
Ile Arg 980 985 990Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met
Met Leu Gly Ser 995 1000 1005Gly Met Pro Glu Leu Gln Ser Phe Asp
Asp Ile Ala Tyr Ile Arg 1010 1015 1020Lys Thr Leu Ala Leu Asp Lys
Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035Phe Met Lys Gln Met
Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050Lys Met Asp
Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060
106533207DNAMus musculus 3atgcctccac gaccatcttc gggtgaactg
tggggcatcc acttgatgcc cccacgaatc 60ctagtggaat gtttactccc caatggaatg
atagtgactt tagaatgcct ccgtgaggcc 120acactcgtca ccatcaaaca
tgaactgttc agagaggcca ggaaataccc tctccatcag 180cttctgcaag
acgaaacttc ttacattttc gtaagtgtca cccaagaagc agaaagggaa
240gaattttttg atgaaacaag acgactttgt gaccttcggc tttttcaacc
ctttttaaaa 300gttattgaac cagtaggcaa ccgtgaagaa aagatcctca
atcgagaaat tggttttgtt 360attggcatgc cagtgtgtga atttgatatg
gttaaagatc cagaagtcca agactttcga 420aggaacattc tgaatgtttg
caaagaagct gtggacctgc gggatctcaa ctcgcctcat 480agcagagcaa
tgtatgtcta ccctccaaat gtcgagtctt ccccagaact gccaaagcac
540atctacaaca agttagataa aggacaaatc atagtggtga tttgggtaat
agtctctcca 600aacaacgaca agcagaagta cactctgaag atcaatcatg
actgtgtgcc agagcaagtc 660attgctgaag ccatcaggaa aaagactcgg
agcatgttgt tgtcctctga gcagctgaaa 720ctctgtgtct tagaatatca
gggcaagtat attctgaaag tgtgtggctg tgacgaatac 780ttcctggaaa
agtaccctct gagtcagtac aagtacataa gaagctgtat aatgctgggg
840aggatgccca acttgatgct gatggccaaa gaaagcctat actctcagct
gccgattgat 900agcttcacca tgccgtcata ctccaggcgc atctccacag
ccacacccta catgaatgga 960gagacatcta cgaaatccct ctgggtcata
aatagtgcgc tcagaataaa aattctttgt 1020gcaacctatg taaatgtaaa
tattcgagac attgataaga tctatgttcg aacaggtatc 1080taccatggag
gagaaccctt atgtgacaat gtgaacactc aaagagtacc ttgttccaat
1140cctaggtgga atgaatggct gaattatgat atatacattc ctgatcttcc
tcgtgctgcg 1200cgcctttgcc tttcaatctg ctctgttaaa ggccgaaagg
gtgctaagga ggagcactgt 1260ccgttggcct ggggaaacat aaacttgttt
gattatacag acaccctagt gtccgggaaa 1320atggctttga atctctggcc
tgtaccgcat gggttagaag atctgctgaa ccctattggt 1380gttactgggt
caaatccaaa taaagaaact ccatgcttag agttggagtt tgattggttc
1440agcagtgtgg tgaagtttcc agacatgtct gtgatcgaag aacatgccaa
ttggtccgtg 1500tcccgagaag ctggattcag ttactcccat acaggactga
gtaacagact agccagagac 1560aatgagttaa gagaaaatga caaggaacag
ctccgagcac tttgcacccg ggacccacta 1620tctgaaatca ctgaacaaga
gaaagacttc ctatggagcc acagacacta ctgcgtaact 1680attcctgaaa
tcctacccaa attgcttctg tctgtcaagt ggaattccag agacgaagtg
1740gcccagatgt actgcttagt aaaagattgg cctccaatca aaccagagca
agccatggaa 1800ctcctggact gtaactatcc agatcctatg gttcggagtt
ttgctgttcg gtgcttagaa 1860aaatatttaa cagatgacaa actttctcag
tacctcattc aacttgtaca ggtcttaaaa 1920tatgaacagt atttggataa
cctgcttgtg agatttttac tcaagaaagc attgacaaat 1980caaaggattg
gccatttttt cttttggcat ttaaaatctg agatgcacaa taagactgtc
2040agtcagaggt ttggcctgct attggagtcc tactgccgtg cctgtgggat
gtatctgaag 2100cacctgaaca gacaagtaga ggccatggag aagctcatca
acctaacgga catccttaag 2160caggagaaga aggatgagac acaaaaggta
cagatgaagt ttttggttga acagatgaga 2220cagccagact tcatggatgc
tttgcagggt tttctgtccc ctctgaatcc tgctcaccaa 2280ctaggaaacc
tcaggcttga agagtgtcga attatgtcct ctgcaaaaag gccactgtgg
2340ttgaattggg agaacccaga catcatgtca gagctactgt ttcagaacaa
tgagatcatc 2400tttaaaaatg gcgacgactt acggcaagat atgttaaccc
ttcagatcat ccgaatcatg 2460gagaacatct ggcaaaacca aggccttgac
cttcgcatgc taccttatgg ctgtctatcc 2520attggggact gtgtgggtct
catcgaggtg gtgagaaact ctcacaccat catgcaaatc 2580cagtgcaaag
gaggcctgaa gggggcgctg cagttcaaca gccacacact gcatcaatgg
2640ctcaaggaca agaacaaggg cgagatatat gatgcagcca ttgacctgtt
cactcggtcc 2700tgcgctgggt actgcgtggc aacctttatc ttgggaattg
gagaccggca caacagcaac 2760atcatggtga aagatgacgg acagctgttt
catatagatt ttgggcactt tttggatcac 2820aagaagaaaa aatttggcta
taagcgggaa cgtgtgccat ttgtgttgac acaggatttc 2880ttgattgtga
ttagtaaggg agcacaagag tacaccaaga ccagagagtt tgagaggttt
2940caggagatgt gttacaaggc ttacctagca attcggcagc atgccaatct
cttcatcaac 3000cttttttcaa tgatgcttgg ctctggaatg ccagaactac
aatcttttga tgacattgca 3060tatatccgaa agactctagc cttggacaaa
actgagcaag aagctttgga atatttcaca 3120aagcaaatga atgatgcaca
tcatggtgga tggacgacaa aaatggattg gatcttccac 3180accatcaagc
agcatgcttt gaactga 320741068PRTMus musculus 4Met Pro Pro Arg Pro
Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met1 5 10 15Pro Pro Arg Ile
Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30Thr Leu Glu
Cys Leu Arg Glu Ala Thr Leu Val Thr Ile Lys His Glu 35 40 45Leu Phe
Arg Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60Glu
Thr Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu65 70 75
80Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln
85 90 95Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys
Ile 100 105 110Leu Asn Arg Glu Ile Gly Phe Val Ile Gly Met Pro Val
Cys Glu Phe 115 120 125Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe
Arg Arg Asn Ile Leu 130 135 140Asn Val Cys Lys Glu Ala Val Asp Leu
Arg Asp Leu Asn Ser Pro His145 150 155 160Ser Arg Ala Met Tyr Val
Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175Leu Pro Lys His
Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190Val Ile
Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200
205Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala
210 215 220Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln
Leu Lys225 230 235 240Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile
Leu Lys Val Cys Gly 245 250 255Cys Asp Glu Tyr Phe Leu Glu Lys Tyr
Pro Leu Ser Gln Tyr Lys Tyr 260 265 270Ile Arg Ser Cys Ile Met Leu
Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285Ala Lys Glu Ser Leu
Tyr Ser Gln Leu Pro Ile Asp Ser Phe Thr Met 290 295 300Pro Ser Tyr
Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly305 310 315
320Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg
Ile
325 330 335Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp
Ile Asp 340 345 350Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly
Glu Pro Leu Cys 355 360 365Asp Asn Val Asn Thr Gln Arg Val Pro Cys
Ser Asn Pro Arg Trp Asn 370 375 380Glu Trp Leu Asn Tyr Asp Ile Tyr
Ile Pro Asp Leu Pro Arg Ala Ala385 390 395 400Arg Leu Cys Leu Ser
Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415Glu Glu His
Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430Thr
Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440
445Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser
450 455 460Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp
Trp Phe465 470 475 480Ser Ser Val Val Lys Phe Pro Asp Met Ser Val
Ile Glu Glu His Ala 485 490 495Asn Trp Ser Val Ser Arg Glu Ala Gly
Phe Ser Tyr Ser His Thr Gly 500 505 510Leu Ser Asn Arg Leu Ala Arg
Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525Glu Gln Leu Arg Ala
Leu Cys Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535 540Glu Gln Glu
Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr545 550 555
560Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser
565 570 575Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp
Pro Pro 580 585 590Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys
Asn Tyr Pro Asp 595 600 605Pro Met Val Arg Ser Phe Ala Val Arg Cys
Leu Glu Lys Tyr Leu Thr 610 615 620Asp Asp Lys Leu Ser Gln Tyr Leu
Ile Gln Leu Val Gln Val Leu Lys625 630 635 640Tyr Glu Gln Tyr Leu
Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655Ala Leu Thr
Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670Ser
Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680
685Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg
690 695 700Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile
Leu Lys705 710 715 720Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln
Met Lys Phe Leu Val 725 730 735Glu Gln Met Arg Gln Pro Asp Phe Met
Asp Ala Leu Gln Gly Phe Leu 740 745 750Ser Pro Leu Asn Pro Ala His
Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765Cys Arg Ile Met Ser
Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780Asn Pro Asp
Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile785 790 795
800Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile
805 810 815Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp
Leu Arg 820 825 830Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys
Val Gly Leu Ile 835 840 845Glu Val Val Arg Asn Ser His Thr Ile Met
Gln Ile Gln Cys Lys Gly 850 855 860Gly Leu Lys Gly Ala Leu Gln Phe
Asn Ser His Thr Leu His Gln Trp865 870 875 880Leu Lys Asp Lys Asn
Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895Phe Thr Arg
Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910Ile
Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920
925Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys
930 935 940Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln
Asp Phe945 950 955 960Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Tyr
Thr Lys Thr Arg Glu 965 970 975Phe Glu Arg Phe Gln Glu Met Cys Tyr
Lys Ala Tyr Leu Ala Ile Arg 980 985 990Gln His Ala Asn Leu Phe Ile
Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005Gly Met Pro Glu
Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020Lys Thr
Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030
1035Phe Thr Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr
1040 1045 1050Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala
Leu Asn 1055 1060 106553207DNAMus musculus 5atgcctccac gaccatcttc
gggtgaactg tggggcatcc acttgatgcc cccacgaatc 60ctagtggaat gtttactccc
caatggaatg atagtgactt tagaatgcct ccgtgaggcc 120acactcgtca
ccatcaaaca tgaactgttc agagaggcca ggaaataccc tctccatcag
180cttctgcaag acgaaacttc ttacattttc gtaagtgtca cccaagaagc
agaaagggaa 240gaattttttg atgaaacaag acgactttgt gaccttcggc
tttttcaacc ctttttaaaa 300gttattgaac cagtaggcaa ccgtgaagaa
aagatcctca atcgagaaat tggttttgtt 360attggcatgc cagtgtgtga
atttgatatg gttaaagatc cagaagtcca agactttcga 420aggaacattc
tgaatgtttg caaagaagct gtggacctgc gggatctcaa ctcgcctcat
480agcagagcaa tgtatgtcta ccctccaaat gtcgagtctt ccccagaact
gccaaagcac 540atctacaaca agttagataa aggacaaatc atagtggtga
tttgggtaat agtctctcca 600aacaacgaca agcagaagta cactctgaag
atcaatcatg actgtgtgcc agagcaagtc 660attgctgaag ccatcaggaa
aaagactcgg agcatgttgt tgtcctctga gcagctgaaa 720ctctgtgtct
tagaatatca gggcaagtat attctgaaag tgtgtggctg tgacgaatac
780ttcctggaaa agtaccctct gagtcagtac aagtacataa gaagctgtat
aatgctgggg 840aggatgccca acttgatgct gatggccaaa gaaagcctat
actctcagct gccgattgat 900agcttcacca tgccgtcata ctccaggcgc
atctccacag ccacacccta catgaatgga 960gagacatcta cgaaatccct
ctgggtcata aatagtgcgc tcagaataaa aattctttgt 1020gcaacctatg
taaatgtaaa tattcgagac attgataaga tctatgttcg aacaggtatc
1080taccatggag gagaaccctt atgtgacaat gtgaacactc aaagagtacc
ttgttccaat 1140cctaggtgga atgaatggct gaattatgat atatacattc
ctgatcttcc tcgtgctgcg 1200cgcctttgcc tttcaatctg ctctgttaaa
ggccgaaagg gtgctaagga ggagcactgt 1260ccgttggcct ggggaaacat
aaacttgttt gattatacag acaccctagt gtccgggaaa 1320atggctttga
atctctggcc tgtaccgcat gggttagaag atctgctgaa ccctattggt
1380gttactgggt caaatccaaa taaagaaact ccatgcttag agttggagtt
tgattggttc 1440agcagtgtgg tgaagtttcc agacatgtct gtgatcgaag
aacatgccaa ttggtccgtg 1500tcccgagaag ctggattcag ttactcccat
acaggactga gtaacagact agccagagac 1560aatgagttaa gagaaaatga
caaggaacag ctccgagcac tttgcacccg ggacccacta 1620tctgaaatca
ctgaacaaga gaaagacttc ctatggagcc acagacacta ctgcgtaact
1680attcctgaaa tcctacccaa attgcttctg tctgtcaagt ggaattccag
agacgaagtg 1740gcccagatgt actgcttagt aaaagattgg cctccaatca
aaccagagca agccatggaa 1800ctcctggact gtaactatcc agatcctatg
gttcggagtt ttgctgttcg gtgcttagaa 1860aaatatttaa cagatgacaa
actttctcag tacctcattc aacttgtaca ggtcttaaaa 1920tatgaacagt
atttggataa cctgcttgtg agatttttac tcaagaaagc attgacaaat
1980caaaggattg gccatttttt cttttggcat ttaaaatctg agatgcacaa
taagactgtc 2040agtcagaggt ttggcctgct attggagtcc tactgccgtg
cctgtgggat gtatctgaag 2100cacctgaaca gacaagtaga ggccatggag
aagctcatca acctaacgga catccttaag 2160caggagaaga aggatgagac
acaaaaggta cagatgaagt ttttggttga acagatgaga 2220cagccagact
tcatggatgc tttgcagggt tttctgtccc ctctgaatcc tgctcaccaa
2280ctaggaaacc tcaggcttga agagtgtcga attatgtcct ctgcaaaaag
gccactgtgg 2340ttgaattggg agaacccaga catcatgtca gagctactgt
ttcagaacaa tgagatcatc 2400tttaaaaatg gcgacgactt acggcaagat
atgttaaccc ttcagatcat ccgaatcatg 2460gagaacatct ggcaaaacca
aggccttgac cttcgcatgc taccttatgg ctgtctatcc 2520attggggact
gtgtgggtct catcgaggtg gtgagaaact ctcacaccat catgcaaatc
2580cagtgcaaag gaggcctgaa gggggcgctg cagttcaaca gccacacact
gcatcaatgg 2640ctcaaggaca agaacaaggg cgagatatat gatgcagcca
ttgacctgtt cactcggtcc 2700tgcgctgggt actgcgtggc aacctttatc
ttgggaattg gagaccggca caacagcaac 2760atcatggtga aagatgacgg
acagctgttt catatagatt ttgggcactt tttggatcac 2820aagaagaaaa
aatttggcta taagcgggaa cgtgtgccat ttgtgttgac acaggatttc
2880ttgattgtga ttagtaaggg agcacaagag tacaccaaga ccagagagtt
tgagaggttt 2940caggagatgt gttacaaggc ttacctagca attcggcagc
atgccaatct cttcatcaac 3000cttttttcaa tgatgcttgg ctctggaatg
ccagaactac aatcttttga tgacattgca 3060tatatccgaa agactctagc
cttggacaaa actgagcaag aagctttgga atatttcaca 3120aagcaaatga
atgatgcaca tcatggtgga tggacgacaa aaatggattg gatcttccac
3180accatcaagc agcatgcttt gaactga 320761068PRTMus musculus 6Met Pro
Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met1 5 10 15Pro
Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25
30Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Val Thr Ile Lys His Glu
35 40 45Leu Phe Arg Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln
Asp 50 55 60Glu Thr Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu
Arg Glu65 70 75 80Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu
Arg Leu Phe Gln 85 90 95Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn
Arg Glu Glu Lys Ile 100 105 110Leu Asn Arg Glu Ile Gly Phe Val Ile
Gly Met Pro Val Cys Glu Phe 115 120 125Asp Met Val Lys Asp Pro Glu
Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135 140Asn Val Cys Lys Glu
Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His145 150 155 160Ser Arg
Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170
175Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val
180 185 190Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys
Tyr Thr 195 200 205Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val
Ile Ala Glu Ala 210 215 220Ile Arg Lys Lys Thr Arg Ser Met Leu Leu
Ser Ser Glu Gln Leu Lys225 230 235 240Leu Cys Val Leu Glu Tyr Gln
Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255Cys Asp Glu Tyr Phe
Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270Ile Arg Ser
Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285Ala
Lys Glu Ser Leu Tyr Ser Gln Leu Pro Ile Asp Ser Phe Thr Met 290 295
300Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn
Gly305 310 315 320Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser
Ala Leu Arg Ile 325 330 335Lys Ile Leu Cys Ala Thr Tyr Val Asn Val
Asn Ile Arg Asp Ile Asp 340 345 350Lys Ile Tyr Val Arg Thr Gly Ile
Tyr His Gly Gly Glu Pro Leu Cys 355 360 365Asp Asn Val Asn Thr Gln
Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380Glu Trp Leu Asn
Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala385 390 395 400Arg
Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410
415Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr
420 425 430Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp
Pro Val 435 440 445Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly
Val Thr Gly Ser 450 455 460Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu
Leu Glu Phe Asp Trp Phe465 470 475 480Ser Ser Val Val Lys Phe Pro
Asp Met Ser Val Ile Glu Glu His Ala 485 490 495Asn Trp Ser Val Ser
Arg Glu Ala Gly Phe Ser Tyr Ser His Thr Gly 500 505 510Leu Ser Asn
Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525Glu
Gln Leu Arg Ala Leu Cys Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535
540Glu Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val
Thr545 550 555 560Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val
Lys Trp Asn Ser 565 570 575Arg Asp Glu Val Ala Gln Met Tyr Cys Leu
Val Lys Asp Trp Pro Pro 580 585 590Ile Lys Pro Glu Gln Ala Met Glu
Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605Pro Met Val Arg Ser Phe
Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620Asp Asp Lys Leu
Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys625 630 635 640Tyr
Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650
655Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys
660 665 670Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu
Leu Leu 675 680 685Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys
His Leu Asn Arg 690 695 700Gln Val Glu Ala Met Glu Lys Leu Ile Asn
Leu Thr Asp Ile Leu Lys705 710 715 720Gln Glu Lys Lys Asp Glu Thr
Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735Glu Gln Met Arg Gln
Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750Ser Pro Leu
Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765Cys
Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775
780Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile
Ile785 790 795 800Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu
Thr Leu Gln Ile 805 810 815Ile Arg Ile Met Glu Asn Ile Trp Gln Asn
Gln Gly Leu Asp Leu Arg 820 825 830Met Leu Pro Tyr Gly Cys Leu Ser
Ile Gly Asp Cys Val Gly Leu Ile 835 840 845Glu Val Val Arg Asn Ser
His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860Gly Leu Lys Gly
Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp865 870 875 880Leu
Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890
895Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly
900 905 910Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp
Gly Gln 915 920 925Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His
Lys Lys Lys Lys 930 935 940Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe
Val Leu Thr Gln Asp Phe945 950 955 960Leu Ile Val Ile Ser Lys Gly
Ala Gln Glu Tyr Thr Lys Thr Arg Glu 965 970 975Phe Glu Arg Phe Gln
Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990Gln His Ala
Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000
1005Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg
1010 1015 1020Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu
Glu Tyr 1025 1030 1035Phe Thr Lys Gln Met Asn Asp Ala His His Gly
Gly Trp Thr Thr 1040 1045 1050Lys Met Asp Trp Ile Phe His Thr Ile
Lys Gln His Ala Leu Asn 1055 1060 106573213DNAHomo sapiens
7atgtgcttca gtttcataat gcctcctgct atggcagaca tccttgacat ctgggcggtg
60gattcacaga tagcatctga tggctccata cctgtggatt tccttttgcc cactgggatt
120tatatccagt tggaggtacc tcgggaagct accatttctt atattaagca
gatgttatgg 180aagcaagttc acaattaccc aatgttcaac ctccttatgg
atattgactc ctatatgttt 240gcatgtgtga atcagactgc tgtatatgag
gagcttgaag atgaaacacg aagactctgt 300gatgtcagac cttttcttcc
agttctcaaa ttagtgacaa gaagttgtga cccaggggaa 360aaattagact
caaaaattgg agtccttata ggaaaaggtc tgcatgaatt tgattccttg
420aaggatcctg aagtaaatga atttcgaaga aaaatgcgca aattcagcga
ggaaaaaatc 480ctgtcacttg tgggattgtc ttggatggac tggctaaaac
aaacatatcc accagagcat 540gaaccatcca tccctgaaaa cttagaagat
aaactttatg ggggaaagct catcgtagct 600gttcattttg aaaactgcca
ggacgtgttt agctttcaag tgtctcctaa tatgaatcct 660atcaaagtaa
atgaattggc aatccaaaaa cgtttgacta ttcatgggaa ggaagatgaa
720gttagcccct atgattatgt gttgcaagtc
agcgggagag tagaatatgt ttttggtgat 780catccactaa ttcagttcca
gtatatccgg aactgtgtga tgaacagagc cctgccccat 840tttatacttg
tggaatgctg caagatcaag aaaatgtatg aacaagaaat gattgccata
900gaggctgcca taaatcgaaa ttcatctaat cttcctcttc cattaccacc
aaagaaaaca 960cgaattattt ctcatgtttg ggaaaataac aaccctttcc
aaattgtctt ggttaaggga 1020aataaactta acacagagga aactgtaaaa
gttcatgtca gggctggtct ttttcatggt 1080actgagctcc tgtgtaaaac
catcgtaagc tcagaggtat cagggaaaaa tgatcatatt 1140tggaatgaac
cactggaatt tgatattaat atttgtgact taccaagaat ggctcgatta
1200tgttttgctg tttatgcagt tttggataaa gtaaaaacga agaaatcaac
gaaaactatt 1260aatccctcta aatatcagac catcaggaaa gctggaaaag
tgcattatcc tgtagcgtgg 1320gtaaatacga tggtttttga ctttaaagga
caattgagaa ctggagacat aatattacac 1380agctggtctt catttcctga
tgaactcgaa gaaatgttga atccaatggg aactgttcaa 1440acaaatccat
atactgaaaa tgcaacagct ttgcatgtta aatttccaga gaataaaaaa
1500caaccttatt attaccctcc cttcgataag attattgaaa aggcagctga
gattgcaagc 1560agtgatagtg ctaatgtgtc aagtcgaggt ggaaaaaagt
ttcttcctgt attgaaagaa 1620atcttggaca gggatccctt gtctcaactg
tgtgaaaatg aaatggatct tatttggact 1680ttgcgacaag actgccgaga
gattttccca caatcactgc caaaattact gctgtcaatc 1740aagtggaata
aacttgagga tgttgctcag cttcaggcgc tgcttcagat ttggcctaaa
1800ctgccccccc gggaggccct agagcttctg gatttcaact atccagacca
gtacgttcga 1860gaatatgctg taggctgcct gcgacagatg agtgatgaag
aactttctca atatctttta 1920caactggtgc aagtgttaaa atatgagcct
tttcttgatt gtgccctctc tagattccta 1980ttagaaagag cacttggtaa
tcggaggata gggcagtttc tattttggca tcttaggtca 2040gaagtgcaca
ttcctgctgt ctcagtacaa tttggtgtca tccttgaagc atactgccgg
2100ggaagtgtgg ggcacatgaa agtgctttct aagcaggttg aagcactcaa
taagttaaaa 2160actttaaata gtttaatcaa actgaatgcc gtgaagttaa
acagagccaa agggaaggag 2220gccatgcata cctgtttaaa acagagtgct
taccgggaag ccctctctga cctgcagtca 2280cccctgaacc catgtgttat
cctctcagaa ctctatgttg aaaagtgcaa atacatggat 2340tccaaaatga
agcctttgtg gctggtatac aataacaagg tatttggtga ggattcagtt
2400ggagtgattt ttaaaaatgg tgatgattta cgacaggata tgttgacact
ccaaatgttg 2460cgcttgatgg atttactctg gaaagaagct ggtttggatc
ttcggatgtt gccttatggc 2520tgtttagcaa caggagatcg ctctggcctc
attgaagttg tgagcacctc tgaaacaatt 2580gctgacattc agctgaacag
tagcaatgtg gctgctgcag cagccttcaa caaagatgcc 2640cttctgaact
ggcttaaaga atacaactct ggggatgacc tggaccgagc cattgaggaa
2700tttacactgt cctgtgctgg ctactgtgta gcttcttatg tccttgggat
tggtgacaga 2760catagtgaca acatcatggt caaaaaaact ggccagctct
tccacattga ctttggacat 2820attcttggaa atttcaaatc taagtttggc
attaaaaggg agcgagtgcc ttttattctt 2880acctatgatt tcatccatgt
cattcaacaa ggaaaaacag gaaatacaga aaagtttggc 2940cggttccgcc
agtgttgtga ggatgcatat ctgattttac gacggcatgg gaatctcttc
3000atcactctct ttgcgctgat gttgactgca gggcttcctg aactcacatc
agtcaaagat 3060atacagtatc ttaaggactc tcttgcatta gggaagagtg
aagaagaagc actcaaacag 3120tttaagcaaa aatttgatga ggcgctcagg
gaaagctgga ctactaaagt gaactggatg 3180gcccacacag ttcggaaaga
ctacagatct taa 321381070PRTHomo sapiens 8Met Cys Phe Ser Phe Ile
Met Pro Pro Ala Met Ala Asp Ile Leu Asp1 5 10 15Ile Trp Ala Val Asp
Ser Gln Ile Ala Ser Asp Gly Ser Ile Pro Val 20 25 30Asp Phe Leu Leu
Pro Thr Gly Ile Tyr Ile Gln Leu Glu Val Pro Arg 35 40 45Glu Ala Thr
Ile Ser Tyr Ile Lys Gln Met Leu Trp Lys Gln Val His 50 55 60Asn Tyr
Pro Met Phe Asn Leu Leu Met Asp Ile Asp Ser Tyr Met Phe65 70 75
80Ala Cys Val Asn Gln Thr Ala Val Tyr Glu Glu Leu Glu Asp Glu Thr
85 90 95Arg Arg Leu Cys Asp Val Arg Pro Phe Leu Pro Val Leu Lys Leu
Val 100 105 110Thr Arg Ser Cys Asp Pro Gly Glu Lys Leu Asp Ser Lys
Ile Gly Val 115 120 125Leu Ile Gly Lys Gly Leu His Glu Phe Asp Ser
Leu Lys Asp Pro Glu 130 135 140Val Asn Glu Phe Arg Arg Lys Met Arg
Lys Phe Ser Glu Glu Lys Ile145 150 155 160Leu Ser Leu Val Gly Leu
Ser Trp Met Asp Trp Leu Lys Gln Thr Tyr 165 170 175Pro Pro Glu His
Glu Pro Ser Ile Pro Glu Asn Leu Glu Asp Lys Leu 180 185 190Tyr Gly
Gly Lys Leu Ile Val Ala Val His Phe Glu Asn Cys Gln Asp 195 200
205Val Phe Ser Phe Gln Val Ser Pro Asn Met Asn Pro Ile Lys Val Asn
210 215 220Glu Leu Ala Ile Gln Lys Arg Leu Thr Ile His Gly Lys Glu
Asp Glu225 230 235 240Val Ser Pro Tyr Asp Tyr Val Leu Gln Val Ser
Gly Arg Val Glu Tyr 245 250 255Val Phe Gly Asp His Pro Leu Ile Gln
Phe Gln Tyr Ile Arg Asn Cys 260 265 270Val Met Asn Arg Ala Leu Pro
His Phe Ile Leu Val Glu Cys Cys Lys 275 280 285Ile Lys Lys Met Tyr
Glu Gln Glu Met Ile Ala Ile Glu Ala Ala Ile 290 295 300Asn Arg Asn
Ser Ser Asn Leu Pro Leu Pro Leu Pro Pro Lys Lys Thr305 310 315
320Arg Ile Ile Ser His Val Trp Glu Asn Asn Asn Pro Phe Gln Ile Val
325 330 335Leu Val Lys Gly Asn Lys Leu Asn Thr Glu Glu Thr Val Lys
Val His 340 345 350Val Arg Ala Gly Leu Phe His Gly Thr Glu Leu Leu
Cys Lys Thr Ile 355 360 365Val Ser Ser Glu Val Ser Gly Lys Asn Asp
His Ile Trp Asn Glu Pro 370 375 380Leu Glu Phe Asp Ile Asn Ile Cys
Asp Leu Pro Arg Met Ala Arg Leu385 390 395 400Cys Phe Ala Val Tyr
Ala Val Leu Asp Lys Val Lys Thr Lys Lys Ser 405 410 415Thr Lys Thr
Ile Asn Pro Ser Lys Tyr Gln Thr Ile Arg Lys Ala Gly 420 425 430Lys
Val His Tyr Pro Val Ala Trp Val Asn Thr Met Val Phe Asp Phe 435 440
445Lys Gly Gln Leu Arg Thr Gly Asp Ile Ile Leu His Ser Trp Ser Ser
450 455 460Phe Pro Asp Glu Leu Glu Glu Met Leu Asn Pro Met Gly Thr
Val Gln465 470 475 480Thr Asn Pro Tyr Thr Glu Asn Ala Thr Ala Leu
His Val Lys Phe Pro 485 490 495Glu Asn Lys Lys Gln Pro Tyr Tyr Tyr
Pro Pro Phe Asp Lys Ile Ile 500 505 510Glu Lys Ala Ala Glu Ile Ala
Ser Ser Asp Ser Ala Asn Val Ser Ser 515 520 525Arg Gly Gly Lys Lys
Phe Leu Pro Val Leu Lys Glu Ile Leu Asp Arg 530 535 540Asp Pro Leu
Ser Gln Leu Cys Glu Asn Glu Met Asp Leu Ile Trp Thr545 550 555
560Leu Arg Gln Asp Cys Arg Glu Ile Phe Pro Gln Ser Leu Pro Lys Leu
565 570 575Leu Leu Ser Ile Lys Trp Asn Lys Leu Glu Asp Val Ala Gln
Leu Gln 580 585 590Ala Leu Leu Gln Ile Trp Pro Lys Leu Pro Pro Arg
Glu Ala Leu Glu 595 600 605Leu Leu Asp Phe Asn Tyr Pro Asp Gln Tyr
Val Arg Glu Tyr Ala Val 610 615 620Gly Cys Leu Arg Gln Met Ser Asp
Glu Glu Leu Ser Gln Tyr Leu Leu625 630 635 640Gln Leu Val Gln Val
Leu Lys Tyr Glu Pro Phe Leu Asp Cys Ala Leu 645 650 655Ser Arg Phe
Leu Leu Glu Arg Ala Leu Gly Asn Arg Arg Ile Gly Gln 660 665 670Phe
Leu Phe Trp His Leu Arg Ser Glu Val His Ile Pro Ala Val Ser 675 680
685Val Gln Phe Gly Val Ile Leu Glu Ala Tyr Cys Arg Gly Ser Val Gly
690 695 700His Met Lys Val Leu Ser Lys Gln Val Glu Ala Leu Asn Lys
Leu Lys705 710 715 720Thr Leu Asn Ser Leu Ile Lys Leu Asn Ala Val
Lys Leu Asn Arg Ala 725 730 735Lys Gly Lys Glu Ala Met His Thr Cys
Leu Lys Gln Ser Ala Tyr Arg 740 745 750Glu Ala Leu Ser Asp Leu Gln
Ser Pro Leu Asn Pro Cys Val Ile Leu 755 760 765Ser Glu Leu Tyr Val
Glu Lys Cys Lys Tyr Met Asp Ser Lys Met Lys 770 775 780Pro Leu Trp
Leu Val Tyr Asn Asn Lys Val Phe Gly Glu Asp Ser Val785 790 795
800Gly Val Ile Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr
805 810 815Leu Gln Met Leu Arg Leu Met Asp Leu Leu Trp Lys Glu Ala
Gly Leu 820 825 830Asp Leu Arg Met Leu Pro Tyr Gly Cys Leu Ala Thr
Gly Asp Arg Ser 835 840 845Gly Leu Ile Glu Val Val Ser Thr Ser Glu
Thr Ile Ala Asp Ile Gln 850 855 860Leu Asn Ser Ser Asn Val Ala Ala
Ala Ala Ala Phe Asn Lys Asp Ala865 870 875 880Leu Leu Asn Trp Leu
Lys Glu Tyr Asn Ser Gly Asp Asp Leu Asp Arg 885 890 895Ala Ile Glu
Glu Phe Thr Leu Ser Cys Ala Gly Tyr Cys Val Ala Ser 900 905 910Tyr
Val Leu Gly Ile Gly Asp Arg His Ser Asp Asn Ile Met Val Lys 915 920
925Lys Thr Gly Gln Leu Phe His Ile Asp Phe Gly His Ile Leu Gly Asn
930 935 940Phe Lys Ser Lys Phe Gly Ile Lys Arg Glu Arg Val Pro Phe
Ile Leu945 950 955 960Thr Tyr Asp Phe Ile His Val Ile Gln Gln Gly
Lys Thr Gly Asn Thr 965 970 975Glu Lys Phe Gly Arg Phe Arg Gln Cys
Cys Glu Asp Ala Tyr Leu Ile 980 985 990Leu Arg Arg His Gly Asn Leu
Phe Ile Thr Leu Phe Ala Leu Met Leu 995 1000 1005Thr Ala Gly Leu
Pro Glu Leu Thr Ser Val Lys Asp Ile Gln Tyr 1010 1015 1020Leu Lys
Asp Ser Leu Ala Leu Gly Lys Ser Glu Glu Glu Ala Leu 1025 1030
1035Lys Gln Phe Lys Gln Lys Phe Asp Glu Ala Leu Arg Glu Ser Trp
1040 1045 1050Thr Thr Lys Val Asn Trp Met Ala His Thr Val Arg Lys
Asp Tyr 1055 1060 1065Arg Ser 107091749DNAHomo sapiens 9atgttgaatc
caatgggaac tgttcaaaca aatccatata ctgaaaatgc aacagctttg 60catgttaaat
ttccagagaa taaaaaacaa ccttattatt accctccctt cgataagagt
120cgaggtggaa aaaagtttct tcctgtattg aaagaaatct tggacaggga
tcccttgtct 180caactgtgtg aaaatgaaat ggatcttatt tggactttgc
gacaagactg ccgagagatt 240ttcccacaat cactgccaaa attactgctg
tcaatcaagt ggaataaact tgaggatgtt 300gctcagcttc aggcgctgct
tcagatttgg cctaaactgc ccccccggga ggccctagag 360cttctggatt
tcaactatcc agaccagtac gttcgagaat atgctgtagg ctgcctgcga
420cagatgagtg atgaagaact ttctcaatat cttttacaac tggtgcaagt
gttaaaatat 480gagccttttc ttgattgtgc cctctctaga ttcctattag
aaagagcact tggtaatcgg 540aggatagggc agtttctatt ttggcatctt
aggtcagaag tgcacattcc tgctgtctca 600gtacaatttg gtgtcatcct
tgaagcatac tgccggggaa gtgtggggca catgaaagtg 660ctttctaagc
aggttgaagc actcaataag ttaaaaactt taaatagttt aatcaaactg
720aatgccgtga agttaaacag agccaaaggg aaggaggcca tgcatacctg
tttaaaacag 780agtgcttacc gggaagccct ctctgacctg cagtcacccc
tgaacccatg tgttatcctc 840tcagaactct atgttgaaaa gtgcaaatac
atggattcca aaatgaagcc tttgtggctg 900gtatacaata acaaggtatt
tggtgaggat tcagttggag tgatttttaa aaatggtgat 960gatttacgac
aggatatgtt gacactccaa atgttgcgct tgatggattt actctggaaa
1020gaagctggtt tggatcttcg gatgttgcct tatggctgtt tagcaacagg
agatcgctct 1080ggcctcattg aagttgtgag cacctctgaa acaattgctg
acattcagct gaacagtagc 1140aatgtggctg ctgcagcagc cttcaacaaa
gatgcccttc tgaactggct taaagaatac 1200aactctgggg atgacctgga
ccgagccatt gaggaattta cactgtcctg tgctggctac 1260tgtgtagctt
cttatgtcct tgggattggt gacagacata gtgacaacat catggtcaaa
1320aaaactggcc agctcttcca cattgacttt ggacatattc ttggaaattt
caaatctaag 1380tttggcatta aaagggagcg agtgcctttt attcttacct
atgatttcat ccatgtcatt 1440caacaaggaa aaacaggaaa tacagaaaag
tttggccggt tccgccagtg ttgtgaggat 1500gcatatctga ttttacgacg
gcatgggaat ctcttcatca ctctctttgc gctgatgttg 1560actgcagggc
ttcctgaact cacatcagtc aaagatatac agtatcttaa ggactctctt
1620gcattaggga agagtgaaga agaagcactc aaacagttta agcaaaaatt
tgatgaggcg 1680ctcagggaaa gctggactac taaagtgaac tggatggccc
acacagttcg gaaagactac 1740agatcttaa 174910582PRTHomo sapiens 10Met
Leu Asn Pro Met Gly Thr Val Gln Thr Asn Pro Tyr Thr Glu Asn1 5 10
15Ala Thr Ala Leu His Val Lys Phe Pro Glu Asn Lys Lys Gln Pro Tyr
20 25 30Tyr Tyr Pro Pro Phe Asp Lys Ser Arg Gly Gly Lys Lys Phe Leu
Pro 35 40 45Val Leu Lys Glu Ile Leu Asp Arg Asp Pro Leu Ser Gln Leu
Cys Glu 50 55 60Asn Glu Met Asp Leu Ile Trp Thr Leu Arg Gln Asp Cys
Arg Glu Ile65 70 75 80Phe Pro Gln Ser Leu Pro Lys Leu Leu Leu Ser
Ile Lys Trp Asn Lys 85 90 95Leu Glu Asp Val Ala Gln Leu Gln Ala Leu
Leu Gln Ile Trp Pro Lys 100 105 110Leu Pro Pro Arg Glu Ala Leu Glu
Leu Leu Asp Phe Asn Tyr Pro Asp 115 120 125Gln Tyr Val Arg Glu Tyr
Ala Val Gly Cys Leu Arg Gln Met Ser Asp 130 135 140Glu Glu Leu Ser
Gln Tyr Leu Leu Gln Leu Val Gln Val Leu Lys Tyr145 150 155 160Glu
Pro Phe Leu Asp Cys Ala Leu Ser Arg Phe Leu Leu Glu Arg Ala 165 170
175Leu Gly Asn Arg Arg Ile Gly Gln Phe Leu Phe Trp His Leu Arg Ser
180 185 190Glu Val His Ile Pro Ala Val Ser Val Gln Phe Gly Val Ile
Leu Glu 195 200 205Ala Tyr Cys Arg Gly Ser Val Gly His Met Lys Val
Leu Ser Lys Gln 210 215 220Val Glu Ala Leu Asn Lys Leu Lys Thr Leu
Asn Ser Leu Ile Lys Leu225 230 235 240Asn Ala Val Lys Leu Asn Arg
Ala Lys Gly Lys Glu Ala Met His Thr 245 250 255Cys Leu Lys Gln Ser
Ala Tyr Arg Glu Ala Leu Ser Asp Leu Gln Ser 260 265 270Pro Leu Asn
Pro Cys Val Ile Leu Ser Glu Leu Tyr Val Glu Lys Cys 275 280 285Lys
Tyr Met Asp Ser Lys Met Lys Pro Leu Trp Leu Val Tyr Asn Asn 290 295
300Lys Val Phe Gly Glu Asp Ser Val Gly Val Ile Phe Lys Asn Gly
Asp305 310 315 320Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Met Leu
Arg Leu Met Asp 325 330 335Leu Leu Trp Lys Glu Ala Gly Leu Asp Leu
Arg Met Leu Pro Tyr Gly 340 345 350Cys Leu Ala Thr Gly Asp Arg Ser
Gly Leu Ile Glu Val Val Ser Thr 355 360 365Ser Glu Thr Ile Ala Asp
Ile Gln Leu Asn Ser Ser Asn Val Ala Ala 370 375 380Ala Ala Ala Phe
Asn Lys Asp Ala Leu Leu Asn Trp Leu Lys Glu Tyr385 390 395 400Asn
Ser Gly Asp Asp Leu Asp Arg Ala Ile Glu Glu Phe Thr Leu Ser 405 410
415Cys Ala Gly Tyr Cys Val Ala Ser Tyr Val Leu Gly Ile Gly Asp Arg
420 425 430His Ser Asp Asn Ile Met Val Lys Lys Thr Gly Gln Leu Phe
His Ile 435 440 445Asp Phe Gly His Ile Leu Gly Asn Phe Lys Ser Lys
Phe Gly Ile Lys 450 455 460Arg Glu Arg Val Pro Phe Ile Leu Thr Tyr
Asp Phe Ile His Val Ile465 470 475 480Gln Gln Gly Lys Thr Gly Asn
Thr Glu Lys Phe Gly Arg Phe Arg Gln 485 490 495Cys Cys Glu Asp Ala
Tyr Leu Ile Leu Arg Arg His Gly Asn Leu Phe 500 505 510Ile Thr Leu
Phe Ala Leu Met Leu Thr Ala Gly Leu Pro Glu Leu Thr 515 520 525Ser
Val Lys Asp Ile Gln Tyr Leu Lys Asp Ser Leu Ala Leu Gly Lys 530 535
540Ser Glu Glu Glu Ala Leu Lys Gln Phe Lys Gln Lys Phe Asp Glu
Ala545 550 555 560Leu Arg Glu Ser Trp Thr Thr Lys Val Asn Trp Met
Ala His Thr Val 565 570 575Arg Lys Asp Tyr Arg Ser 580113195DNAMus
musculus 11atgcctcctg ctatggcaga caaccttgac atctgggcag tggactcaca
gattgcatcc 60gatggcgcca tatccgtcga tttccttctg cccaccggga tttatatcca
gttggaagta 120cctcgggaag ctaccatttc ttatattaaa cagatgttat
ggaagcaagt tcacaactac 180ccgatgttta acctcctcat ggacattgac
tcgtatatgt ttgcatgtgt gaatcaaact 240gctgtatatg aggaactgga
agacgaaaca cgaagacttt gtgatgtcag accttttctt 300ccagttctca
aactagtgac tagaagctgt gaccccgcag aaaaattgga ctcaaaaatt
360ggggttctta taggaaaagg tcttcatgag tttgatgcct tgaaggatcc
cgaagtgaat 420gaatttagaa gaaaaatgcg caaattcagt gaggccaaga
ttcagtctct ggtagggttg 480tcttggatcg actggctaaa gcacacgtat
ccgcctgagc acgagccgtc cgtcctggag 540aacttggaag ataaacttta
tggaggaaag ctggttgtgg ctgtgcactt tgaaaatagc 600caggatgtat
ttagttttca
agtgtctccc aatttgaatc ctataaaaat aaatgaattg 660gcaatccaga
aacgcctcac tattcgtgga aaggaagatg aagctagccc ctgtgactat
720gtgttacagg tcagtgggag agtggagtat gtgtttggcg atcatccact
aattcagttc 780cagtacatcc ggaattgtgt gatgaataga accctgcccc
acttcatcct tgtggaatgt 840tgtaagatca agaaaatgta tgaacaagaa
atgattgcca tagaggctgc catcaaccga 900aactcatcca accttcctct
ccctttacca ccaaagaaaa cgcgagttat ttctcatatc 960tgggacaaca
acaacccttt ccaaattacc ttggttaaag gaaataagct taatacagaa
1020gaaactgtga aagttcatgt ccgagctggg ctttttcacg gaaccgagct
cctgtgtaaa 1080accgtcgtaa gctcagagat atcaggaaag aacgaccata
tttggaatga acaactggaa 1140tttgatatta atatttgtga cttaccaaga
atggctcgat tatgttttgc tgtttatgca 1200gttttggata aagtaaaaac
gaagaaatca acaaagacta ttaatccctc taagtatcag 1260accatcagga
aagccgggaa agtgcattat cctgtcgcat gggtaaatac catggttttt
1320gacttcaaag gacagctgag gtctggagac gtcatattgc atagctggtc
ttcgtttcct 1380gatgagctgg aagaaatgct gaatcccatg gggactgtgc
agacgaaccc atatgctgag 1440aacgccaccg ccttgcacat tacgttccca
gagaataaga agcagccgtg ttattatccc 1500cccttcgata agatcattga
gaaggcagct gagcttgcca gcggagacag tgctaatgtg 1560tcaagtcgtg
gtggaaaaaa atttcttgct gtgctgaaag aaatcttgga cagggacccc
1620ctgtctcagc tgtgtgagaa cgaaatggac cttatttgga ctctacggca
agactgccga 1680gaaaatttcc ctcagtcact gccaaaacta ctcttgtcaa
tcaagtggaa taaacttgaa 1740gatgttgctc agcttcaggc gctcctgcag
atatggccca aactgccccc cagggaagcc 1800ctggaactcc tggatttcaa
ctatccagac cagtatgtcc gggaatacgc tgtaggctgc 1860cttcgacaga
tgagtgatga agaactctct cagtatcttt tacaattggt gcaagttttg
1920aaatatgagc cttttctcga ttgtgccctc tccagattcc tattagaaag
agcacttgat 1980aatcggagga ttgggcagtt tctgttttgg catcttaggt
cagaggtgca cactcctgct 2040gtgtccgtac agtttggtgt catcctggaa
gcatactgtc gaggaagcgt ggggcacatg 2100aaagtgcttt ccaaacaggt
ggaagcactc aataagttaa aaactttaaa tagcttaatc 2160aaactgaatg
cggtgaagct gagcagagct aagggaaagg aggccatgca cacgtgcctg
2220aaacagagtg cttaccggga ggcgctctct gacctgcagt cgccgctgaa
cccctgcgtc 2280atcctctcag agctctatgt tgaaaagtgc aaatacatgg
actccaagat gaagcccctg 2340tggctggtct acagcagcag agcctttgga
gaggactcgg ttggagtgat ctttaaaaat 2400ggtgacgatt tgcggcagga
catgctgacg ctgcagatgt tgcgcctgat ggatctgctt 2460tggaaagaag
ctggcttgga cctgcggatg ctcccctatg gctgcttagc aacaggagat
2520cgctctggcc tcattgaggt tgtgagcacc tctgagacaa tcgctgacat
tcagctgaac 2580agtagtaacg tggctgccac ggcagccttc aacaaagacg
cactcctgaa ctggctcaag 2640gagtacaact ctggggatga cctggaccga
gcgattgagg agtttacctt gtcctgtgct 2700ggctactgtg tagcctctta
tgtcctcggc attggtgaca ggcacagtga caacatcatg 2760gtgaagaaaa
ccggccagct cttccacata gattttgggc atattcttgg aaatttcaaa
2820tctaaatttg gcattaaaag ggagcgagta ccttttattc ttacttatga
cttcattcat 2880gtcattcaac aaggaaaaac gggaaacact gaaaaatttg
gcagattccg ccagtgctgt 2940gaagatgcgt atctgatttt acggcggcat
gggaatctct tcatcaccct gtttgccctg 3000atgttgactg cagggctgcc
tgagctcaca tcggtcaaag atatacagta tcttaaggac 3060tcgcttgcct
tagggaagag cgaggaggaa gcactgaagc agttcaagca gaagtttgac
3120gaggccctca gggaaagctg gactactaaa gtgaactgga tggctcacac
agtacggaaa 3180gactacaggt cctag 3195121064PRTMus musculus 12Met Pro
Pro Ala Met Ala Asp Asn Leu Asp Ile Trp Ala Val Asp Ser1 5 10 15Gln
Ile Ala Ser Asp Gly Ala Ile Ser Val Asp Phe Leu Leu Pro Thr 20 25
30Gly Ile Tyr Ile Gln Leu Glu Val Pro Arg Glu Ala Thr Ile Ser Tyr
35 40 45Ile Lys Gln Met Leu Trp Lys Gln Val His Asn Tyr Pro Met Phe
Asn 50 55 60Leu Leu Met Asp Ile Asp Ser Tyr Met Phe Ala Cys Val Asn
Gln Thr65 70 75 80Ala Val Tyr Glu Glu Leu Glu Asp Glu Thr Arg Arg
Leu Cys Asp Val 85 90 95Arg Pro Phe Leu Pro Val Leu Lys Leu Val Thr
Arg Ser Cys Asp Pro 100 105 110Ala Glu Lys Leu Asp Ser Lys Ile Gly
Val Leu Ile Gly Lys Gly Leu 115 120 125His Glu Phe Asp Ala Leu Lys
Asp Pro Glu Val Asn Glu Phe Arg Arg 130 135 140Lys Met Arg Lys Phe
Ser Glu Ala Lys Ile Gln Ser Leu Val Gly Leu145 150 155 160Ser Trp
Ile Asp Trp Leu Lys His Thr Tyr Pro Pro Glu His Glu Pro 165 170
175Ser Val Leu Glu Asn Leu Glu Asp Lys Leu Tyr Gly Gly Lys Leu Val
180 185 190Val Ala Val His Phe Glu Asn Ser Gln Asp Val Phe Ser Phe
Gln Val 195 200 205Ser Pro Asn Leu Asn Pro Ile Lys Ile Asn Glu Leu
Ala Ile Gln Lys 210 215 220Arg Leu Thr Ile Arg Gly Lys Glu Asp Glu
Ala Ser Pro Cys Asp Tyr225 230 235 240Val Leu Gln Val Ser Gly Arg
Val Glu Tyr Val Phe Gly Asp His Pro 245 250 255Leu Ile Gln Phe Gln
Tyr Ile Arg Asn Cys Val Met Asn Arg Thr Leu 260 265 270Pro His Phe
Ile Leu Val Glu Cys Cys Lys Ile Lys Lys Met Tyr Glu 275 280 285Gln
Glu Met Ile Ala Ile Glu Ala Ala Ile Asn Arg Asn Ser Ser Asn 290 295
300Leu Pro Leu Pro Leu Pro Pro Lys Lys Thr Arg Val Ile Ser His
Ile305 310 315 320Trp Asp Asn Asn Asn Pro Phe Gln Ile Thr Leu Val
Lys Gly Asn Lys 325 330 335Leu Asn Thr Glu Glu Thr Val Lys Val His
Val Arg Ala Gly Leu Phe 340 345 350His Gly Thr Glu Leu Leu Cys Lys
Thr Val Val Ser Ser Glu Ile Ser 355 360 365Gly Lys Asn Asp His Ile
Trp Asn Glu Gln Leu Glu Phe Asp Ile Asn 370 375 380Ile Cys Asp Leu
Pro Arg Met Ala Arg Leu Cys Phe Ala Val Tyr Ala385 390 395 400Val
Leu Asp Lys Val Lys Thr Lys Lys Ser Thr Lys Thr Ile Asn Pro 405 410
415Ser Lys Tyr Gln Thr Ile Arg Lys Ala Gly Lys Val His Tyr Pro Val
420 425 430Ala Trp Val Asn Thr Met Val Phe Asp Phe Lys Gly Gln Leu
Arg Ser 435 440 445Gly Asp Val Ile Leu His Ser Trp Ser Ser Phe Pro
Asp Glu Leu Glu 450 455 460Glu Met Leu Asn Pro Met Gly Thr Val Gln
Thr Asn Pro Tyr Ala Glu465 470 475 480Asn Ala Thr Ala Leu His Ile
Thr Phe Pro Glu Asn Lys Lys Gln Pro 485 490 495Cys Tyr Tyr Pro Pro
Phe Asp Lys Ile Ile Glu Lys Ala Ala Glu Leu 500 505 510Ala Ser Gly
Asp Ser Ala Asn Val Ser Ser Arg Gly Gly Lys Lys Phe 515 520 525Leu
Ala Val Leu Lys Glu Ile Leu Asp Arg Asp Pro Leu Ser Gln Leu 530 535
540Cys Glu Asn Glu Met Asp Leu Ile Trp Thr Leu Arg Gln Asp Cys
Arg545 550 555 560Glu Asn Phe Pro Gln Ser Leu Pro Lys Leu Leu Leu
Ser Ile Lys Trp 565 570 575Asn Lys Leu Glu Asp Val Ala Gln Leu Gln
Ala Leu Leu Gln Ile Trp 580 585 590Pro Lys Leu Pro Pro Arg Glu Ala
Leu Glu Leu Leu Asp Phe Asn Tyr 595 600 605Pro Asp Gln Tyr Val Arg
Glu Tyr Ala Val Gly Cys Leu Arg Gln Met 610 615 620Ser Asp Glu Glu
Leu Ser Gln Tyr Leu Leu Gln Leu Val Gln Val Leu625 630 635 640Lys
Tyr Glu Pro Phe Leu Asp Cys Ala Leu Ser Arg Phe Leu Leu Glu 645 650
655Arg Ala Leu Asp Asn Arg Arg Ile Gly Gln Phe Leu Phe Trp His Leu
660 665 670Arg Ser Glu Val His Thr Pro Ala Val Ser Val Gln Phe Gly
Val Ile 675 680 685Leu Glu Ala Tyr Cys Arg Gly Ser Val Gly His Met
Lys Val Leu Ser 690 695 700Lys Gln Val Glu Ala Leu Asn Lys Leu Lys
Thr Leu Asn Ser Leu Ile705 710 715 720Lys Leu Asn Ala Val Lys Leu
Ser Arg Ala Lys Gly Lys Glu Ala Met 725 730 735His Thr Cys Leu Lys
Gln Ser Ala Tyr Arg Glu Ala Leu Ser Asp Leu 740 745 750Gln Ser Pro
Leu Asn Pro Cys Val Ile Leu Ser Glu Leu Tyr Val Glu 755 760 765Lys
Cys Lys Tyr Met Asp Ser Lys Met Lys Pro Leu Trp Leu Val Tyr 770 775
780Ser Ser Arg Ala Phe Gly Glu Asp Ser Val Gly Val Ile Phe Lys
Asn785 790 795 800Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln
Met Leu Arg Leu 805 810 815Met Asp Leu Leu Trp Lys Glu Ala Gly Leu
Asp Leu Arg Met Leu Pro 820 825 830Tyr Gly Cys Leu Ala Thr Gly Asp
Arg Ser Gly Leu Ile Glu Val Val 835 840 845Ser Thr Ser Glu Thr Ile
Ala Asp Ile Gln Leu Asn Ser Ser Asn Val 850 855 860Ala Ala Thr Ala
Ala Phe Asn Lys Asp Ala Leu Leu Asn Trp Leu Lys865 870 875 880Glu
Tyr Asn Ser Gly Asp Asp Leu Asp Arg Ala Ile Glu Glu Phe Thr 885 890
895Leu Ser Cys Ala Gly Tyr Cys Val Ala Ser Tyr Val Leu Gly Ile Gly
900 905 910Asp Arg His Ser Asp Asn Ile Met Val Lys Lys Thr Gly Gln
Leu Phe 915 920 925His Ile Asp Phe Gly His Ile Leu Gly Asn Phe Lys
Ser Lys Phe Gly 930 935 940Ile Lys Arg Glu Arg Val Pro Phe Ile Leu
Thr Tyr Asp Phe Ile His945 950 955 960Val Ile Gln Gln Gly Lys Thr
Gly Asn Thr Glu Lys Phe Gly Arg Phe 965 970 975Arg Gln Cys Cys Glu
Asp Ala Tyr Leu Ile Leu Arg Arg His Gly Asn 980 985 990Leu Phe Ile
Thr Leu Phe Ala Leu Met Leu Thr Ala Gly Leu Pro Glu 995 1000
1005Leu Thr Ser Val Lys Asp Ile Gln Tyr Leu Lys Asp Ser Leu Ala
1010 1015 1020Leu Gly Lys Ser Glu Glu Glu Ala Leu Lys Gln Phe Lys
Gln Lys 1025 1030 1035Phe Asp Glu Ala Leu Arg Glu Ser Trp Thr Thr
Lys Val Asn Trp 1040 1045 1050Met Ala His Thr Val Arg Lys Asp Tyr
Arg Ser 1055 1060133309DNAHomo sapiens 13atggagctgg agaactataa
acagcccgtg gtgctgagag aggacaactg ccgaaggcgc 60cggaggatga agccgcgcag
tgctgcggcc agcctgtcct ccatggagct catccccatc 120gagttcgtgc
tgcccaccag ccagcgcaaa tgcaagagcc ccgaaacggc gctgctgcac
180gtggccggcc acggcaacgt ggagcagatg aaggcccagg tgtggctgcg
agcgctggag 240accagcgtgg cggcggactt ctaccaccgg ctgggaccgc
atcacttcct cctgctctat 300cagaagaagg ggcagtggta cgagatctac
gacaagtacc aggtggtgca gactctggac 360tgcctgcgct actggaaggc
cacgcaccgg agcccgggcc agatccacct ggtgcagcgg 420cacccgccct
ccgaggagtc ccaagccttc cagcggcagc tcacggcgct gattggctat
480gacgtcactg acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg
ccgtggcttg 540gtgaccccgc gcatggcgga ggtggccagc cgcgacccca
agctctacgc catgcacccg 600tgggtgacgt ccaagcccct cccggagtac
ctgtggaaga agattgccaa caactgcatc 660ttcatcgtca ttcaccgcag
caccaccagc cagaccatta aggtctcacc cgacgacacc 720cccggcgcca
tcctgcagag cttcttcacc aagatggcca agaagaaatc tctgatggat
780attcccgaaa gccaaagcga acaggatttt gtgctgcgcg tctgtggccg
ggatgagtac 840ctggtgggcg aaacgcccat caaaaacttc cagtgggtga
ggcactgcct caagaacgga 900gaagagattc acgtggtact ggacacgcct
ccagacccgg ccctagacga ggtgaggaag 960gaagagtggc cactggtgga
tgactgcacg ggagtcaccg gctaccatga gcagcttacc 1020atccacggca
aggaccacga gagtgtgttc accgtgtccc tgtgggactg cgaccgcaag
1080ttcagggtca agatcagagg cattgatatc cccgtcctgc ctcggaacac
cgacctcaca 1140gtttttgtag aggcaaacat ccagcatggg caacaagtcc
tttgccaaag gagaaccagc 1200cccaaaccct tcacagagga ggtgctgtgg
aatgtgtggc ttgagttcag tatcaaaatc 1260aaagacttgc ccaaaggggc
tctactgaac ctccagatct actgcggtaa agctccagca 1320ctgtccagca
aggcctctgc agagtccccc agttctgagt ccaagggcaa agttcagctt
1380ctctattatg tgaacctgct gctgatagac caccgtttcc tcctgcgccg
tggagaatac 1440gtcctccaca tgtggcagat atctgggaag ggagaagacc
aaggaagctt caatgctgac 1500aaactcacgt ctgcaactaa cccagacaag
gagaactcaa tgtccatctc cattcttctg 1560gacaattact gccacccgat
agccctgcct aagcatcagc ccacccctga cccggaaggg 1620gaccgggttc
gagcagaaat gcccaaccag cttcgcaagc aattggaggc gatcatagcc
1680actgatccac ttaaccctct cacagcagag gacaaagaat tgctctggca
ttttagatac 1740gaaagcctta agcacccaaa agcatatcct aagctattta
gttcagtgaa atggggacag 1800caagaaattg tggccaaaac ataccaattg
ttggccagaa gggaagtctg ggatcaaagt 1860gctttggatg ttgggttaac
aatgcagctc ctggactgca acttctcaga tgaaaatgta 1920agagccattg
cagttcagaa actggagagc ttggaggacg atgatgttct gcattacctt
1980ctacaattgg tccaggctgt gaaatttgaa ccataccatg atagcgccct
tgccagattt 2040ctgctgaagc gtggtttaag aaacaaaaga attggtcact
ttttgttttg gttcttgaga 2100agtgagatag cccagtccag acactatcag
cagaggttcg ctgtgattct ggaagcctat 2160ctgaggggct gtggcacagc
catgctgcac gactttaccc aacaagtcca agtaatcgag 2220atgttacaaa
aagtcaccct tgatattaaa tcgctctctg ctgaaaagta tgacgtcagt
2280tcccaagtta tttcacaact taaacaaaag cttgaaaacc tgcagaattc
tcaactcccc 2340gaaagcttta gagttccata tgatcctgga ctgaaagcag
gagcgctggc aattgaaaaa 2400tgtaaagtaa tggcctccaa gaaaaaacca
ctatggcttg agtttaaatg tgccgatcct 2460acagccctat caaatgaaac
aattggaatt atctttaaac atggtgatga tctgcgccaa 2520gacatgctta
ttttacagat tctacgaatc atggagtcta tttgggagac tgaatctttg
2580gatctatgcc tcctgccata tggttgcatt tcaactggtg acaaaatagg
aatgatcgag 2640attgtgaaag acgccacgac aattgccaaa attcagcaaa
gcacagtggg caacacggga 2700gcatttaaag atgaagtcct gaatcactgg
ctcaaagaaa aatcccctac tgaagaaaag 2760tttcaggcag cagtggagag
atttgtttat tcctgtgcag gctactgtgt ggcaaccttt 2820gttcttggaa
taggcgacag acacaatgac aatattatga tcaccgagac aggaaaccta
2880tttcatattg acttcgggca cattcttggg aattacaaaa gtttcctggg
cattaataaa 2940gagagagtgc catttgtgct aacccctgac ttcctctttg
tgatgggaac ttctggaaag 3000aagacaagcc cacacttcca gaaatttcag
gacatctgtg ttaaggctta tctagccctt 3060cgtcatcaca caaacctact
gatcatcctg ttctccatga tgctgatgac aggaatgccc 3120cagttaacaa
gcaaagaaga cattgaatat atccgggatg ccctcacagt ggggaaaaat
3180gaggaggatg ctaaaaagta ttttcttgat cagatcgaag tttgcagaga
caaaggatgg 3240actgtgcagt ttaattggtt tctacatctt gttcttggca
tcaaacaagg agagaaacat 3300tcagcctaa 3309141102PRTHomo sapiens 14Met
Glu Leu Glu Asn Tyr Lys Gln Pro Val Val Leu Arg Glu Asp Asn1 5 10
15Cys Arg Arg Arg Arg Arg Met Lys Pro Arg Ser Ala Ala Ala Ser Leu
20 25 30Ser Ser Met Glu Leu Ile Pro Ile Glu Phe Val Leu Pro Thr Ser
Gln 35 40 45Arg Lys Cys Lys Ser Pro Glu Thr Ala Leu Leu His Val Ala
Gly His 50 55 60Gly Asn Val Glu Gln Met Lys Ala Gln Val Trp Leu Arg
Ala Leu Glu65 70 75 80Thr Ser Val Ala Ala Asp Phe Tyr His Arg Leu
Gly Pro His His Phe 85 90 95Leu Leu Leu Tyr Gln Lys Lys Gly Gln Trp
Tyr Glu Ile Tyr Asp Lys 100 105 110Tyr Gln Val Val Gln Thr Leu Asp
Cys Leu Arg Tyr Trp Lys Ala Thr 115 120 125His Arg Ser Pro Gly Gln
Ile His Leu Val Gln Arg His Pro Pro Ser 130 135 140Glu Glu Ser Gln
Ala Phe Gln Arg Gln Leu Thr Ala Leu Ile Gly Tyr145 150 155 160Asp
Val Thr Asp Val Ser Asn Val His Asp Asp Glu Leu Glu Phe Thr 165 170
175Arg Arg Gly Leu Val Thr Pro Arg Met Ala Glu Val Ala Ser Arg Asp
180 185 190Pro Lys Leu Tyr Ala Met His Pro Trp Val Thr Ser Lys Pro
Leu Pro 195 200 205Glu Tyr Leu Trp Lys Lys Ile Ala Asn Asn Cys Ile
Phe Ile Val Ile 210 215 220His Arg Ser Thr Thr Ser Gln Thr Ile Lys
Val Ser Pro Asp Asp Thr225 230 235 240Pro Gly Ala Ile Leu Gln Ser
Phe Phe Thr Lys Met Ala Lys Lys Lys 245 250 255Ser Leu Met Asp Ile
Pro Glu Ser Gln Ser Glu Gln Asp Phe Val Leu 260 265 270Arg Val Cys
Gly Arg Asp Glu Tyr Leu Val Gly Glu Thr Pro Ile Lys 275 280 285Asn
Phe Gln Trp Val Arg His Cys Leu Lys Asn Gly Glu Glu Ile His 290 295
300Val Val Leu Asp Thr Pro Pro Asp Pro Ala Leu Asp Glu Val Arg
Lys305 310 315 320Glu Glu Trp Pro Leu Val Asp Asp Cys Thr Gly Val
Thr Gly Tyr His 325 330 335Glu Gln Leu Thr Ile His Gly Lys Asp His
Glu Ser Val Phe Thr Val 340 345 350Ser Leu Trp Asp Cys Asp Arg Lys
Phe Arg Val Lys Ile Arg Gly Ile 355 360 365Asp Ile Pro Val Leu Pro
Arg Asn Thr Asp Leu Thr Val Phe Val Glu 370 375 380Ala Asn Ile Gln
His Gly Gln Gln Val Leu Cys Gln Arg Arg Thr Ser385 390 395 400Pro
Lys Pro Phe Thr Glu Glu Val Leu Trp Asn Val Trp Leu Glu Phe 405 410
415Ser Ile Lys
Ile Lys Asp Leu Pro Lys Gly Ala Leu Leu Asn Leu Gln 420 425 430Ile
Tyr Cys Gly Lys Ala Pro Ala Leu Ser Ser Lys Ala Ser Ala Glu 435 440
445Ser Pro Ser Ser Glu Ser Lys Gly Lys Val Gln Leu Leu Tyr Tyr Val
450 455 460Asn Leu Leu Leu Ile Asp His Arg Phe Leu Leu Arg Arg Gly
Glu Tyr465 470 475 480Val Leu His Met Trp Gln Ile Ser Gly Lys Gly
Glu Asp Gln Gly Ser 485 490 495Phe Asn Ala Asp Lys Leu Thr Ser Ala
Thr Asn Pro Asp Lys Glu Asn 500 505 510Ser Met Ser Ile Ser Ile Leu
Leu Asp Asn Tyr Cys His Pro Ile Ala 515 520 525Leu Pro Lys His Gln
Pro Thr Pro Asp Pro Glu Gly Asp Arg Val Arg 530 535 540Ala Glu Met
Pro Asn Gln Leu Arg Lys Gln Leu Glu Ala Ile Ile Ala545 550 555
560Thr Asp Pro Leu Asn Pro Leu Thr Ala Glu Asp Lys Glu Leu Leu Trp
565 570 575His Phe Arg Tyr Glu Ser Leu Lys His Pro Lys Ala Tyr Pro
Lys Leu 580 585 590Phe Ser Ser Val Lys Trp Gly Gln Gln Glu Ile Val
Ala Lys Thr Tyr 595 600 605Gln Leu Leu Ala Arg Arg Glu Val Trp Asp
Gln Ser Ala Leu Asp Val 610 615 620Gly Leu Thr Met Gln Leu Leu Asp
Cys Asn Phe Ser Asp Glu Asn Val625 630 635 640Arg Ala Ile Ala Val
Gln Lys Leu Glu Ser Leu Glu Asp Asp Asp Val 645 650 655Leu His Tyr
Leu Leu Gln Leu Val Gln Ala Val Lys Phe Glu Pro Tyr 660 665 670His
Asp Ser Ala Leu Ala Arg Phe Leu Leu Lys Arg Gly Leu Arg Asn 675 680
685Lys Arg Ile Gly His Phe Leu Phe Trp Phe Leu Arg Ser Glu Ile Ala
690 695 700Gln Ser Arg His Tyr Gln Gln Arg Phe Ala Val Ile Leu Glu
Ala Tyr705 710 715 720Leu Arg Gly Cys Gly Thr Ala Met Leu His Asp
Phe Thr Gln Gln Val 725 730 735Gln Val Ile Glu Met Leu Gln Lys Val
Thr Leu Asp Ile Lys Ser Leu 740 745 750Ser Ala Glu Lys Tyr Asp Val
Ser Ser Gln Val Ile Ser Gln Leu Lys 755 760 765Gln Lys Leu Glu Asn
Leu Gln Asn Ser Gln Leu Pro Glu Ser Phe Arg 770 775 780Val Pro Tyr
Asp Pro Gly Leu Lys Ala Gly Ala Leu Ala Ile Glu Lys785 790 795
800Cys Lys Val Met Ala Ser Lys Lys Lys Pro Leu Trp Leu Glu Phe Lys
805 810 815Cys Ala Asp Pro Thr Ala Leu Ser Asn Glu Thr Ile Gly Ile
Ile Phe 820 825 830Lys His Gly Asp Asp Leu Arg Gln Asp Met Leu Ile
Leu Gln Ile Leu 835 840 845Arg Ile Met Glu Ser Ile Trp Glu Thr Glu
Ser Leu Asp Leu Cys Leu 850 855 860Leu Pro Tyr Gly Cys Ile Ser Thr
Gly Asp Lys Ile Gly Met Ile Glu865 870 875 880Ile Val Lys Asp Ala
Thr Thr Ile Ala Lys Ile Gln Gln Ser Thr Val 885 890 895Gly Asn Thr
Gly Ala Phe Lys Asp Glu Val Leu Asn His Trp Leu Lys 900 905 910Glu
Lys Ser Pro Thr Glu Glu Lys Phe Gln Ala Ala Val Glu Arg Phe 915 920
925Val Tyr Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Val Leu Gly Ile
930 935 940Gly Asp Arg His Asn Asp Asn Ile Met Ile Thr Glu Thr Gly
Asn Leu945 950 955 960Phe His Ile Asp Phe Gly His Ile Leu Gly Asn
Tyr Lys Ser Phe Leu 965 970 975Gly Ile Asn Lys Glu Arg Val Pro Phe
Val Leu Thr Pro Asp Phe Leu 980 985 990Phe Val Met Gly Thr Ser Gly
Lys Lys Thr Ser Pro His Phe Gln Lys 995 1000 1005Phe Gln Asp Ile
Cys Val Lys Ala Tyr Leu Ala Leu Arg His His 1010 1015 1020Thr Asn
Leu Leu Ile Ile Leu Phe Ser Met Met Leu Met Thr Gly 1025 1030
1035Met Pro Gln Leu Thr Ser Lys Glu Asp Ile Glu Tyr Ile Arg Asp
1040 1045 1050Ala Leu Thr Val Gly Lys Asn Glu Glu Asp Ala Lys Lys
Tyr Phe 1055 1060 1065Leu Asp Gln Ile Glu Val Cys Arg Asp Lys Gly
Trp Thr Val Gln 1070 1075 1080Phe Asn Trp Phe Leu His Leu Val Leu
Gly Ile Lys Gln Gly Glu 1085 1090 1095Lys His Ser Ala
1100153309DNAHomo sapiens 15atggagctgg agaactataa acagcccgtg
gtgctgagag aggacaactg ccgaaggcgc 60cggaggatga agccgcgcag tgctgcggcc
agcctgtcct ccatggagct catccccatc 120gagttcgtgc tgcccaccag
ccagcgcaaa tgcaagagcc ccgaaacggc gctgctgcac 180gtggccggcc
acggcaacgt ggagcagatg aaggcccagg tgtggctgcg agcgctggag
240accagcgtgg cggcggactt ctaccaccgg ctgggaccgc atcacttcct
cctgctctat 300cagaagaagg ggcagtggta cgagatctac gacaagtacc
aggtggtgca gactctggac 360tgcctgcgct actggaaggc cacgcaccgg
agcccgggcc agatccacct ggtgcagcgg 420cacccgccct ccgaggagtc
ccaagccttc cagcggcagc tcacggcgct gattggctat 480gacgtcactg
acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg ccgtggcttg
540gtgaccccgc gcatggcgga ggtggccagc cgcgacccca agctctacgc
catgcacccg 600tgggtgacgt ccaagcccct cccggagtac ctgtggaaga
agattgccaa caactgcatc 660ttcatcgtca ttcaccgcag caccaccagc
cagaccatta aggtctcacc cgacgacacc 720cccggcgcca tcctgcagag
cttcttcacc aagatggcca agaagaaatc tctgatggat 780attcccgaaa
gccaaagcga acaggatttt gtgctgcgcg tctgtggccg ggatgagtac
840ctggtgggcg aaacgcccat caaaaacttc cagtgggtga ggcactgcct
caagaacgga 900gaagagattc acgtggtact ggacacgcct ccagacccgg
ccctagacga ggtgaggaag 960gaagagtggc cactggtgga tgactgcacg
ggagtcaccg gctaccatga gcagcttacc 1020atccacggca aggaccacga
gagtgtgttc accgtgtccc tgtgggactg cgaccgcaag 1080ttcagggtca
agatcagagg cattgatatc cccgtcctgc ctcggaacac cgacctcaca
1140gtttttgtag aggcaaacat ccagcatggg caacaagtcc tttgccaaag
gagaaccagc 1200cccaaaccct tcacagagga ggtgctgtgg aatgtgtggc
ttgagttcag tatcaaaatc 1260aaagacttgc ccaaaggggc tctactgaac
ctccagatct actgcggtaa agctccagca 1320ctgtccagca aggcctctgc
agagtccccc agttctgagt ccaagggcaa agttcagctt 1380ctctattatg
tgaacctgct gctgatagac caccgtttcc tcctgcgccg tggagaatac
1440gtcctccaca tgtggcagat atctgggaag ggagaagacc aaggaagctt
caatgctgac 1500aaactcacgt ctgcaactaa cccagacaag gagaactcaa
tgtccatctc cattcttctg 1560gacaattact gccacccgat agccctgcct
aagcatcagc ccacccctga cccggaaggg 1620gaccgggttc gagcagaaat
gcccaaccag cttcgcaagc aattggaggc gatcatagcc 1680actgatccac
ttaaccctct cacagcagag gacaaagaat tgctctggca ttttagatac
1740gaaagcctta agcacccaaa agcatatcct aagctattta gttcagtgaa
atggggacag 1800caagaaattg tggccaaaac ataccaattg ttggccagaa
gggaagtctg ggatcaaagt 1860gctttggatg ttgggttaac aatgcagctc
ctggactgca acttctcaga tgaaaatgta 1920agagccattg cagttcagaa
actggagagc ttggaggacg atgatgttct gcattacctt 1980ctacaattgg
tccaggctgt gaaatttgaa ccataccatg atagcgccct tgccagattt
2040ctgctgaagc gtggtttaag aaacaaaaga attggtcact ttttgttttg
gttcttgaga 2100agtgagatag cccagtccag acactatcag cagaggttcg
ctgtgattct ggaagcctat 2160ctgaggggct gtggcacagc catgctgcac
gactttaccc aacaagtcca agtaatcgag 2220atgttacaaa aagtcaccct
tgatattaaa tcgctctctg ctgaaaagta tgacgtcagt 2280tcccaagtta
tttcacaact taaacaaaag cttgaaaacc tgcagaattc tcaactcccc
2340gaaagcttta gagttccata tgatcctgga ctgaaagcag gagcgctggc
aattgaaaaa 2400tgtaaagtaa tggcctccaa gaaaaaacca ctatggcttg
agtttaaatg tgccgatcct 2460acagccctat caaatgaaac aattggaatt
atctttaaac atggtgatga tctgcgccaa 2520gacatgctta ttttacagat
tctacgaatc atggagtcta tttgggagac tgaatctttg 2580gatctatgcc
tcctgccata tggttgcatt tcaactggtg acaaaatagg aatgatcgag
2640attgtgaaag acgccacgac aattgccaaa attcagcaaa gcacagtggg
caacacggga 2700gcatttaaag atgaagtcct gaatcactgg ctcaaagaaa
aatcccctac tgaagaaaag 2760tttcaggcag cagtggagag atttgtttat
tcctgtgcag gctactgtgt ggcaaccttt 2820gttcttggaa taggcgacag
acacaatgac aatattatga tcaccgagac aggaaaccta 2880tttcatattg
acttcgggca cattcttggg aattacaaaa gtttcctggg cattaataaa
2940gagagagtgc catttgtgct aacccctgac ttcctctttg tgatgggaac
ttctggaaag 3000aagacaagcc cacacttcca gaaatttcag gacatctgtg
ttaaggctta tctagccctt 3060cgtcatcaca caaacctact gatcatcctg
ttctccatga tgctgatgac aggaatgccc 3120cagttaacaa gcaaagaaga
cattgaatat atccgggatg ccctcacagt ggggaaaaat 3180gaggaggatg
ctaaaaagta ttttcttgat cagatcgaag tttgcagaga caaaggatgg
3240actgtgcagt ttaattggtt tctacatctt gttcttggca tcaaacaagg
agagaaacat 3300tcagcctaa 3309161102PRTHomo sapiens 16Met Glu Leu
Glu Asn Tyr Lys Gln Pro Val Val Leu Arg Glu Asp Asn1 5 10 15Cys Arg
Arg Arg Arg Arg Met Lys Pro Arg Ser Ala Ala Ala Ser Leu 20 25 30Ser
Ser Met Glu Leu Ile Pro Ile Glu Phe Val Leu Pro Thr Ser Gln 35 40
45Arg Lys Cys Lys Ser Pro Glu Thr Ala Leu Leu His Val Ala Gly His
50 55 60Gly Asn Val Glu Gln Met Lys Ala Gln Val Trp Leu Arg Ala Leu
Glu65 70 75 80Thr Ser Val Ala Ala Asp Phe Tyr His Arg Leu Gly Pro
His His Phe 85 90 95Leu Leu Leu Tyr Gln Lys Lys Gly Gln Trp Tyr Glu
Ile Tyr Asp Lys 100 105 110Tyr Gln Val Val Gln Thr Leu Asp Cys Leu
Arg Tyr Trp Lys Ala Thr 115 120 125His Arg Ser Pro Gly Gln Ile His
Leu Val Gln Arg His Pro Pro Ser 130 135 140Glu Glu Ser Gln Ala Phe
Gln Arg Gln Leu Thr Ala Leu Ile Gly Tyr145 150 155 160Asp Val Thr
Asp Val Ser Asn Val His Asp Asp Glu Leu Glu Phe Thr 165 170 175Arg
Arg Gly Leu Val Thr Pro Arg Met Ala Glu Val Ala Ser Arg Asp 180 185
190Pro Lys Leu Tyr Ala Met His Pro Trp Val Thr Ser Lys Pro Leu Pro
195 200 205Glu Tyr Leu Trp Lys Lys Ile Ala Asn Asn Cys Ile Phe Ile
Val Ile 210 215 220His Arg Ser Thr Thr Ser Gln Thr Ile Lys Val Ser
Pro Asp Asp Thr225 230 235 240Pro Gly Ala Ile Leu Gln Ser Phe Phe
Thr Lys Met Ala Lys Lys Lys 245 250 255Ser Leu Met Asp Ile Pro Glu
Ser Gln Ser Glu Gln Asp Phe Val Leu 260 265 270Arg Val Cys Gly Arg
Asp Glu Tyr Leu Val Gly Glu Thr Pro Ile Lys 275 280 285Asn Phe Gln
Trp Val Arg His Cys Leu Lys Asn Gly Glu Glu Ile His 290 295 300Val
Val Leu Asp Thr Pro Pro Asp Pro Ala Leu Asp Glu Val Arg Lys305 310
315 320Glu Glu Trp Pro Leu Val Asp Asp Cys Thr Gly Val Thr Gly Tyr
His 325 330 335Glu Gln Leu Thr Ile His Gly Lys Asp His Glu Ser Val
Phe Thr Val 340 345 350Ser Leu Trp Asp Cys Asp Arg Lys Phe Arg Val
Lys Ile Arg Gly Ile 355 360 365Asp Ile Pro Val Leu Pro Arg Asn Thr
Asp Leu Thr Val Phe Val Glu 370 375 380Ala Asn Ile Gln His Gly Gln
Gln Val Leu Cys Gln Arg Arg Thr Ser385 390 395 400Pro Lys Pro Phe
Thr Glu Glu Val Leu Trp Asn Val Trp Leu Glu Phe 405 410 415Ser Ile
Lys Ile Lys Asp Leu Pro Lys Gly Ala Leu Leu Asn Leu Gln 420 425
430Ile Tyr Cys Gly Lys Ala Pro Ala Leu Ser Ser Lys Ala Ser Ala Glu
435 440 445Ser Pro Ser Ser Glu Ser Lys Gly Lys Val Gln Leu Leu Tyr
Tyr Val 450 455 460Asn Leu Leu Leu Ile Asp His Arg Phe Leu Leu Arg
Arg Gly Glu Tyr465 470 475 480Val Leu His Met Trp Gln Ile Ser Gly
Lys Gly Glu Asp Gln Gly Ser 485 490 495Phe Asn Ala Asp Lys Leu Thr
Ser Ala Thr Asn Pro Asp Lys Glu Asn 500 505 510Ser Met Ser Ile Ser
Ile Leu Leu Asp Asn Tyr Cys His Pro Ile Ala 515 520 525Leu Pro Lys
His Gln Pro Thr Pro Asp Pro Glu Gly Asp Arg Val Arg 530 535 540Ala
Glu Met Pro Asn Gln Leu Arg Lys Gln Leu Glu Ala Ile Ile Ala545 550
555 560Thr Asp Pro Leu Asn Pro Leu Thr Ala Glu Asp Lys Glu Leu Leu
Trp 565 570 575His Phe Arg Tyr Glu Ser Leu Lys His Pro Lys Ala Tyr
Pro Lys Leu 580 585 590Phe Ser Ser Val Lys Trp Gly Gln Gln Glu Ile
Val Ala Lys Thr Tyr 595 600 605Gln Leu Leu Ala Arg Arg Glu Val Trp
Asp Gln Ser Ala Leu Asp Val 610 615 620Gly Leu Thr Met Gln Leu Leu
Asp Cys Asn Phe Ser Asp Glu Asn Val625 630 635 640Arg Ala Ile Ala
Val Gln Lys Leu Glu Ser Leu Glu Asp Asp Asp Val 645 650 655Leu His
Tyr Leu Leu Gln Leu Val Gln Ala Val Lys Phe Glu Pro Tyr 660 665
670His Asp Ser Ala Leu Ala Arg Phe Leu Leu Lys Arg Gly Leu Arg Asn
675 680 685Lys Arg Ile Gly His Phe Leu Phe Trp Phe Leu Arg Ser Glu
Ile Ala 690 695 700Gln Ser Arg His Tyr Gln Gln Arg Phe Ala Val Ile
Leu Glu Ala Tyr705 710 715 720Leu Arg Gly Cys Gly Thr Ala Met Leu
His Asp Phe Thr Gln Gln Val 725 730 735Gln Val Ile Glu Met Leu Gln
Lys Val Thr Leu Asp Ile Lys Ser Leu 740 745 750Ser Ala Glu Lys Tyr
Asp Val Ser Ser Gln Val Ile Ser Gln Leu Lys 755 760 765Gln Lys Leu
Glu Asn Leu Gln Asn Ser Gln Leu Pro Glu Ser Phe Arg 770 775 780Val
Pro Tyr Asp Pro Gly Leu Lys Ala Gly Ala Leu Ala Ile Glu Lys785 790
795 800Cys Lys Val Met Ala Ser Lys Lys Lys Pro Leu Trp Leu Glu Phe
Lys 805 810 815Cys Ala Asp Pro Thr Ala Leu Ser Asn Glu Thr Ile Gly
Ile Ile Phe 820 825 830Lys His Gly Asp Asp Leu Arg Gln Asp Met Leu
Ile Leu Gln Ile Leu 835 840 845Arg Ile Met Glu Ser Ile Trp Glu Thr
Glu Ser Leu Asp Leu Cys Leu 850 855 860Leu Pro Tyr Gly Cys Ile Ser
Thr Gly Asp Lys Ile Gly Met Ile Glu865 870 875 880Ile Val Lys Asp
Ala Thr Thr Ile Ala Lys Ile Gln Gln Ser Thr Val 885 890 895Gly Asn
Thr Gly Ala Phe Lys Asp Glu Val Leu Asn His Trp Leu Lys 900 905
910Glu Lys Ser Pro Thr Glu Glu Lys Phe Gln Ala Ala Val Glu Arg Phe
915 920 925Val Tyr Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Val Leu
Gly Ile 930 935 940Gly Asp Arg His Asn Asp Asn Ile Met Ile Thr Glu
Thr Gly Asn Leu945 950 955 960Phe His Ile Asp Phe Gly His Ile Leu
Gly Asn Tyr Lys Ser Phe Leu 965 970 975Gly Ile Asn Lys Glu Arg Val
Pro Phe Val Leu Thr Pro Asp Phe Leu 980 985 990Phe Val Met Gly Thr
Ser Gly Lys Lys Thr Ser Pro His Phe Gln Lys 995 1000 1005Phe Gln
Asp Ile Cys Val Lys Ala Tyr Leu Ala Leu Arg His His 1010 1015
1020Thr Asn Leu Leu Ile Ile Leu Phe Ser Met Met Leu Met Thr Gly
1025 1030 1035Met Pro Gln Leu Thr Ser Lys Glu Asp Ile Glu Tyr Ile
Arg Asp 1040 1045 1050Ala Leu Thr Val Gly Lys Asn Glu Glu Asp Ala
Lys Lys Tyr Phe 1055 1060 1065Leu Asp Gln Ile Glu Val Cys Arg Asp
Lys Gly Trp Thr Val Gln 1070 1075 1080Phe Asn Trp Phe Leu His Leu
Val Leu Gly Ile Lys Gln Gly Glu 1085 1090 1095Lys His Ser Ala
1100173309DNAHomo sapiens 17atggagctgg agaactataa acagcccgtg
gtgctgagag aggacaactg ccgaaggcgc 60cggaggatga agccgcgcag tgctgcggcc
agcctgtcct ccatggagct catccccatc 120gagttcgtgc tgcccaccag
ccagcgcaaa tgcaagagcc ccgaaacggc gctgctgcac 180gtggccggcc
acggcaacgt ggagcagatg aaggcccagg tgtggctgcg agcgctggag
240accagcgtgg cggcggactt ctaccaccgg ctgggaccgc atcacttcct
cctgctctat 300cagaagaagg ggcagtggta cgagatctac gacaagtacc
aggtggtgca gactctggac 360tgcctgcgct actggaaggc cacgcaccgg
agcccgggcc agatccacct ggtgcagcgg 420cacccgccct ccgaggagtc
ccaagccttc cagcggcagc tcacggcgct gattggctat 480gacgtcactg
acgtcagcaa cgtgcacgac gatgagctgg agttcacgcg ccgtggcttg
540gtgaccccgc gcatggcgga ggtggccagc cgcgacccca agctctacgc
catgcacccg 600tgggtgacgt ccaagcccct cccggagtac ctgtggaaga
agattgccaa caactgcatc 660ttcatcgtca ttcaccgcag caccaccagc
cagaccatta aggtctcacc cgacgacacc 720cccggcgcca tcctgcagag
cttcttcacc
aagatggcca agaagaaatc tctgatggat 780attcccgaaa gccaaagcga
acaggatttt gtgctgcgcg tctgtggccg ggatgagtac 840ctggtgggcg
aaacgcccat caaaaacttc cagtgggtga ggcactgcct caagaacgga
900gaagagattc acgtggtact ggacacgcct ccagacccgg ccctagacga
ggtgaggaag 960gaagagtggc cactggtgga tgactgcacg ggagtcaccg
gctaccatga gcagcttacc 1020atccacggca aggaccacga gagtgtgttc
accgtgtccc tgtgggactg cgaccgcaag 1080ttcagggtca agatcagagg
cattgatatc cccgtcctgc ctcggaacac cgacctcaca 1140gtttttgtag
aggcaaacat ccagcatggg caacaagtcc tttgccaaag gagaaccagc
1200cccaaaccct tcacagagga ggtgctgtgg aatgtgtggc ttgagttcag
tatcaaaatc 1260aaagacttgc ccaaaggggc tctactgaac ctccagatct
actgcggtaa agctccagca 1320ctgtccagca aggcctctgc agagtccccc
agttctgagt ccaagggcaa agttcagctt 1380ctctattatg tgaacctgct
gctgatagac caccgtttcc tcctgcgccg tggagaatac 1440gtcctccaca
tgtggcagat atctgggaag ggagaagacc aaggaagctt caatgctgac
1500aaactcacgt ctgcaactaa cccagacaag gagaactcaa tgtccatctc
cattcttctg 1560gacaattact gccacccgat agccctgcct aagcatcagc
ccacccctga cccggaaggg 1620gaccgggttc gagcagaaat gcccaaccag
cttcgcaagc aattggaggc gatcatagcc 1680actgatccac ttaaccctct
cacagcagag gacaaagaat tgctctggca ttttagatac 1740gaaagcctta
agcacccaaa agcatatcct aagctattta gttcagtgaa atggggacag
1800caagaaattg tggccaaaac ataccaattg ttggccagaa gggaagtctg
ggatcaaagt 1860gctttggatg ttgggttaac aatgcagctc ctggactgca
acttctcaga tgaaaatgta 1920agagccattg cagttcagaa actggagagc
ttggaggacg atgatgttct gcattacctt 1980ctacaattgg tccaggctgt
gaaatttgaa ccataccatg atagcgccct tgccagattt 2040ctgctgaagc
gtggtttaag aaacaaaaga attggtcact ttttgttttg gttcttgaga
2100agtgagatag cccagtccag acactatcag cagaggttcg ctgtgattct
ggaagcctat 2160ctgaggggct gtggcacagc catgctgcac gactttaccc
aacaagtcca agtaatcgag 2220atgttacaaa aagtcaccct tgatattaaa
tcgctctctg ctgaaaagta tgacgtcagt 2280tcccaagtta tttcacaact
taaacaaaag cttgaaaacc tgcagaattc tcaactcccc 2340gaaagcttta
gagttccata tgatcctgga ctgaaagcag gagcgctggc aattgaaaaa
2400tgtaaagtaa tggcctccaa gaaaaaacca ctatggcttg agtttaaatg
tgccgatcct 2460acagccctat caaatgaaac aattggaatt atctttaaac
atggtgatga tctgcgccaa 2520gacatgctta ttttacagat tctacgaatc
atggagtcta tttgggagac tgaatctttg 2580gatctatgcc tcctgccata
tggttgcatt tcaactggtg acaaaatagg aatgatcgag 2640attgtgaaag
acgccacgac aattgccaaa attcagcaaa gcacagtggg caacacggga
2700gcatttaaag atgaagtcct gaatcactgg ctcaaagaaa aatcccctac
tgaagaaaag 2760tttcaggcag cagtggagag atttgtttat tcctgtgcag
gctactgtgt ggcaaccttt 2820gttcttggaa taggcgacag acacaatgac
aatattatga tcaccgagac aggaaaccta 2880tttcatattg acttcgggca
cattcttggg aattacaaaa gtttcctggg cattaataaa 2940gagagagtgc
catttgtgct aacccctgac ttcctctttg tgatgggaac ttctggaaag
3000aagacaagcc cacacttcca gaaatttcag gacatctgtg ttaaggctta
tctagccctt 3060cgtcatcaca caaacctact gatcatcctg ttctccatga
tgctgatgac aggaatgccc 3120cagttaacaa gcaaagaaga cattgaatat
atccgggatg ccctcacagt ggggaaaaat 3180gaggaggatg ctaaaaagta
ttttcttgat cagatcgaag tttgcagaga caaaggatgg 3240actgtgcagt
ttaattggtt tctacatctt gttcttggca tcaaacaagg agagaaacat
3300tcagcctaa 3309181102PRTHomo sapiens 18Met Glu Leu Glu Asn Tyr
Lys Gln Pro Val Val Leu Arg Glu Asp Asn1 5 10 15Cys Arg Arg Arg Arg
Arg Met Lys Pro Arg Ser Ala Ala Ala Ser Leu 20 25 30Ser Ser Met Glu
Leu Ile Pro Ile Glu Phe Val Leu Pro Thr Ser Gln 35 40 45Arg Lys Cys
Lys Ser Pro Glu Thr Ala Leu Leu His Val Ala Gly His 50 55 60Gly Asn
Val Glu Gln Met Lys Ala Gln Val Trp Leu Arg Ala Leu Glu65 70 75
80Thr Ser Val Ala Ala Asp Phe Tyr His Arg Leu Gly Pro His His Phe
85 90 95Leu Leu Leu Tyr Gln Lys Lys Gly Gln Trp Tyr Glu Ile Tyr Asp
Lys 100 105 110Tyr Gln Val Val Gln Thr Leu Asp Cys Leu Arg Tyr Trp
Lys Ala Thr 115 120 125His Arg Ser Pro Gly Gln Ile His Leu Val Gln
Arg His Pro Pro Ser 130 135 140Glu Glu Ser Gln Ala Phe Gln Arg Gln
Leu Thr Ala Leu Ile Gly Tyr145 150 155 160Asp Val Thr Asp Val Ser
Asn Val His Asp Asp Glu Leu Glu Phe Thr 165 170 175Arg Arg Gly Leu
Val Thr Pro Arg Met Ala Glu Val Ala Ser Arg Asp 180 185 190Pro Lys
Leu Tyr Ala Met His Pro Trp Val Thr Ser Lys Pro Leu Pro 195 200
205Glu Tyr Leu Trp Lys Lys Ile Ala Asn Asn Cys Ile Phe Ile Val Ile
210 215 220His Arg Ser Thr Thr Ser Gln Thr Ile Lys Val Ser Pro Asp
Asp Thr225 230 235 240Pro Gly Ala Ile Leu Gln Ser Phe Phe Thr Lys
Met Ala Lys Lys Lys 245 250 255Ser Leu Met Asp Ile Pro Glu Ser Gln
Ser Glu Gln Asp Phe Val Leu 260 265 270Arg Val Cys Gly Arg Asp Glu
Tyr Leu Val Gly Glu Thr Pro Ile Lys 275 280 285Asn Phe Gln Trp Val
Arg His Cys Leu Lys Asn Gly Glu Glu Ile His 290 295 300Val Val Leu
Asp Thr Pro Pro Asp Pro Ala Leu Asp Glu Val Arg Lys305 310 315
320Glu Glu Trp Pro Leu Val Asp Asp Cys Thr Gly Val Thr Gly Tyr His
325 330 335Glu Gln Leu Thr Ile His Gly Lys Asp His Glu Ser Val Phe
Thr Val 340 345 350Ser Leu Trp Asp Cys Asp Arg Lys Phe Arg Val Lys
Ile Arg Gly Ile 355 360 365Asp Ile Pro Val Leu Pro Arg Asn Thr Asp
Leu Thr Val Phe Val Glu 370 375 380Ala Asn Ile Gln His Gly Gln Gln
Val Leu Cys Gln Arg Arg Thr Ser385 390 395 400Pro Lys Pro Phe Thr
Glu Glu Val Leu Trp Asn Val Trp Leu Glu Phe 405 410 415Ser Ile Lys
Ile Lys Asp Leu Pro Lys Gly Ala Leu Leu Asn Leu Gln 420 425 430Ile
Tyr Cys Gly Lys Ala Pro Ala Leu Ser Ser Lys Ala Ser Ala Glu 435 440
445Ser Pro Ser Ser Glu Ser Lys Gly Lys Val Gln Leu Leu Tyr Tyr Val
450 455 460Asn Leu Leu Leu Ile Asp His Arg Phe Leu Leu Arg Arg Gly
Glu Tyr465 470 475 480Val Leu His Met Trp Gln Ile Ser Gly Lys Gly
Glu Asp Gln Gly Ser 485 490 495Phe Asn Ala Asp Lys Leu Thr Ser Ala
Thr Asn Pro Asp Lys Glu Asn 500 505 510Ser Met Ser Ile Ser Ile Leu
Leu Asp Asn Tyr Cys His Pro Ile Ala 515 520 525Leu Pro Lys His Gln
Pro Thr Pro Asp Pro Glu Gly Asp Arg Val Arg 530 535 540Ala Glu Met
Pro Asn Gln Leu Arg Lys Gln Leu Glu Ala Ile Ile Ala545 550 555
560Thr Asp Pro Leu Asn Pro Leu Thr Ala Glu Asp Lys Glu Leu Leu Trp
565 570 575His Phe Arg Tyr Glu Ser Leu Lys His Pro Lys Ala Tyr Pro
Lys Leu 580 585 590Phe Ser Ser Val Lys Trp Gly Gln Gln Glu Ile Val
Ala Lys Thr Tyr 595 600 605Gln Leu Leu Ala Arg Arg Glu Val Trp Asp
Gln Ser Ala Leu Asp Val 610 615 620Gly Leu Thr Met Gln Leu Leu Asp
Cys Asn Phe Ser Asp Glu Asn Val625 630 635 640Arg Ala Ile Ala Val
Gln Lys Leu Glu Ser Leu Glu Asp Asp Asp Val 645 650 655Leu His Tyr
Leu Leu Gln Leu Val Gln Ala Val Lys Phe Glu Pro Tyr 660 665 670His
Asp Ser Ala Leu Ala Arg Phe Leu Leu Lys Arg Gly Leu Arg Asn 675 680
685Lys Arg Ile Gly His Phe Leu Phe Trp Phe Leu Arg Ser Glu Ile Ala
690 695 700Gln Ser Arg His Tyr Gln Gln Arg Phe Ala Val Ile Leu Glu
Ala Tyr705 710 715 720Leu Arg Gly Cys Gly Thr Ala Met Leu His Asp
Phe Thr Gln Gln Val 725 730 735Gln Val Ile Glu Met Leu Gln Lys Val
Thr Leu Asp Ile Lys Ser Leu 740 745 750Ser Ala Glu Lys Tyr Asp Val
Ser Ser Gln Val Ile Ser Gln Leu Lys 755 760 765Gln Lys Leu Glu Asn
Leu Gln Asn Ser Gln Leu Pro Glu Ser Phe Arg 770 775 780Val Pro Tyr
Asp Pro Gly Leu Lys Ala Gly Ala Leu Ala Ile Glu Lys785 790 795
800Cys Lys Val Met Ala Ser Lys Lys Lys Pro Leu Trp Leu Glu Phe Lys
805 810 815Cys Ala Asp Pro Thr Ala Leu Ser Asn Glu Thr Ile Gly Ile
Ile Phe 820 825 830Lys His Gly Asp Asp Leu Arg Gln Asp Met Leu Ile
Leu Gln Ile Leu 835 840 845Arg Ile Met Glu Ser Ile Trp Glu Thr Glu
Ser Leu Asp Leu Cys Leu 850 855 860Leu Pro Tyr Gly Cys Ile Ser Thr
Gly Asp Lys Ile Gly Met Ile Glu865 870 875 880Ile Val Lys Asp Ala
Thr Thr Ile Ala Lys Ile Gln Gln Ser Thr Val 885 890 895Gly Asn Thr
Gly Ala Phe Lys Asp Glu Val Leu Asn His Trp Leu Lys 900 905 910Glu
Lys Ser Pro Thr Glu Glu Lys Phe Gln Ala Ala Val Glu Arg Phe 915 920
925Val Tyr Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Val Leu Gly Ile
930 935 940Gly Asp Arg His Asn Asp Asn Ile Met Ile Thr Glu Thr Gly
Asn Leu945 950 955 960Phe His Ile Asp Phe Gly His Ile Leu Gly Asn
Tyr Lys Ser Phe Leu 965 970 975Gly Ile Asn Lys Glu Arg Val Pro Phe
Val Leu Thr Pro Asp Phe Leu 980 985 990Phe Val Met Gly Thr Ser Gly
Lys Lys Thr Ser Pro His Phe Gln Lys 995 1000 1005Phe Gln Asp Ile
Cys Val Lys Ala Tyr Leu Ala Leu Arg His His 1010 1015 1020Thr Asn
Leu Leu Ile Ile Leu Phe Ser Met Met Leu Met Thr Gly 1025 1030
1035Met Pro Gln Leu Thr Ser Lys Glu Asp Ile Glu Tyr Ile Arg Asp
1040 1045 1050Ala Leu Thr Val Gly Lys Asn Glu Glu Asp Ala Lys Lys
Tyr Phe 1055 1060 1065Leu Asp Gln Ile Glu Val Cys Arg Asp Lys Gly
Trp Thr Val Gln 1070 1075 1080Phe Asn Trp Phe Leu His Leu Val Leu
Gly Ile Lys Gln Gly Glu 1085 1090 1095Lys His Ser Ala
1100193309DNAMus musculus 19atggagctgg agaactatga acaaccggtg
gttctaagag aggacaacct ccgccggcgc 60cggaggatga agccacgcag cgcagcaggc
agcctgtctt ccatggagct catccccatt 120gagttcgtac tgcccaccag
ccagcgcatc agcaagactc cagaaacagc gctgctgcat 180gtggctggcc
atggcaatgt ggaacagatg aaagctcagg tgtggctgcg cgcactggag
240accagtgtgg ctgcggagtt ctaccaccga ttgggcccgg accaattcct
cctgctctac 300cagaagaaag gacaatggta tgagatctat gacaggtacc
aagtggtgca gaccctagac 360tgcctgcatt actggaagtt gatgcacaag
agccctggcc agatccacgt ggtacagcga 420cacgtacctt ctgaggagac
cttggctttc cagaagcagc tcacctccct gattggctat 480gacgtcactg
acatcagcaa tgtgcacgat gatgagctag agttcactcg ccgccgtctg
540gttacgcccc gcatggctga agtggctggc cgggatgcca aactctatgc
tatgcaccct 600tgggtaacgt ccaaacctct cccagactac ctgtcaaaaa
agattgccaa caactgcatc 660ttcatcgtca tccaccgcgg taccaccagc
caaaccatca aggtctccgc agatgatact 720cctggtacca tcctccagag
cttcttcacc aagatggcca agaagaagtc cctaatgaat 780atctcagaaa
gtcaaagtga gcaggatttt gtattgcggg tttgtggccg cgatgagtac
840ctggtgggtg aaacacccct caaaaatttc cagtgggtga ggcagtgcct
caagaacgga 900gatgaaatac acctggtgct cgacacgcct ccagacccag
cccttgatga ggtgaggaag 960gaagaatggc cgctggtgga tgactgcact
ggagtcaccg gctaccacga gcagctgacc 1020atccatggca aggaccacga
gagtgtgttc acagtgtctt tgtgggactg cgaccgaaag 1080ttcagggtca
agatcagagg cattgatatc cctgtcctgc ctcggaacac cgacctcact
1140gtgtttgtgg aagcgaacat ccagcacggg caacaagtcc tctgccaaag
gagaaccagc 1200cctaagccct tcgcagaaga ggtactctgg aatgtgtggc
tggagtttgg catcaaaatc 1260aaagacttgc ccaaaggggc tctattgaac
ctacagatct actgctgcaa aaccccatca 1320ctgtccagca aggcttctgc
agagactcca ggctccgagt ccaagggcaa agcccagctt 1380ctctattacg
tgaacttgct gttaatagac caccgtttcc tcctccgcca cggggactat
1440gtgctccaca tgtggcagat atctggcaag gcagaggagc agggcagctt
caatgctgac 1500aagctcacat ccgcaaccaa tcctgacaag gagaactcaa
tgtccatttc catcctgctg 1560gacaattact gtcaccccat agctttgcct
aagcaccggc ccacccctga cccagaggga 1620gacagggttc gggctgaaat
gcccaatcag cttcgaaagc aattggaggc gatcatagcc 1680acagatccac
ttaaccccct cacagcagag gacaaagaat tgctctggca ttttcgatat
1740gaaagcctga agcatccgaa ggcttaccct aagctattca gctcagtgaa
atgggggcag 1800caagaaattg ttgccaaaac gtaccagctg ttagccagaa
gggagatctg ggatcaaagt 1860gctttggacg ttggcttaac catgcagctc
ctggactgca acttttcaga cgagaatgtc 1920cgggccattg cagttcagaa
actggagagc ttagaggacg atgacgtttt acattacctt 1980ctccagctgg
tacaggctgt gaaatttgaa ccgtaccacg acagtgcgct ggccagattc
2040ctgctgaagc gtggcttgag gaacaaaaga atcggtcact tcttgttctg
gttcctgcga 2100agtgagatcg cacagtccag acactatcag cagaggttcg
ctgtgatcct ggaggcgtac 2160ctgcgaggct gtggcacagc catgttgcag
gacttcacac agcaggtcca tgtgattgag 2220atgttacaga aagtcaccat
tgatattaaa tcgctctcgg cagagaagta tgacgtcagt 2280tcccaagtta
tttcacagct taagcaaaag cttgaaagcc ttcagaactc caatctcccc
2340gagagcttta gagttcccta tgatcctgga ctaaaagccg gtaccctggt
gatcgagaaa 2400tgcaaagtga tggcctccaa gaagaagccc ctgtggcttg
agtttaagtg tgctgatccc 2460acagtcctat ccaacgaaac cattggaatc
atctttaaac atggtgatga tctgcgccaa 2520gacatgttga tcttgcagat
tctacgcatc atggagtcca tttgggagac tgaatctctg 2580gacctgtgcc
ttctgcctta cggttgcatc tcaactggtg acaaaatagg aatgatcgag
2640attgtaaagg atgccacaac gatcgctcaa attcagcaaa gcacagtggg
taacacgggg 2700gcattcaaag atgaagtcct gaatcactgg ctcaaggaaa
aatgtcctat tgaagaaaag 2760tttcaggccg cagtggaaag gtttgtttac
tcctgtgcag gctactgtgt ggccacattt 2820gttcttggga tcggtgacag
gcacaacgac aacattatga tctcagagac aggaaaccta 2880tttcatatag
acttcggaca cattcttggg aattacaaga gtttcctggg catcaataaa
2940gagagagtgc ccttcgtcct aaccccagac ttcttgtttg tgatgggatc
ttctggaaaa 3000aagacaagtc cacacttcca gaaattccag gatgtctgtg
ttagagctta cctagctctt 3060cgccatcaca caaacctgtt gatcatcttg
ttctccatga tgctgatgac aggaatgccc 3120cagctgacaa gcaaagagga
cattgaatat atccgggatg ccctcaccgt gggaaaaagc 3180gaggaggacg
ctaagaaata tttccttgat cagatcgaag tctgcagaga caaaggatgg
3240actgtgcagt ttaactggtt cctacatctt gttcttggca tcaaacaagg
agaaaagcac 3300tccgcttga 3309201102PRTMus musculus 20Met Glu Leu
Glu Asn Tyr Glu Gln Pro Val Val Leu Arg Glu Asp Asn1 5 10 15Leu Arg
Arg Arg Arg Arg Met Lys Pro Arg Ser Ala Ala Gly Ser Leu 20 25 30Ser
Ser Met Glu Leu Ile Pro Ile Glu Phe Val Leu Pro Thr Ser Gln 35 40
45Arg Ile Ser Lys Thr Pro Glu Thr Ala Leu Leu His Val Ala Gly His
50 55 60Gly Asn Val Glu Gln Met Lys Ala Gln Val Trp Leu Arg Ala Leu
Glu65 70 75 80Thr Ser Val Ala Ala Glu Phe Tyr His Arg Leu Gly Pro
Asp Gln Phe 85 90 95Leu Leu Leu Tyr Gln Lys Lys Gly Gln Trp Tyr Glu
Ile Tyr Asp Arg 100 105 110Tyr Gln Val Val Gln Thr Leu Asp Cys Leu
His Tyr Trp Lys Leu Met 115 120 125His Lys Ser Pro Gly Gln Ile His
Val Val Gln Arg His Val Pro Ser 130 135 140Glu Glu Thr Leu Ala Phe
Gln Lys Gln Leu Thr Ser Leu Ile Gly Tyr145 150 155 160Asp Val Thr
Asp Ile Ser Asn Val His Asp Asp Glu Leu Glu Phe Thr 165 170 175Arg
Arg Arg Leu Val Thr Pro Arg Met Ala Glu Val Ala Gly Arg Asp 180 185
190Ala Lys Leu Tyr Ala Met His Pro Trp Val Thr Ser Lys Pro Leu Pro
195 200 205Asp Tyr Leu Ser Lys Lys Ile Ala Asn Asn Cys Ile Phe Ile
Val Ile 210 215 220His Arg Gly Thr Thr Ser Gln Thr Ile Lys Val Ser
Ala Asp Asp Thr225 230 235 240Pro Gly Thr Ile Leu Gln Ser Phe Phe
Thr Lys Met Ala Lys Lys Lys 245 250 255Ser Leu Met Asn Ile Ser Glu
Ser Gln Ser Glu Gln Asp Phe Val Leu 260 265 270Arg Val Cys Gly Arg
Asp Glu Tyr Leu Val Gly Glu Thr Pro Leu Lys 275 280 285Asn Phe Gln
Trp Val Arg Gln Cys Leu Lys Asn Gly Asp Glu Ile His 290 295 300Leu
Val Leu Asp Thr Pro Pro Asp Pro Ala Leu Asp Glu Val Arg Lys305 310
315 320Glu Glu Trp Pro Leu Val Asp Asp Cys Thr Gly Val Thr Gly Tyr
His 325 330 335Glu Gln Leu Thr Ile His Gly Lys Asp His Glu Ser Val
Phe Thr Val 340 345 350Ser Leu Trp Asp Cys Asp Arg Lys Phe Arg Val
Lys Ile Arg Gly Ile 355 360 365Asp Ile Pro Val Leu Pro Arg Asn Thr
Asp Leu Thr Val Phe Val Glu 370 375
380Ala Asn Ile Gln His Gly Gln Gln Val Leu Cys Gln Arg Arg Thr
Ser385 390 395 400Pro Lys Pro Phe Ala Glu Glu Val Leu Trp Asn Val
Trp Leu Glu Phe 405 410 415Gly Ile Lys Ile Lys Asp Leu Pro Lys Gly
Ala Leu Leu Asn Leu Gln 420 425 430Ile Tyr Cys Cys Lys Thr Pro Ser
Leu Ser Ser Lys Ala Ser Ala Glu 435 440 445Thr Pro Gly Ser Glu Ser
Lys Gly Lys Ala Gln Leu Leu Tyr Tyr Val 450 455 460Asn Leu Leu Leu
Ile Asp His Arg Phe Leu Leu Arg His Gly Asp Tyr465 470 475 480Val
Leu His Met Trp Gln Ile Ser Gly Lys Ala Glu Glu Gln Gly Ser 485 490
495Phe Asn Ala Asp Lys Leu Thr Ser Ala Thr Asn Pro Asp Lys Glu Asn
500 505 510Ser Met Ser Ile Ser Ile Leu Leu Asp Asn Tyr Cys His Pro
Ile Ala 515 520 525Leu Pro Lys His Arg Pro Thr Pro Asp Pro Glu Gly
Asp Arg Val Arg 530 535 540Ala Glu Met Pro Asn Gln Leu Arg Lys Gln
Leu Glu Ala Ile Ile Ala545 550 555 560Thr Asp Pro Leu Asn Pro Leu
Thr Ala Glu Asp Lys Glu Leu Leu Trp 565 570 575His Phe Arg Tyr Glu
Ser Leu Lys His Pro Lys Ala Tyr Pro Lys Leu 580 585 590Phe Ser Ser
Val Lys Trp Gly Gln Gln Glu Ile Val Ala Lys Thr Tyr 595 600 605Gln
Leu Leu Ala Arg Arg Glu Ile Trp Asp Gln Ser Ala Leu Asp Val 610 615
620Gly Leu Thr Met Gln Leu Leu Asp Cys Asn Phe Ser Asp Glu Asn
Val625 630 635 640Arg Ala Ile Ala Val Gln Lys Leu Glu Ser Leu Glu
Asp Asp Asp Val 645 650 655Leu His Tyr Leu Leu Gln Leu Val Gln Ala
Val Lys Phe Glu Pro Tyr 660 665 670His Asp Ser Ala Leu Ala Arg Phe
Leu Leu Lys Arg Gly Leu Arg Asn 675 680 685Lys Arg Ile Gly His Phe
Leu Phe Trp Phe Leu Arg Ser Glu Ile Ala 690 695 700Gln Ser Arg His
Tyr Gln Gln Arg Phe Ala Val Ile Leu Glu Ala Tyr705 710 715 720Leu
Arg Gly Cys Gly Thr Ala Met Leu Gln Asp Phe Thr Gln Gln Val 725 730
735His Val Ile Glu Met Leu Gln Lys Val Thr Ile Asp Ile Lys Ser Leu
740 745 750Ser Ala Glu Lys Tyr Asp Val Ser Ser Gln Val Ile Ser Gln
Leu Lys 755 760 765Gln Lys Leu Glu Ser Leu Gln Asn Ser Asn Leu Pro
Glu Ser Phe Arg 770 775 780Val Pro Tyr Asp Pro Gly Leu Lys Ala Gly
Thr Leu Val Ile Glu Lys785 790 795 800Cys Lys Val Met Ala Ser Lys
Lys Lys Pro Leu Trp Leu Glu Phe Lys 805 810 815Cys Ala Asp Pro Thr
Val Leu Ser Asn Glu Thr Ile Gly Ile Ile Phe 820 825 830Lys His Gly
Asp Asp Leu Arg Gln Asp Met Leu Ile Leu Gln Ile Leu 835 840 845Arg
Ile Met Glu Ser Ile Trp Glu Thr Glu Ser Leu Asp Leu Cys Leu 850 855
860Leu Pro Tyr Gly Cys Ile Ser Thr Gly Asp Lys Ile Gly Met Ile
Glu865 870 875 880Ile Val Lys Asp Ala Thr Thr Ile Ala Gln Ile Gln
Gln Ser Thr Val 885 890 895Gly Asn Thr Gly Ala Phe Lys Asp Glu Val
Leu Asn His Trp Leu Lys 900 905 910Glu Lys Cys Pro Ile Glu Glu Lys
Phe Gln Ala Ala Val Glu Arg Phe 915 920 925Val Tyr Ser Cys Ala Gly
Tyr Cys Val Ala Thr Phe Val Leu Gly Ile 930 935 940Gly Asp Arg His
Asn Asp Asn Ile Met Ile Ser Glu Thr Gly Asn Leu945 950 955 960Phe
His Ile Asp Phe Gly His Ile Leu Gly Asn Tyr Lys Ser Phe Leu 965 970
975Gly Ile Asn Lys Glu Arg Val Pro Phe Val Leu Thr Pro Asp Phe Leu
980 985 990Phe Val Met Gly Ser Ser Gly Lys Lys Thr Ser Pro His Phe
Gln Lys 995 1000 1005Phe Gln Asp Val Cys Val Arg Ala Tyr Leu Ala
Leu Arg His His 1010 1015 1020Thr Asn Leu Leu Ile Ile Leu Phe Ser
Met Met Leu Met Thr Gly 1025 1030 1035Met Pro Gln Leu Thr Ser Lys
Glu Asp Ile Glu Tyr Ile Arg Asp 1040 1045 1050Ala Leu Thr Val Gly
Lys Ser Glu Glu Asp Ala Lys Lys Tyr Phe 1055 1060 1065Leu Asp Gln
Ile Glu Val Cys Arg Asp Lys Gly Trp Thr Val Gln 1070 1075 1080Phe
Asn Trp Phe Leu His Leu Val Leu Gly Ile Lys Gln Gly Glu 1085 1090
1095Lys His Ser Ala 1100213309DNAMus musculus 21atggagctgg
agaactatga acaaccggtg gttctaagag aggacaacct ccgccggcgc 60cggaggatga
agccacgcag cgcagcaggc agcctgtctt ccatggagct catccccatt
120gagttcgtac tgcccaccag ccagcgcatc agcaagactc cagaaacagc
gctgctgcat 180gtggctggcc atggcaatgt ggaacagatg aaagctcagg
tgtggctgcg cgcactggag 240accagtgtgg ctgcggagtt ctaccaccga
ttgggcccgg accaattcct cctgctctac 300cagaagaaag gacaatggta
tgagatctat gacaggtacc aagtggtgca gaccctagac 360tgcctgcatt
actggaagtt gatgcacaag agccctggcc agatccacgt ggtacagcga
420cacgtacctt ctgaggagac cttggctttc cagaagcagc tcacctccct
gattggctat 480gacgtcactg acatcagcaa tgtgcacgat gatgagctag
agttcactcg ccgccgtctg 540gttacgcccc gcatggctga agtggctggc
cgggatgcca aactctatgc tatgcaccct 600tgggtaacgt ccaaacctct
cccagactac ctgtcaaaaa agattgccaa caactgcatc 660ttcatcgtca
tccaccgcgg taccaccagc caaaccatca aggtctccgc agatgatact
720cctggtacca tcctccagag cttcttcacc aagatggcca agaagaagtc
cctaatgaat 780atctcagaaa gtcaaagtga gcaggatttt gtattgcggg
tttgtggccg cgatgagtac 840ctggtgggtg aaacacccct caaaaatttc
cagtgggtga ggcagtgcct caagaacgga 900gatgaaatac acctggtgct
cgacacgcct ccagacccag cccttgatga ggtgaggaag 960gaagaatggc
cgctggtgga tgactgcact ggagtcaccg gctaccacga gcagctgacc
1020atccatggca aggaccacga gagtgtgttc acagtgtctt tgtgggactg
cgaccgaaag 1080ttcagggtca agatcagagg cattgatatc cctgtcctgc
ctcggaacac cgacctcact 1140gtgtttgtgg aagcgaacat ccagcacggg
caacaagtcc tctgccaaag gagaaccagc 1200cctaagccct tcgcagaaga
ggtactctgg aatgtgtggc tggagtttgg catcaaaatc 1260aaagacttgc
ccaaaggggc tctattgaac ctacagatct actgctgcaa aaccccatca
1320ctgtccagca aggcttctgc agagactcca ggctccgagt ccaagggcaa
agcccagctt 1380ctctattacg tgaacttgct gttaatagac caccgtttcc
tcctccgcca cggggactat 1440gtgctccaca tgtggcagat atctggcaag
gcagaggagc agggcagctt caatgctgac 1500aagctcacat ccgcaaccaa
tcctgacaag gagaactcaa tgtccatttc catcctgctg 1560gacaattact
gtcaccccat agctttgcct aagcaccggc ccacccctga cccagaggga
1620gacagggttc gggctgaaat gcccaatcag cttcgaaagc aattggaggc
gatcatagcc 1680acagatccac ttaaccccct cacagcagag gacaaagaat
tgctctggca ttttcgatat 1740gaaagcctga agcatccgaa ggcttaccct
aagctattca gctcagtgaa atgggggcag 1800caagaaattg ttgccaaaac
gtaccagctg ttagccagaa gggagatctg ggatcaaagt 1860gctttggacg
ttggcttaac catgcagctc ctggactgca acttttcaga cgagaatgtc
1920cgggccattg cagttcagaa actggagagc ttagaggacg atgacgtttt
acattacctt 1980ctccagctgg tacaggctgt gaaatttgaa ccgtaccacg
acagtgcgct ggccagattc 2040ctgctgaagc gtggcttgag gaacaaaaga
atcggtcact tcttgttctg gttcctgcga 2100agtgagatcg cacagtccag
acactatcag cagaggttcg ctgtgatcct ggaggcgtac 2160ctgcgaggct
gtggcacagc catgttgcag gacttcacac agcaggtcca tgtgattgag
2220atgttacaga aagtcaccat tgatattaaa tcgctctcgg cagagaagta
tgacgtcagt 2280tcccaagtta tttcacagct taagcaaaag cttgaaagcc
ttcagaactc caatctcccc 2340gagagcttta gagttcccta tgatcctgga
ctaaaagccg gtaccctggt gatcgagaaa 2400tgcaaagtga tggcctccaa
gaagaagccc ctgtggcttg agtttaagtg tgctgatccc 2460acagtcctat
ccaacgaaac cattggaatc atctttaaac atggtgatga tctgcgccaa
2520gacatgttga tcttgcagat tctacgcatc atggagtcca tttgggagac
tgaatctctg 2580gacctgtgcc ttctgcctta cggttgcatc tcaactggtg
acaaaatagg aatgatcgag 2640attgtaaagg atgccacaac gatcgctcaa
attcagcaaa gcacagtggg taacacgggg 2700gcattcaaag atgaagtcct
gaatcactgg ctcaaggaaa aatgtcctat tgaagaaaag 2760tttcaggccg
cagtggaaag gtttgtttac tcctgtgcag gctactgtgt ggccacattt
2820gttcttggga tcggtgacag gcacaacgac aacattatga tctcagagac
aggaaaccta 2880tttcatatag acttcggaca cattcttggg aattacaaga
gtttcctggg catcaataaa 2940gagagagtgc ccttcgtcct aaccccagac
ttcttgtttg tgatgggatc ttctggaaaa 3000aagacaagtc cacacttcca
gaaattccag gatgtctgtg ttagagctta cctagctctt 3060cgccatcaca
caaacctgtt gatcatcttg ttctccatga tgctgatgac aggaatgccc
3120cagctgacaa gcaaagagga cattgaatat atccgggatg ccctcaccgt
gggaaaaagc 3180gaggaggacg ctaagaaata tttccttgat cagatcgaag
tctgcagaga caaaggatgg 3240actgtgcagt ttaactggtt cctacatctt
gttcttggca tcaaacaagg agaaaagcac 3300tccgcttga 3309221102PRTMus
musculus 22Met Glu Leu Glu Asn Tyr Glu Gln Pro Val Val Leu Arg Glu
Asp Asn1 5 10 15Leu Arg Arg Arg Arg Arg Met Lys Pro Arg Ser Ala Ala
Gly Ser Leu 20 25 30Ser Ser Met Glu Leu Ile Pro Ile Glu Phe Val Leu
Pro Thr Ser Gln 35 40 45Arg Ile Ser Lys Thr Pro Glu Thr Ala Leu Leu
His Val Ala Gly His 50 55 60Gly Asn Val Glu Gln Met Lys Ala Gln Val
Trp Leu Arg Ala Leu Glu65 70 75 80Thr Ser Val Ala Ala Glu Phe Tyr
His Arg Leu Gly Pro Asp Gln Phe 85 90 95Leu Leu Leu Tyr Gln Lys Lys
Gly Gln Trp Tyr Glu Ile Tyr Asp Arg 100 105 110Tyr Gln Val Val Gln
Thr Leu Asp Cys Leu His Tyr Trp Lys Leu Met 115 120 125His Lys Ser
Pro Gly Gln Ile His Val Val Gln Arg His Val Pro Ser 130 135 140Glu
Glu Thr Leu Ala Phe Gln Lys Gln Leu Thr Ser Leu Ile Gly Tyr145 150
155 160Asp Val Thr Asp Ile Ser Asn Val His Asp Asp Glu Leu Glu Phe
Thr 165 170 175Arg Arg Arg Leu Val Thr Pro Arg Met Ala Glu Val Ala
Gly Arg Asp 180 185 190Ala Lys Leu Tyr Ala Met His Pro Trp Val Thr
Ser Lys Pro Leu Pro 195 200 205Asp Tyr Leu Ser Lys Lys Ile Ala Asn
Asn Cys Ile Phe Ile Val Ile 210 215 220His Arg Gly Thr Thr Ser Gln
Thr Ile Lys Val Ser Ala Asp Asp Thr225 230 235 240Pro Gly Thr Ile
Leu Gln Ser Phe Phe Thr Lys Met Ala Lys Lys Lys 245 250 255Ser Leu
Met Asn Ile Ser Glu Ser Gln Ser Glu Gln Asp Phe Val Leu 260 265
270Arg Val Cys Gly Arg Asp Glu Tyr Leu Val Gly Glu Thr Pro Leu Lys
275 280 285Asn Phe Gln Trp Val Arg Gln Cys Leu Lys Asn Gly Asp Glu
Ile His 290 295 300Leu Val Leu Asp Thr Pro Pro Asp Pro Ala Leu Asp
Glu Val Arg Lys305 310 315 320Glu Glu Trp Pro Leu Val Asp Asp Cys
Thr Gly Val Thr Gly Tyr His 325 330 335Glu Gln Leu Thr Ile His Gly
Lys Asp His Glu Ser Val Phe Thr Val 340 345 350Ser Leu Trp Asp Cys
Asp Arg Lys Phe Arg Val Lys Ile Arg Gly Ile 355 360 365Asp Ile Pro
Val Leu Pro Arg Asn Thr Asp Leu Thr Val Phe Val Glu 370 375 380Ala
Asn Ile Gln His Gly Gln Gln Val Leu Cys Gln Arg Arg Thr Ser385 390
395 400Pro Lys Pro Phe Ala Glu Glu Val Leu Trp Asn Val Trp Leu Glu
Phe 405 410 415Gly Ile Lys Ile Lys Asp Leu Pro Lys Gly Ala Leu Leu
Asn Leu Gln 420 425 430Ile Tyr Cys Cys Lys Thr Pro Ser Leu Ser Ser
Lys Ala Ser Ala Glu 435 440 445Thr Pro Gly Ser Glu Ser Lys Gly Lys
Ala Gln Leu Leu Tyr Tyr Val 450 455 460Asn Leu Leu Leu Ile Asp His
Arg Phe Leu Leu Arg His Gly Asp Tyr465 470 475 480Val Leu His Met
Trp Gln Ile Ser Gly Lys Ala Glu Glu Gln Gly Ser 485 490 495Phe Asn
Ala Asp Lys Leu Thr Ser Ala Thr Asn Pro Asp Lys Glu Asn 500 505
510Ser Met Ser Ile Ser Ile Leu Leu Asp Asn Tyr Cys His Pro Ile Ala
515 520 525Leu Pro Lys His Arg Pro Thr Pro Asp Pro Glu Gly Asp Arg
Val Arg 530 535 540Ala Glu Met Pro Asn Gln Leu Arg Lys Gln Leu Glu
Ala Ile Ile Ala545 550 555 560Thr Asp Pro Leu Asn Pro Leu Thr Ala
Glu Asp Lys Glu Leu Leu Trp 565 570 575His Phe Arg Tyr Glu Ser Leu
Lys His Pro Lys Ala Tyr Pro Lys Leu 580 585 590Phe Ser Ser Val Lys
Trp Gly Gln Gln Glu Ile Val Ala Lys Thr Tyr 595 600 605Gln Leu Leu
Ala Arg Arg Glu Ile Trp Asp Gln Ser Ala Leu Asp Val 610 615 620Gly
Leu Thr Met Gln Leu Leu Asp Cys Asn Phe Ser Asp Glu Asn Val625 630
635 640Arg Ala Ile Ala Val Gln Lys Leu Glu Ser Leu Glu Asp Asp Asp
Val 645 650 655Leu His Tyr Leu Leu Gln Leu Val Gln Ala Val Lys Phe
Glu Pro Tyr 660 665 670His Asp Ser Ala Leu Ala Arg Phe Leu Leu Lys
Arg Gly Leu Arg Asn 675 680 685Lys Arg Ile Gly His Phe Leu Phe Trp
Phe Leu Arg Ser Glu Ile Ala 690 695 700Gln Ser Arg His Tyr Gln Gln
Arg Phe Ala Val Ile Leu Glu Ala Tyr705 710 715 720Leu Arg Gly Cys
Gly Thr Ala Met Leu Gln Asp Phe Thr Gln Gln Val 725 730 735His Val
Ile Glu Met Leu Gln Lys Val Thr Ile Asp Ile Lys Ser Leu 740 745
750Ser Ala Glu Lys Tyr Asp Val Ser Ser Gln Val Ile Ser Gln Leu Lys
755 760 765Gln Lys Leu Glu Ser Leu Gln Asn Ser Asn Leu Pro Glu Ser
Phe Arg 770 775 780Val Pro Tyr Asp Pro Gly Leu Lys Ala Gly Thr Leu
Val Ile Glu Lys785 790 795 800Cys Lys Val Met Ala Ser Lys Lys Lys
Pro Leu Trp Leu Glu Phe Lys 805 810 815Cys Ala Asp Pro Thr Val Leu
Ser Asn Glu Thr Ile Gly Ile Ile Phe 820 825 830Lys His Gly Asp Asp
Leu Arg Gln Asp Met Leu Ile Leu Gln Ile Leu 835 840 845Arg Ile Met
Glu Ser Ile Trp Glu Thr Glu Ser Leu Asp Leu Cys Leu 850 855 860Leu
Pro Tyr Gly Cys Ile Ser Thr Gly Asp Lys Ile Gly Met Ile Glu865 870
875 880Ile Val Lys Asp Ala Thr Thr Ile Ala Gln Ile Gln Gln Ser Thr
Val 885 890 895Gly Asn Thr Gly Ala Phe Lys Asp Glu Val Leu Asn His
Trp Leu Lys 900 905 910Glu Lys Cys Pro Ile Glu Glu Lys Phe Gln Ala
Ala Val Glu Arg Phe 915 920 925Val Tyr Ser Cys Ala Gly Tyr Cys Val
Ala Thr Phe Val Leu Gly Ile 930 935 940Gly Asp Arg His Asn Asp Asn
Ile Met Ile Ser Glu Thr Gly Asn Leu945 950 955 960Phe His Ile Asp
Phe Gly His Ile Leu Gly Asn Tyr Lys Ser Phe Leu 965 970 975Gly Ile
Asn Lys Glu Arg Val Pro Phe Val Leu Thr Pro Asp Phe Leu 980 985
990Phe Val Met Gly Ser Ser Gly Lys Lys Thr Ser Pro His Phe Gln Lys
995 1000 1005Phe Gln Asp Val Cys Val Arg Ala Tyr Leu Ala Leu Arg
His His 1010 1015 1020Thr Asn Leu Leu Ile Ile Leu Phe Ser Met Met
Leu Met Thr Gly 1025 1030 1035Met Pro Gln Leu Thr Ser Lys Glu Asp
Ile Glu Tyr Ile Arg Asp 1040 1045 1050Ala Leu Thr Val Gly Lys Ser
Glu Glu Asp Ala Lys Lys Tyr Phe 1055 1060 1065Leu Asp Gln Ile Glu
Val Cys Arg Asp Lys Gly Trp Thr Val Gln 1070 1075 1080Phe Asn Trp
Phe Leu His Leu Val Leu Gly Ile Lys Gln Gly Glu 1085 1090 1095Lys
His Ser Ala 1100233309DNAMus musculus 23atggagctgg agaactatga
acaaccggtg gttctaagag aggacaacct ccgccggcgc 60cggaggatga agccacgcag
cgcagcaggc agcctgtctt ccatggagct catccccatt 120gagttcgtac
tgcccaccag ccagcgcatc agcaagactc cagaaacagc gctgctgcat
180gtggctggcc atggcaatgt ggaacagatg aaagctcagg tgtggctgcg
cgcactggag 240accagtgtgg ctgcggagtt ctaccaccga ttgggcccgg
accaattcct cctgctctac 300cagaagaaag gacaatggta tgagatctat
gacaggtacc aagtggtgca gaccctagac 360tgcctgcatt actggaagtt
gatgcacaag agccctggcc agatccacgt ggtacagcga 420cacgtacctt
ctgaggagac cttggctttc cagaagcagc tcacctccct gattggctat
480gacgtcactg acatcagcaa tgtgcacgat gatgagctag agttcactcg
ccgccgtctg
540gttacgcccc gcatggctga agtggctggc cgggatgcca aactctatgc
tatgcaccct 600tgggtaacgt ccaaacctct cccagactac ctgtcaaaaa
agattgccaa caactgcatc 660ttcatcgtca tccaccgcgg taccaccagc
caaaccatca aggtctccgc agatgatact 720cctggtacca tcctccagag
cttcttcacc aagatggcca agaagaagtc cctaatgaat 780atctcagaaa
gtcaaagtga gcaggatttt gtattgcggg tttgtggccg cgatgagtac
840ctggtgggtg aaacacccct caaaaatttc cagtgggtga ggcagtgcct
caagaacgga 900gatgaaatac acctggtgct cgacacgcct ccagacccag
cccttgatga ggtgaggaag 960gaagaatggc cgctggtgga tgactgcact
ggagtcaccg gctaccacga gcagctgacc 1020atccatggca aggaccacga
gagtgtgttc acagtgtctt tgtgggactg cgaccgaaag 1080ttcagggtca
agatcagagg cattgatatc cctgtcctgc ctcggaacac cgacctcact
1140gtgtttgtgg aagcgaacat ccagcacggg caacaagtcc tctgccaaag
gagaaccagc 1200cctaagccct tcgcagaaga ggtactctgg aatgtgtggc
tggagtttgg catcaaaatc 1260aaagacttgc ccaaaggggc tctattgaac
ctacagatct actgctgcaa aaccccatca 1320ctgtccagca aggcttctgc
agagactcca ggctccgagt ccaagggcaa agcccagctt 1380ctctattacg
tgaacttgct gttaatagac caccgtttcc tcctccgcca cggggactat
1440gtgctccaca tgtggcagat atctggcaag gcagaggagc agggcagctt
caatgctgac 1500aagctcacat ccgcaaccaa tcctgacaag gagaactcaa
tgtccatttc catcctgctg 1560gacaattact gtcaccccat agctttgcct
aagcaccggc ccacccctga cccagaggga 1620gacagggttc gggctgaaat
gcccaatcag cttcgaaagc aattggaggc gatcatagcc 1680acagatccac
ttaaccccct cacagcagag gacaaagaat tgctctggca ttttcgatat
1740gaaagcctga agcatccgaa ggcttaccct aagctattca gctcagtgaa
atgggggcag 1800caagaaattg ttgccaaaac gtaccagctg ttagccagaa
gggagatctg ggatcaaagt 1860gctttggacg ttggcttaac catgcagctc
ctggactgca acttttcaga cgagaatgtc 1920cgggccattg cagttcagaa
actggagagc ttagaggacg atgacgtttt acattacctt 1980ctccagctgg
tacaggctgt gaaatttgaa ccgtaccacg acagtgcgct ggccagattc
2040ctgctgaagc gtggcttgag gaacaaaaga atcggtcact tcttgttctg
gttcctgcga 2100agtgagatcg cacagtccag acactatcag cagaggttcg
ctgtgatcct ggaggcgtac 2160ctgcgaggct gtggcacagc catgttgcag
gacttcacac agcaggtcca tgtgattgag 2220atgttacaga aagtcaccat
tgatattaaa tcgctctcgg cagagaagta tgacgtcagt 2280tcccaagtta
tttcacagct taagcaaaag cttgaaagcc ttcagaactc caatctcccc
2340gagagcttta gagttcccta tgatcctgga ctaaaagccg gtaccctggt
gatcgagaaa 2400tgcaaagtga tggcctccaa gaagaagccc ctgtggcttg
agtttaagtg tgctgatccc 2460acagtcctat ccaacgaaac cattggaatc
atctttaaac atggtgatga tctgcgccaa 2520gacatgttga tcttgcagat
tctacgcatc atggagtcca tttgggagac tgaatctctg 2580gacctgtgcc
ttctgcctta cggttgcatc tcaactggtg acaaaatagg aatgatcgag
2640attgtaaagg atgccacaac gatcgctcaa attcagcaaa gcacagtggg
taacacgggg 2700gcattcaaag atgaagtcct gaatcactgg ctcaaggaaa
aatgtcctat tgaagaaaag 2760tttcaggccg cagtggaaag gtttgtttac
tcctgtgcag gctactgtgt ggccacattt 2820gttcttggga tcggtgacag
gcacaacgac aacattatga tctcagagac aggaaaccta 2880tttcatatag
acttcggaca cattcttggg aattacaaga gtttcctggg catcaataaa
2940gagagagtgc ccttcgtcct aaccccagac ttcttgtttg tgatgggatc
ttctggaaaa 3000aagacaagtc cacacttcca gaaattccag gatgtctgtg
ttagagctta cctagctctt 3060cgccatcaca caaacctgtt gatcatcttg
ttctccatga tgctgatgac aggaatgccc 3120cagctgacaa gcaaagagga
cattgaatat atccgggatg ccctcaccgt gggaaaaagc 3180gaggaggacg
ctaagaaata tttccttgat cagatcgaag tctgcagaga caaaggatgg
3240actgtgcagt ttaactggtt cctacatctt gttcttggca tcaaacaagg
agaaaagcac 3300tccgcttga 3309241102PRTMus musculus 24Met Glu Leu
Glu Asn Tyr Glu Gln Pro Val Val Leu Arg Glu Asp Asn1 5 10 15Leu Arg
Arg Arg Arg Arg Met Lys Pro Arg Ser Ala Ala Gly Ser Leu 20 25 30Ser
Ser Met Glu Leu Ile Pro Ile Glu Phe Val Leu Pro Thr Ser Gln 35 40
45Arg Ile Ser Lys Thr Pro Glu Thr Ala Leu Leu His Val Ala Gly His
50 55 60Gly Asn Val Glu Gln Met Lys Ala Gln Val Trp Leu Arg Ala Leu
Glu65 70 75 80Thr Ser Val Ala Ala Glu Phe Tyr His Arg Leu Gly Pro
Asp Gln Phe 85 90 95Leu Leu Leu Tyr Gln Lys Lys Gly Gln Trp Tyr Glu
Ile Tyr Asp Arg 100 105 110Tyr Gln Val Val Gln Thr Leu Asp Cys Leu
His Tyr Trp Lys Leu Met 115 120 125His Lys Ser Pro Gly Gln Ile His
Val Val Gln Arg His Val Pro Ser 130 135 140Glu Glu Thr Leu Ala Phe
Gln Lys Gln Leu Thr Ser Leu Ile Gly Tyr145 150 155 160Asp Val Thr
Asp Ile Ser Asn Val His Asp Asp Glu Leu Glu Phe Thr 165 170 175Arg
Arg Arg Leu Val Thr Pro Arg Met Ala Glu Val Ala Gly Arg Asp 180 185
190Ala Lys Leu Tyr Ala Met His Pro Trp Val Thr Ser Lys Pro Leu Pro
195 200 205Asp Tyr Leu Ser Lys Lys Ile Ala Asn Asn Cys Ile Phe Ile
Val Ile 210 215 220His Arg Gly Thr Thr Ser Gln Thr Ile Lys Val Ser
Ala Asp Asp Thr225 230 235 240Pro Gly Thr Ile Leu Gln Ser Phe Phe
Thr Lys Met Ala Lys Lys Lys 245 250 255Ser Leu Met Asn Ile Ser Glu
Ser Gln Ser Glu Gln Asp Phe Val Leu 260 265 270Arg Val Cys Gly Arg
Asp Glu Tyr Leu Val Gly Glu Thr Pro Leu Lys 275 280 285Asn Phe Gln
Trp Val Arg Gln Cys Leu Lys Asn Gly Asp Glu Ile His 290 295 300Leu
Val Leu Asp Thr Pro Pro Asp Pro Ala Leu Asp Glu Val Arg Lys305 310
315 320Glu Glu Trp Pro Leu Val Asp Asp Cys Thr Gly Val Thr Gly Tyr
His 325 330 335Glu Gln Leu Thr Ile His Gly Lys Asp His Glu Ser Val
Phe Thr Val 340 345 350Ser Leu Trp Asp Cys Asp Arg Lys Phe Arg Val
Lys Ile Arg Gly Ile 355 360 365Asp Ile Pro Val Leu Pro Arg Asn Thr
Asp Leu Thr Val Phe Val Glu 370 375 380Ala Asn Ile Gln His Gly Gln
Gln Val Leu Cys Gln Arg Arg Thr Ser385 390 395 400Pro Lys Pro Phe
Ala Glu Glu Val Leu Trp Asn Val Trp Leu Glu Phe 405 410 415Gly Ile
Lys Ile Lys Asp Leu Pro Lys Gly Ala Leu Leu Asn Leu Gln 420 425
430Ile Tyr Cys Cys Lys Thr Pro Ser Leu Ser Ser Lys Ala Ser Ala Glu
435 440 445Thr Pro Gly Ser Glu Ser Lys Gly Lys Ala Gln Leu Leu Tyr
Tyr Val 450 455 460Asn Leu Leu Leu Ile Asp His Arg Phe Leu Leu Arg
His Gly Asp Tyr465 470 475 480Val Leu His Met Trp Gln Ile Ser Gly
Lys Ala Glu Glu Gln Gly Ser 485 490 495Phe Asn Ala Asp Lys Leu Thr
Ser Ala Thr Asn Pro Asp Lys Glu Asn 500 505 510Ser Met Ser Ile Ser
Ile Leu Leu Asp Asn Tyr Cys His Pro Ile Ala 515 520 525Leu Pro Lys
His Arg Pro Thr Pro Asp Pro Glu Gly Asp Arg Val Arg 530 535 540Ala
Glu Met Pro Asn Gln Leu Arg Lys Gln Leu Glu Ala Ile Ile Ala545 550
555 560Thr Asp Pro Leu Asn Pro Leu Thr Ala Glu Asp Lys Glu Leu Leu
Trp 565 570 575His Phe Arg Tyr Glu Ser Leu Lys His Pro Lys Ala Tyr
Pro Lys Leu 580 585 590Phe Ser Ser Val Lys Trp Gly Gln Gln Glu Ile
Val Ala Lys Thr Tyr 595 600 605Gln Leu Leu Ala Arg Arg Glu Ile Trp
Asp Gln Ser Ala Leu Asp Val 610 615 620Gly Leu Thr Met Gln Leu Leu
Asp Cys Asn Phe Ser Asp Glu Asn Val625 630 635 640Arg Ala Ile Ala
Val Gln Lys Leu Glu Ser Leu Glu Asp Asp Asp Val 645 650 655Leu His
Tyr Leu Leu Gln Leu Val Gln Ala Val Lys Phe Glu Pro Tyr 660 665
670His Asp Ser Ala Leu Ala Arg Phe Leu Leu Lys Arg Gly Leu Arg Asn
675 680 685Lys Arg Ile Gly His Phe Leu Phe Trp Phe Leu Arg Ser Glu
Ile Ala 690 695 700Gln Ser Arg His Tyr Gln Gln Arg Phe Ala Val Ile
Leu Glu Ala Tyr705 710 715 720Leu Arg Gly Cys Gly Thr Ala Met Leu
Gln Asp Phe Thr Gln Gln Val 725 730 735His Val Ile Glu Met Leu Gln
Lys Val Thr Ile Asp Ile Lys Ser Leu 740 745 750Ser Ala Glu Lys Tyr
Asp Val Ser Ser Gln Val Ile Ser Gln Leu Lys 755 760 765Gln Lys Leu
Glu Ser Leu Gln Asn Ser Asn Leu Pro Glu Ser Phe Arg 770 775 780Val
Pro Tyr Asp Pro Gly Leu Lys Ala Gly Thr Leu Val Ile Glu Lys785 790
795 800Cys Lys Val Met Ala Ser Lys Lys Lys Pro Leu Trp Leu Glu Phe
Lys 805 810 815Cys Ala Asp Pro Thr Val Leu Ser Asn Glu Thr Ile Gly
Ile Ile Phe 820 825 830Lys His Gly Asp Asp Leu Arg Gln Asp Met Leu
Ile Leu Gln Ile Leu 835 840 845Arg Ile Met Glu Ser Ile Trp Glu Thr
Glu Ser Leu Asp Leu Cys Leu 850 855 860Leu Pro Tyr Gly Cys Ile Ser
Thr Gly Asp Lys Ile Gly Met Ile Glu865 870 875 880Ile Val Lys Asp
Ala Thr Thr Ile Ala Gln Ile Gln Gln Ser Thr Val 885 890 895Gly Asn
Thr Gly Ala Phe Lys Asp Glu Val Leu Asn His Trp Leu Lys 900 905
910Glu Lys Cys Pro Ile Glu Glu Lys Phe Gln Ala Ala Val Glu Arg Phe
915 920 925Val Tyr Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Val Leu
Gly Ile 930 935 940Gly Asp Arg His Asn Asp Asn Ile Met Ile Ser Glu
Thr Gly Asn Leu945 950 955 960Phe His Ile Asp Phe Gly His Ile Leu
Gly Asn Tyr Lys Ser Phe Leu 965 970 975Gly Ile Asn Lys Glu Arg Val
Pro Phe Val Leu Thr Pro Asp Phe Leu 980 985 990Phe Val Met Gly Ser
Ser Gly Lys Lys Thr Ser Pro His Phe Gln Lys 995 1000 1005Phe Gln
Asp Val Cys Val Arg Ala Tyr Leu Ala Leu Arg His His 1010 1015
1020Thr Asn Leu Leu Ile Ile Leu Phe Ser Met Met Leu Met Thr Gly
1025 1030 1035Met Pro Gln Leu Thr Ser Lys Glu Asp Ile Glu Tyr Ile
Arg Asp 1040 1045 1050Ala Leu Thr Val Gly Lys Ser Glu Glu Asp Ala
Lys Lys Tyr Phe 1055 1060 1065Leu Asp Gln Ile Glu Val Cys Arg Asp
Lys Gly Trp Thr Val Gln 1070 1075 1080Phe Asn Trp Phe Leu His Leu
Val Leu Gly Ile Lys Gln Gly Glu 1085 1090 1095Lys His Ser Ala
1100253135DNAHomo sapiens 25atgccccctg gggtggactg ccccatggaa
ttctggacca aggaggagaa tcagagcgtt 60gtggttgact tcctgctgcc cacaggggtc
tacctgaact tccctgtgtc ccgcaatgcc 120aacctcagca ccatcaagca
gctgctgtgg caccgcgccc agtatgagcc gctcttccac 180atgctcagtg
gccccgaggc ctatgtgttc acctgcatca accagacagc ggagcagcaa
240gagctggagg acgagcaacg gcgtctgtgt gacgtgcagc ccttcctgcc
cgtcctgcgc 300ctggtggccc gtgagggcga ccgcgtgaag aagctcatca
actcacagat cagcctcctc 360atcggcaaag gcctccacga gtttgactcc
ttgtgcgacc cagaagtgaa cgactttcgc 420gccaagatgt gccaattctg
cgaggaggcg gccgcccgcc ggcagcagct gggctgggag 480gcctggctgc
agtacagttt ccccctgcag ctggagccct cggctcaaac ctgggggcct
540ggtaccctgc ggctcccgaa ccgggccctt ctggtcaacg ttaagtttga
gggcagcgag 600gagagcttca ccttccaggt gtccaccaag gacgtgccgc
tggcgctgat ggcctgtgcc 660ctgcggaaga aggccacagt gttccggcag
ccgctggtgg agcagccgga agactacacg 720ctgcaggtga acggcaggca
tgagtacctg tatggcagct acccgctctg ccagttccag 780tacatctgca
gctgcctgca cagtgggttg acccctcacc tgaccatggt ccattcctcc
840tccatcctcg ccatgcggga tgagcagagc aaccctgccc cccaggtcca
gaaaccgcgt 900gccaaaccac ctcccattcc tgcgaagaag ccttcctctg
tgtccctgtg gtccctggag 960cagccgttcc gcatcgagct catccagggc
agcaaagtga acgccgacga gcggatgaag 1020ctggtggtgc aggccgggct
tttccacggc aacgagatgc tgtgcaagac ggtgtccagc 1080tcggaggtga
gcgtgtgctc ggagcccgtg tggaagcagc ggctggagtt cgacatcaac
1140atctgcgacc tgccccgcat ggcccgtctc tgctttgcgc tgtacgccgt
gatcgagaaa 1200gccaagaagg ctcgctccac caagaagaag tccaagaagg
cggactgccc cattgcctgg 1260gccaacctca tgctgtttga ctacaaggac
cagcttaaga ccggggaacg ctgcctctac 1320atgtggccct ccgtcccaga
tgagaagggc gagctgctga accccacggg cactgtgcgc 1380agtaacccca
acacggatag cgccgctgcc ctgctcatct gcctgcccga ggtggccccg
1440caccccgtgt actaccccgc cctggagaag atcttggagc tggggcgaca
cagcgagtgt 1500gtgcatgtca ccgaggagga gcagctgcag ctgcgggaaa
tcctggagcg gcgggggtct 1560ggggagctgt atgagcacga gaaggacctg
gtgtggaagc tgcggcatga agtccaggag 1620cacttcccgg aggcgctagc
ccggctgctg ctggtcacca agtggaacaa gcatgaggat 1680gtggcccaga
tgctctacct gctgtgctcc tggccggagc tgcccgtcct gagcgccctg
1740gagctgctag acttcagctt ccccgattgc cacgtaggct ccttcgccat
caagtcgctg 1800cggaaactga cggacgatga gctgttccag tacctgctgc
agctggtgca ggtgctcaag 1860tacgagtcct acctggactg cgagctgacc
aaattcctgc tggaccgggc cctggccaac 1920cgcaagatcg gccacttcct
tttctggcac ctccgctccg agatgcacgt gccgtcggtg 1980gccctgcgct
tcggcctcat cctggaggcc tactgcaggg gcagcaccca ccacatgaag
2040gtgctgatga agcaggggga agcactgagc aaactgaagg ccctgaatga
cttcgtcaag 2100ctgagctctc agaagacccc caagccccag accaaggagc
tgatgcactt gtgcatgcgg 2160caggaggcct acctagaggc cctctcccac
ctgcagtccc cactcgaccc cagcaccctg 2220ctggctgaag tctgcgtgga
gcagtgcacc ttcatggact ccaagatgaa gcccctgtgg 2280atcatgtaca
gcaacgagga ggcaggcagc ggcggcagcg tgggcatcat ctttaagaac
2340ggggatgacc tccggcagga catgctgacc ctgcagatga tccagctcat
ggacgtcctg 2400tggaagcagg aggggctgga cctgaggatg accccctatg
gctgcctccc caccggggac 2460cgcacaggcc tcattgaggt ggtactccgt
tcagacacca tcgccaacat ccaactcaac 2520aagagcaaca tggcagccac
agccgccttc aacaaggatg ccctgctcaa ctggctgaag 2580tccaagaacc
cgggggaggc cctggatcga gccattgagg agttcaccct ctcctgtgct
2640ggctattgtg tggccacata tgtgctgggc attggcgatc ggcacagcga
caacatcatg 2700atccgagaga gtgggcagct gttccacatt gattttggcc
actttctggg gaatttcaag 2760accaagtttg gaatcaaccg cgagcgtgtc
ccattcatcc tcacctacga ctttgtccat 2820gtgattcagc aggggaagac
taataatagt gagaaatttg aacggttccg gggctactgt 2880gaaagggcct
acaccatcct gcggcgccac gggcttctct tcctccacct ctttgccctg
2940atgcgggcgg caggcctgcc tgagctcagc tgctccaaag acatccagta
tctcaaggac 3000tccctggcac tggggaaaac agaggaggag gcactgaagc
acttccgagt gaagtttaac 3060gaagccctcc gtgagagctg gaaaaccaaa
gtgaactggc tggcccacaa cgtgtccaaa 3120gacaacaggc agtag
3135261044PRTHomo sapiens 26Met Pro Pro Gly Val Asp Cys Pro Met Glu
Phe Trp Thr Lys Glu Glu1 5 10 15Asn Gln Ser Val Val Val Asp Phe Leu
Leu Pro Thr Gly Val Tyr Leu 20 25 30Asn Phe Pro Val Ser Arg Asn Ala
Asn Leu Ser Thr Ile Lys Gln Leu 35 40 45Leu Trp His Arg Ala Gln Tyr
Glu Pro Leu Phe His Met Leu Ser Gly 50 55 60Pro Glu Ala Tyr Val Phe
Thr Cys Ile Asn Gln Thr Ala Glu Gln Gln65 70 75 80Glu Leu Glu Asp
Glu Gln Arg Arg Leu Cys Asp Val Gln Pro Phe Leu 85 90 95Pro Val Leu
Arg Leu Val Ala Arg Glu Gly Asp Arg Val Lys Lys Leu 100 105 110Ile
Asn Ser Gln Ile Ser Leu Leu Ile Gly Lys Gly Leu His Glu Phe 115 120
125Asp Ser Leu Cys Asp Pro Glu Val Asn Asp Phe Arg Ala Lys Met Cys
130 135 140Gln Phe Cys Glu Glu Ala Ala Ala Arg Arg Gln Gln Leu Gly
Trp Glu145 150 155 160Ala Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu
Glu Pro Ser Ala Gln 165 170 175Thr Trp Gly Pro Gly Thr Leu Arg Leu
Pro Asn Arg Ala Leu Leu Val 180 185 190Asn Val Lys Phe Glu Gly Ser
Glu Glu Ser Phe Thr Phe Gln Val Ser 195 200 205Thr Lys Asp Val Pro
Leu Ala Leu Met Ala Cys Ala Leu Arg Lys Lys 210 215 220Ala Thr Val
Phe Arg Gln Pro Leu Val Glu Gln Pro Glu Asp Tyr Thr225 230 235
240Leu Gln Val Asn Gly Arg His Glu Tyr Leu Tyr Gly Ser Tyr Pro Leu
245 250 255Cys Gln Phe Gln Tyr Ile Cys Ser Cys Leu His Ser Gly Leu
Thr Pro 260 265 270His Leu Thr Met Val His Ser Ser Ser Ile Leu Ala
Met Arg Asp Glu 275 280 285Gln Ser Asn Pro Ala Pro Gln Val Gln Lys
Pro Arg Ala Lys Pro Pro 290 295 300Pro Ile Pro Ala Lys Lys Pro Ser
Ser Val Ser Leu Trp Ser Leu Glu305 310 315 320Gln Pro Phe Arg Ile
Glu Leu Ile Gln Gly Ser Lys Val Asn Ala Asp 325 330 335Glu Arg Met
Lys Leu Val Val Gln Ala Gly Leu Phe His Gly Asn Glu 340 345 350Met
Leu Cys Lys Thr Val Ser Ser Ser Glu Val Ser Val Cys Ser Glu 355 360
365Pro Val Trp Lys Gln Arg Leu Glu Phe Asp Ile Asn Ile Cys Asp Leu
370
375 380Pro Arg Met Ala Arg Leu Cys Phe Ala Leu Tyr Ala Val Ile Glu
Lys385 390 395 400Ala Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser Lys
Lys Ala Asp Cys 405 410 415Pro Ile Ala Trp Ala Asn Leu Met Leu Phe
Asp Tyr Lys Asp Gln Leu 420 425 430Lys Thr Gly Glu Arg Cys Leu Tyr
Met Trp Pro Ser Val Pro Asp Glu 435 440 445Lys Gly Glu Leu Leu Asn
Pro Thr Gly Thr Val Arg Ser Asn Pro Asn 450 455 460Thr Asp Ser Ala
Ala Ala Leu Leu Ile Cys Leu Pro Glu Val Ala Pro465 470 475 480His
Pro Val Tyr Tyr Pro Ala Leu Glu Lys Ile Leu Glu Leu Gly Arg 485 490
495His Ser Glu Cys Val His Val Thr Glu Glu Glu Gln Leu Gln Leu Arg
500 505 510Glu Ile Leu Glu Arg Arg Gly Ser Gly Glu Leu Tyr Glu His
Glu Lys 515 520 525Asp Leu Val Trp Lys Leu Arg His Glu Val Gln Glu
His Phe Pro Glu 530 535 540Ala Leu Ala Arg Leu Leu Leu Val Thr Lys
Trp Asn Lys His Glu Asp545 550 555 560Val Ala Gln Met Leu Tyr Leu
Leu Cys Ser Trp Pro Glu Leu Pro Val 565 570 575Leu Ser Ala Leu Glu
Leu Leu Asp Phe Ser Phe Pro Asp Cys His Val 580 585 590Gly Ser Phe
Ala Ile Lys Ser Leu Arg Lys Leu Thr Asp Asp Glu Leu 595 600 605Phe
Gln Tyr Leu Leu Gln Leu Val Gln Val Leu Lys Tyr Glu Ser Tyr 610 615
620Leu Asp Cys Glu Leu Thr Lys Phe Leu Leu Asp Arg Ala Leu Ala
Asn625 630 635 640Arg Lys Ile Gly His Phe Leu Phe Trp His Leu Arg
Ser Glu Met His 645 650 655Val Pro Ser Val Ala Leu Arg Phe Gly Leu
Ile Leu Glu Ala Tyr Cys 660 665 670Arg Gly Ser Thr His His Met Lys
Val Leu Met Lys Gln Gly Glu Ala 675 680 685Leu Ser Lys Leu Lys Ala
Leu Asn Asp Phe Val Lys Leu Ser Ser Gln 690 695 700Lys Thr Pro Lys
Pro Gln Thr Lys Glu Leu Met His Leu Cys Met Arg705 710 715 720Gln
Glu Ala Tyr Leu Glu Ala Leu Ser His Leu Gln Ser Pro Leu Asp 725 730
735Pro Ser Thr Leu Leu Ala Glu Val Cys Val Glu Gln Cys Thr Phe Met
740 745 750Asp Ser Lys Met Lys Pro Leu Trp Ile Met Tyr Ser Asn Glu
Glu Ala 755 760 765Gly Ser Gly Gly Ser Val Gly Ile Ile Phe Lys Asn
Gly Asp Asp Leu 770 775 780Arg Gln Asp Met Leu Thr Leu Gln Met Ile
Gln Leu Met Asp Val Leu785 790 795 800Trp Lys Gln Glu Gly Leu Asp
Leu Arg Met Thr Pro Tyr Gly Cys Leu 805 810 815Pro Thr Gly Asp Arg
Thr Gly Leu Ile Glu Val Val Leu Arg Ser Asp 820 825 830Thr Ile Ala
Asn Ile Gln Leu Asn Lys Ser Asn Met Ala Ala Thr Ala 835 840 845Ala
Phe Asn Lys Asp Ala Leu Leu Asn Trp Leu Lys Ser Lys Asn Pro 850 855
860Gly Glu Ala Leu Asp Arg Ala Ile Glu Glu Phe Thr Leu Ser Cys
Ala865 870 875 880Gly Tyr Cys Val Ala Thr Tyr Val Leu Gly Ile Gly
Asp Arg His Ser 885 890 895Asp Asn Ile Met Ile Arg Glu Ser Gly Gln
Leu Phe His Ile Asp Phe 900 905 910Gly His Phe Leu Gly Asn Phe Lys
Thr Lys Phe Gly Ile Asn Arg Glu 915 920 925Arg Val Pro Phe Ile Leu
Thr Tyr Asp Phe Val His Val Ile Gln Gln 930 935 940Gly Lys Thr Asn
Asn Ser Glu Lys Phe Glu Arg Phe Arg Gly Tyr Cys945 950 955 960Glu
Arg Ala Tyr Thr Ile Leu Arg Arg His Gly Leu Leu Phe Leu His 965 970
975Leu Phe Ala Leu Met Arg Ala Ala Gly Leu Pro Glu Leu Ser Cys Ser
980 985 990Lys Asp Ile Gln Tyr Leu Lys Asp Ser Leu Ala Leu Gly Lys
Thr Glu 995 1000 1005Glu Glu Ala Leu Lys His Phe Arg Val Lys Phe
Asn Glu Ala Leu 1010 1015 1020Arg Glu Ser Trp Lys Thr Lys Val Asn
Trp Leu Ala His Asn Val 1025 1030 1035Ser Lys Asp Asn Arg Gln
1040273132DNAMus musculus 27atgccccctg gggtggactg ccccatggag
ttctggacca aagaggagag ccagagcgtg 60gttgttgact tcttgctgcc cacaggggtc
tacttgaact tccccgtgtc ccgcaatgcc 120aacctcagca ccatcaagca
ggtgctgtgg caccgtgcac agtatgagcc actcttccac 180atgctcagtg
accccgaggc ctatgtgttc acctgtgtga accagacggc ggagcagcag
240gagttggagg atgagcagcg gaggctgtgc gacatccagc ccttcctgcc
cgtgctgcgc 300ctcgtggccc gagaggggga ccgcgtgaag aagctcatta
actcccagat cagcctcctc 360attggcaaag gtctccatga gtttgattcc
ctgcgggacc cggaagtaaa cgacttccgc 420actaagatgc gccagttttg
tgaagaggct gctgctcacc gccagcagct gggctgggtg 480gaatggctgc
agtacagctt ccccctgcag ctggagccct cagcaagggg ttggcgggcc
540ggcttattgc gtgtcagcaa ccgagccctg ctggtcaacg tgaagttcga
gggcagtgag 600gagagcttca ccttccaggt atccaccaag gacatgcccc
tggcactgat ggcctgtgcc 660ctccgaaaaa aggccacagt gttccggcag
cctctggtgg agcagcctga ggaatatgcc 720ctgcaggtga acgggaggca
cgaatacctc tacggcaact acccgctctg ccactttcag 780tacatctgca
gctgcctaca cagcgggctg acccctcatc tgaccatggt ccactcctcc
840tccatccttg ctatgcggga tgagcagagc aatcctgccc cccaagtaca
gaaaccacgt 900gccaaacctc ccccgatccc tgccaagaag ccctcctctg
tgtccctgtg gtccctggaa 960cagccattct ccattgagct gatcgagggc
cgaaaagtga atgctgacga gcggatgaag 1020ctggttgttc aggccgggct
cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc 1080tcggaggtga
atgtatgctc agagcccgtg tggaagcagc gactggagtt cgatatcagc
1140gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc tctatgccgt
cgtggagaag 1200gctaagaagg cacgctccac aaagaagaag tctaagaagg
cggactgccc catcgcttgg 1260gccaacctca tgctattcga ctacaaagat
cagctcaaga cgggggagcg ctgcctctac 1320atgtggccct ctgtcccaga
tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1380gggaacccca
acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc
1440caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca
cggggagcgt 1500gggcgcatca cggaggagga gctgcagctg cgggagatcc
tggaacggcg gggatccggg 1560gaactgtacg aacatgagaa ggacctggtg
tggaagatgc gccacgaagt ccaggagcat 1620ttcccagagg cgctggcccg
cctgctgctg gtcaccaagt ggaataaaca cgaggatgtg 1680gcccagatgc
tctatttgct gtgctcctgg cccgagctgc ctgtgctgag cgccctggaa
1740cttctggact ttagctttcc cgactgctac gtgggctcct tcgccatcaa
gtcccttcgg 1800aagctgacgg acgatgagct cttccagtac cttctgcagc
tggtgcaagt gctcaaatat 1860gagtcctacc tggactgcga gctgaccaaa
ttcttgctgg gccgagccct ggctaaccgc 1920aagatcggac acttcctgtt
ctggcacctc cgctctgaga tgcacgtacc atcagtggct 1980ctgcggtttg
gtctcatcat ggaagcctac tgcagaggca gcacccacca catgaaggtg
2040ctgatgaagc agggggaagc actgagcaag cttaaggcac tgaatgactt
tgtgaaggtg 2100agttcccaga agaccaccaa gccccaaacc aaggagatga
tgcatatgtg catgcgccag 2160gagacctaca tggaggccct gtcccacctg
cagtctccac tcgaccccag caccctgctg 2220gaggaagtct gtgtggagca
gtgcaccttc atggactcca aaatgaagcc cctgtggatc 2280atgtacagca
gcgaggaggc gggcagtgct ggcaacgtgg gcatcatctt taagaacggg
2340gatgacctcc gccaggacat gctgactctg cagatgatcc agctcatgga
cgtcctgtgg 2400aagcaggagg gcctggacct gaggatgacg ccctacggct
gcctccccac cggggaccgc 2460acaggtctca tcgaggtggt cctccactcg
gacaccatcg ccaacatcca gctgaacaaa 2520agcaacatgg cggccacagc
tgccttcaac aaggacgccc tgctcaactg gctcaagtcc 2580aagaaccctg
gggaggccct ggatcgggcc attgaggaat tcaccctctc ctgtgctggc
2640tactgtgtgg ccacatatgt tctgggcatc ggtgaccggc acagcgacaa
catcatgatc 2700agagagagtg ggcagctctt ccacattgat tttggccact
ttctggggaa cttcaagacc 2760aagtttggaa tcaaccgaga gcgcgtcccc
ttcattctca cctacgactt tgtccacgtg 2820atccagcagg ggaagactaa
caacagtgag aagtttgaaa ggttccgcgg ctactgtgaa 2880cgagcctata
ccatcctgcg gcgccacggg ctgcttttcc tccatctctt cgccctgatg
2940cgggccgcag gtctgcctga gcttagctgc tccaaagata tccagtatct
caaggactct 3000ctggcactgg ggaagacgga ggaagaggcg ctaaagcact
tccgggtgaa gttcaacgaa 3060gctctccgag aaagctggaa aaccaaagtc
aactggctgg cgcacaatgt gtccaaggat 3120aaccgacagt ag 3132281043PRTMus
musculus 28Met Pro Pro Gly Val Asp Cys Pro Met Glu Phe Trp Thr Lys
Glu Glu1 5 10 15Ser Gln Ser Val Val Val Asp Phe Leu Leu Pro Thr Gly
Val Tyr Leu 20 25 30Asn Phe Pro Val Ser Arg Asn Ala Asn Leu Ser Thr
Ile Lys Gln Val 35 40 45Leu Trp His Arg Ala Gln Tyr Glu Pro Leu Phe
His Met Leu Ser Asp 50 55 60Pro Glu Ala Tyr Val Phe Thr Cys Val Asn
Gln Thr Ala Glu Gln Gln65 70 75 80Glu Leu Glu Asp Glu Gln Arg Arg
Leu Cys Asp Ile Gln Pro Phe Leu 85 90 95Pro Val Leu Arg Leu Val Ala
Arg Glu Gly Asp Arg Val Lys Lys Leu 100 105 110Ile Asn Ser Gln Ile
Ser Leu Leu Ile Gly Lys Gly Leu His Glu Phe 115 120 125Asp Ser Leu
Arg Asp Pro Glu Val Asn Asp Phe Arg Thr Lys Met Arg 130 135 140Gln
Phe Cys Glu Glu Ala Ala Ala His Arg Gln Gln Leu Gly Trp Val145 150
155 160Glu Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu Glu Pro Ser Ala
Arg 165 170 175Gly Trp Arg Ala Gly Leu Leu Arg Val Ser Asn Arg Ala
Leu Leu Val 180 185 190Asn Val Lys Phe Glu Gly Ser Glu Glu Ser Phe
Thr Phe Gln Val Ser 195 200 205Thr Lys Asp Met Pro Leu Ala Leu Met
Ala Cys Ala Leu Arg Lys Lys 210 215 220Ala Thr Val Phe Arg Gln Pro
Leu Val Glu Gln Pro Glu Glu Tyr Ala225 230 235 240Leu Gln Val Asn
Gly Arg His Glu Tyr Leu Tyr Gly Asn Tyr Pro Leu 245 250 255Cys His
Phe Gln Tyr Ile Cys Ser Cys Leu His Ser Gly Leu Thr Pro 260 265
270His Leu Thr Met Val His Ser Ser Ser Ile Leu Ala Met Arg Asp Glu
275 280 285Gln Ser Asn Pro Ala Pro Gln Val Gln Lys Pro Arg Ala Lys
Pro Pro 290 295 300Pro Ile Pro Ala Lys Lys Pro Ser Ser Val Ser Leu
Trp Ser Leu Glu305 310 315 320Gln Pro Phe Ser Ile Glu Leu Ile Glu
Gly Arg Lys Val Asn Ala Asp 325 330 335Glu Arg Met Lys Leu Val Val
Gln Ala Gly Leu Phe His Gly Asn Glu 340 345 350Met Leu Cys Lys Thr
Val Ser Ser Ser Glu Val Asn Val Cys Ser Glu 355 360 365Pro Val Trp
Lys Gln Arg Leu Glu Phe Asp Ile Ser Val Cys Asp Leu 370 375 380Pro
Arg Met Ala Arg Leu Cys Phe Ala Leu Tyr Ala Val Val Glu Lys385 390
395 400Ala Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser Lys Lys Ala Asp
Cys 405 410 415Pro Ile Ala Trp Ala Asn Leu Met Leu Phe Asp Tyr Lys
Asp Gln Leu 420 425 430Lys Thr Gly Glu Arg Cys Leu Tyr Met Trp Pro
Ser Val Pro Asp Glu 435 440 445Lys Gly Glu Leu Leu Asn Pro Ala Gly
Thr Val Arg Gly Asn Pro Asn 450 455 460Thr Glu Ser Ala Ala Ala Leu
Val Ile Tyr Leu Pro Glu Val Ala Pro465 470 475 480His Pro Val Tyr
Phe Pro Ala Leu Glu Lys Ile Leu Glu Leu Gly Arg 485 490 495His Gly
Glu Arg Gly Arg Ile Thr Glu Glu Glu Leu Gln Leu Arg Glu 500 505
510Ile Leu Glu Arg Arg Gly Ser Gly Glu Leu Tyr Glu His Glu Lys Asp
515 520 525Leu Val Trp Lys Met Arg His Glu Val Gln Glu His Phe Pro
Glu Ala 530 535 540Leu Ala Arg Leu Leu Leu Val Thr Lys Trp Asn Lys
His Glu Asp Val545 550 555 560Ala Gln Met Leu Tyr Leu Leu Cys Ser
Trp Pro Glu Leu Pro Val Leu 565 570 575Ser Ala Leu Glu Leu Leu Asp
Phe Ser Phe Pro Asp Cys Tyr Val Gly 580 585 590Ser Phe Ala Ile Lys
Ser Leu Arg Lys Leu Thr Asp Asp Glu Leu Phe 595 600 605Gln Tyr Leu
Leu Gln Leu Val Gln Val Leu Lys Tyr Glu Ser Tyr Leu 610 615 620Asp
Cys Glu Leu Thr Lys Phe Leu Leu Gly Arg Ala Leu Ala Asn Arg625 630
635 640Lys Ile Gly His Phe Leu Phe Trp His Leu Arg Ser Glu Met His
Val 645 650 655Pro Ser Val Ala Leu Arg Phe Gly Leu Ile Met Glu Ala
Tyr Cys Arg 660 665 670Gly Ser Thr His His Met Lys Val Leu Met Lys
Gln Gly Glu Ala Leu 675 680 685Ser Lys Leu Lys Ala Leu Asn Asp Phe
Val Lys Val Ser Ser Gln Lys 690 695 700Thr Thr Lys Pro Gln Thr Lys
Glu Met Met His Met Cys Met Arg Gln705 710 715 720Glu Thr Tyr Met
Glu Ala Leu Ser His Leu Gln Ser Pro Leu Asp Pro 725 730 735Ser Thr
Leu Leu Glu Glu Val Cys Val Glu Gln Cys Thr Phe Met Asp 740 745
750Ser Lys Met Lys Pro Leu Trp Ile Met Tyr Ser Ser Glu Glu Ala Gly
755 760 765Ser Ala Gly Asn Val Gly Ile Ile Phe Lys Asn Gly Asp Asp
Leu Arg 770 775 780Gln Asp Met Leu Thr Leu Gln Met Ile Gln Leu Met
Asp Val Leu Trp785 790 795 800Lys Gln Glu Gly Leu Asp Leu Arg Met
Thr Pro Tyr Gly Cys Leu Pro 805 810 815Thr Gly Asp Arg Thr Gly Leu
Ile Glu Val Val Leu His Ser Asp Thr 820 825 830Ile Ala Asn Ile Gln
Leu Asn Lys Ser Asn Met Ala Ala Thr Ala Ala 835 840 845Phe Asn Lys
Asp Ala Leu Leu Asn Trp Leu Lys Ser Lys Asn Pro Gly 850 855 860Glu
Ala Leu Asp Arg Ala Ile Glu Glu Phe Thr Leu Ser Cys Ala Gly865 870
875 880Tyr Cys Val Ala Thr Tyr Val Leu Gly Ile Gly Asp Arg His Ser
Asp 885 890 895Asn Ile Met Ile Arg Glu Ser Gly Gln Leu Phe His Ile
Asp Phe Gly 900 905 910His Phe Leu Gly Asn Phe Lys Thr Lys Phe Gly
Ile Asn Arg Glu Arg 915 920 925Val Pro Phe Ile Leu Thr Tyr Asp Phe
Val His Val Ile Gln Gln Gly 930 935 940Lys Thr Asn Asn Ser Glu Lys
Phe Glu Arg Phe Arg Gly Tyr Cys Glu945 950 955 960Arg Ala Tyr Thr
Ile Leu Arg Arg His Gly Leu Leu Phe Leu His Leu 965 970 975Phe Ala
Leu Met Arg Ala Ala Gly Leu Pro Glu Leu Ser Cys Ser Lys 980 985
990Asp Ile Gln Tyr Leu Lys Asp Ser Leu Ala Leu Gly Lys Thr Glu Glu
995 1000 1005Glu Ala Leu Lys His Phe Arg Val Lys Phe Asn Glu Ala
Leu Arg 1010 1015 1020Glu Ser Trp Lys Thr Lys Val Asn Trp Leu Ala
His Asn Val Ser 1025 1030 1035Lys Asp Asn Arg Gln 1040293144DNAMus
musculus 29atgccccctg gggtggactg ccccatggag ttctggacca aagaggagag
ccagagcgtg 60gttgttgact tcttgctgcc cacaggggtc tacttgaact tccccgtgtc
ccgcaatgcc 120aacctcagca ccatcaagca ggtgctgtgg caccgtgcac
agtatgagcc actcttccac 180atgctcagtg accccgaggc ctatgtgttc
acctgtgtga accagacggc ggagcagcag 240gagttggagg atgagcagcg
gaggctgtgc gacatccagc ccttcctgcc cgtgctgcgc 300ctcgtggccc
gagaggggga ccgcgtgaag aagctcatta actcccagat cagcctcctc
360attggcaaag gtctccatga gtttgattcc ctgcgggacc cggaagtaaa
cgacttccgc 420actaagatgc gccagttttg tgaagaggct gctgctcacc
gccagcagct gggctgggtg 480gaatggctgc agtacagctt ccccctgcag
ctggagccct cagcaagggg ttggcgggcc 540ggcttattgc gtgtcagcaa
ccgagccctg ctggtcaacg tgaagttcga gggcagtgag 600gagagcttca
ccttccaggt atccaccaag gacatgcccc tggcactgat ggcctgtgcc
660ctccgaaaaa aggccacagt gttccggcag cctctggtgg agcagcctga
ggaatatgcc 720ctgcaggtga acgggaggca cgaatacctc tacggcaact
acccgctctg ccactttcag 780tacatctgca gctgcctaca cagcgggctg
acccctcatc tgaccatggt ccactcctcc 840tccatccttg ctatgcggga
tgagcagagc aatcctgccc cccaagtaca gaaaccacgt 900gccaaacctc
ccccgatccc tgccaagaag ccctcctctg tgtccctgtg gtccctggaa
960cagccattct ccattgagct gatcgagggc cgaaaagtga atgctgacga
gcggatgaag 1020ctggttgttc aggccgggct cttccatggc aatgagatgc
tgtgcaagac tgtgtcaagc 1080tcggaggtga atgtatgctc agagcccgtg
tggaagcagc gactggagtt cgatatcagc 1140gtctgtgacc tcccgcgcat
ggctcgactc tgttttgctc tctatgccgt cgtggagaag 1200gctaagaagg
cacgctccac aaagaagaag tctaagaagg cggactgccc catcgcttgg
1260gccaacctca tgctattcga ctacaaagat cagctcaaga cgggggagcg
ctgcctctac 1320atgtggccct ctgtcccaga tgagaaggga gagctgctga
atcctgcggg tacagtgcgc 1380gggaacccca acacggagag tgccgctgcc
ctggtcatct acctgcctga ggtggccccc 1440caccctgtgt acttccccgc
tctggagaag atcctggagc tggggcgtca cggggagcgt 1500gggcgcatca
cggaggagga gcagctgcag ctgcgggaga tcctggaacg gcggggatcc
1560ggggaactgt acgaacatga gaaggacctg gtgtggaaga tgcgccacga
agtccaggag 1620catttcccag aggcgctggc ccgcctgctg ctggtcacca
agtggaataa acacgaggat 1680gtggcccagc tgtcccagat gctctatttg
ctgtgctcct ggcccgagct gcctgtgctg 1740agcgccctgg aacttctgga
ctttagcttt cccgactgct acgtgggctc cttcgccatc 1800aagtcccttc
ggaagctgac ggacgatgag ctcttccagt accttctgca gctggtgcaa
1860gtgctcaaat atgagtccta cctggactgc gagctgacca aattcttgct
gggccgagcc 1920ctggctaacc gcaagatcgg acacttcctg ttctggcacc
tccgctctga gatgcacgta 1980ccatcagtgg ctctgcggtt tggtctcatc
atggaagcct actgcagagg cagcacccac 2040cacatgaagg tgctgatgaa
gcagggggaa gcactgagca agcttaaggc actgaatgac 2100tttgtgaagg
tgagttccca gaagaccacc aagccccaaa ccaaggagat gatgcatatg
2160tgcatgcgcc aggagaccta catggaggcc ctgtcccacc tgcagtctcc
actcgacccc 2220agcaccctgc tggaggaagt ctgtgtggag cagtgcacct
tcatggactc caaaatgaag 2280cccctgtgga tcatgtacag cagcgaggag
gcgggcagtg ctggcaacgt gggcatcatc 2340tttaagaacg gggatgacct
ccgccaggac atgctgactc tgcagatgat ccagctcatg 2400gacgtcctgt
ggaagcagga gggcctggac ctgaggatga cgccctacgg ctgcctcccc
2460accggggacc gcacaggtct catcgaggtg gtcctccact cggacaccat
cgccaacatc 2520cagctgaaca aaagcaacat ggcggccaca gctgccttca
acaaggacgc cctgctcaac 2580tggctcaagt ccaagaaccc tggggaggcc
ctggatcggg ccattgagga attcaccctc 2640tcctgtgctg gctactgtgt
ggccacatat gttctgggca tcggtgaccg gcacagcgac 2700aacatcatga
tcagagagag tgggcagctc ttccacattg attttggcca ctttctgggg
2760aacttcaaga ccaagtttgg aatcaaccga gagcgcgtcc ccttcattct
cacctacgac 2820tttgtccacg tgatccagca ggggaagact aacaacagtg
agaagtttga aaggttccgc 2880ggctactgtg aacgagccta taccatcctg
cggcgccacg ggctgctttt cctccatctc 2940ttcgccctga tgcgggccgc
aggtctgcct gagcttagct gctccaaaga tatccagtat 3000ctcaaggact
ctctggcact ggggaagacg gaggaagagg cgctaaagca cttccgggtg
3060aagttcaacg aagctctccg agaaagctgg aaaaccaaag tcaactggct
ggcgcacaat 3120gtgtccaagg ataaccgaca gtag 3144301047PRTMus musculus
30Met Pro Pro Gly Val Asp Cys Pro Met Glu Phe Trp Thr Lys Glu Glu1
5 10 15Ser Gln Ser Val Val Val Asp Phe Leu Leu Pro Thr Gly Val Tyr
Leu 20 25 30Asn Phe Pro Val Ser Arg Asn Ala Asn Leu Ser Thr Ile Lys
Gln Val 35 40 45Leu Trp His Arg Ala Gln Tyr Glu Pro Leu Phe His Met
Leu Ser Asp 50 55 60Pro Glu Ala Tyr Val Phe Thr Cys Val Asn Gln Thr
Ala Glu Gln Gln65 70 75 80Glu Leu Glu Asp Glu Gln Arg Arg Leu Cys
Asp Ile Gln Pro Phe Leu 85 90 95Pro Val Leu Arg Leu Val Ala Arg Glu
Gly Asp Arg Val Lys Lys Leu 100 105 110Ile Asn Ser Gln Ile Ser Leu
Leu Ile Gly Lys Gly Leu His Glu Phe 115 120 125Asp Ser Leu Arg Asp
Pro Glu Val Asn Asp Phe Arg Thr Lys Met Arg 130 135 140Gln Phe Cys
Glu Glu Ala Ala Ala His Arg Gln Gln Leu Gly Trp Val145 150 155
160Glu Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu Glu Pro Ser Ala Arg
165 170 175Gly Trp Arg Ala Gly Leu Leu Arg Val Ser Asn Arg Ala Leu
Leu Val 180 185 190Asn Val Lys Phe Glu Gly Ser Glu Glu Ser Phe Thr
Phe Gln Val Ser 195 200 205Thr Lys Asp Met Pro Leu Ala Leu Met Ala
Cys Ala Leu Arg Lys Lys 210 215 220Ala Thr Val Phe Arg Gln Pro Leu
Val Glu Gln Pro Glu Glu Tyr Ala225 230 235 240Leu Gln Val Asn Gly
Arg His Glu Tyr Leu Tyr Gly Asn Tyr Pro Leu 245 250 255Cys His Phe
Gln Tyr Ile Cys Ser Cys Leu His Ser Gly Leu Thr Pro 260 265 270His
Leu Thr Met Val His Ser Ser Ser Ile Leu Ala Met Arg Asp Glu 275 280
285Gln Ser Asn Pro Ala Pro Gln Val Gln Lys Pro Arg Ala Lys Pro Pro
290 295 300Pro Ile Pro Ala Lys Lys Pro Ser Ser Val Ser Leu Trp Ser
Leu Glu305 310 315 320Gln Pro Phe Ser Ile Glu Leu Ile Glu Gly Arg
Lys Val Asn Ala Asp 325 330 335Glu Arg Met Lys Leu Val Val Gln Ala
Gly Leu Phe His Gly Asn Glu 340 345 350Met Leu Cys Lys Thr Val Ser
Ser Ser Glu Val Asn Val Cys Ser Glu 355 360 365Pro Val Trp Lys Gln
Arg Leu Glu Phe Asp Ile Ser Val Cys Asp Leu 370 375 380Pro Arg Met
Ala Arg Leu Cys Phe Ala Leu Tyr Ala Val Val Glu Lys385 390 395
400Ala Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser Lys Lys Ala Asp Cys
405 410 415Pro Ile Ala Trp Ala Asn Leu Met Leu Phe Asp Tyr Lys Asp
Gln Leu 420 425 430Lys Thr Gly Glu Arg Cys Leu Tyr Met Trp Pro Ser
Val Pro Asp Glu 435 440 445Lys Gly Glu Leu Leu Asn Pro Ala Gly Thr
Val Arg Gly Asn Pro Asn 450 455 460Thr Glu Ser Ala Ala Ala Leu Val
Ile Tyr Leu Pro Glu Val Ala Pro465 470 475 480His Pro Val Tyr Phe
Pro Ala Leu Glu Lys Ile Leu Glu Leu Gly Arg 485 490 495His Gly Glu
Arg Gly Arg Ile Thr Glu Glu Glu Gln Leu Gln Leu Arg 500 505 510Glu
Ile Leu Glu Arg Arg Gly Ser Gly Glu Leu Tyr Glu His Glu Lys 515 520
525Asp Leu Val Trp Lys Met Arg His Glu Val Gln Glu His Phe Pro Glu
530 535 540Ala Leu Ala Arg Leu Leu Leu Val Thr Lys Trp Asn Lys His
Glu Asp545 550 555 560Val Ala Gln Leu Ser Gln Met Leu Tyr Leu Leu
Cys Ser Trp Pro Glu 565 570 575Leu Pro Val Leu Ser Ala Leu Glu Leu
Leu Asp Phe Ser Phe Pro Asp 580 585 590Cys Tyr Val Gly Ser Phe Ala
Ile Lys Ser Leu Arg Lys Leu Thr Asp 595 600 605Asp Glu Leu Phe Gln
Tyr Leu Leu Gln Leu Val Gln Val Leu Lys Tyr 610 615 620Glu Ser Tyr
Leu Asp Cys Glu Leu Thr Lys Phe Leu Leu Gly Arg Ala625 630 635
640Leu Ala Asn Arg Lys Ile Gly His Phe Leu Phe Trp His Leu Arg Ser
645 650 655Glu Met His Val Pro Ser Val Ala Leu Arg Phe Gly Leu Ile
Met Glu 660 665 670Ala Tyr Cys Arg Gly Ser Thr His His Met Lys Val
Leu Met Lys Gln 675 680 685Gly Glu Ala Leu Ser Lys Leu Lys Ala Leu
Asn Asp Phe Val Lys Val 690 695 700Ser Ser Gln Lys Thr Thr Lys Pro
Gln Thr Lys Glu Met Met His Met705 710 715 720Cys Met Arg Gln Glu
Thr Tyr Met Glu Ala Leu Ser His Leu Gln Ser 725 730 735Pro Leu Asp
Pro Ser Thr Leu Leu Glu Glu Val Cys Val Glu Gln Cys 740 745 750Thr
Phe Met Asp Ser Lys Met Lys Pro Leu Trp Ile Met Tyr Ser Ser 755 760
765Glu Glu Ala Gly Ser Ala Gly Asn Val Gly Ile Ile Phe Lys Asn Gly
770 775 780Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Met Ile Gln
Leu Met785 790 795 800Asp Val Leu Trp Lys Gln Glu Gly Leu Asp Leu
Arg Met Thr Pro Tyr 805 810 815Gly Cys Leu Pro Thr Gly Asp Arg Thr
Gly Leu Ile Glu Val Val Leu 820 825 830His Ser Asp Thr Ile Ala Asn
Ile Gln Leu Asn Lys Ser Asn Met Ala 835 840 845Ala Thr Ala Ala Phe
Asn Lys Asp Ala Leu Leu Asn Trp Leu Lys Ser 850 855 860Lys Asn Pro
Gly Glu Ala Leu Asp Arg Ala Ile Glu Glu Phe Thr Leu865 870 875
880Ser Cys Ala Gly Tyr Cys Val Ala Thr Tyr Val Leu Gly Ile Gly Asp
885 890 895Arg His Ser Asp Asn Ile Met Ile Arg Glu Ser Gly Gln Leu
Phe His 900 905 910Ile Asp Phe Gly His Phe Leu Gly Asn Phe Lys Thr
Lys Phe Gly Ile 915 920 925Asn Arg Glu Arg Val Pro Phe Ile Leu Thr
Tyr Asp Phe Val His Val 930 935 940Ile Gln Gln Gly Lys Thr Asn Asn
Ser Glu Lys Phe Glu Arg Phe Arg945 950 955 960Gly Tyr Cys Glu Arg
Ala Tyr Thr Ile Leu Arg Arg His Gly Leu Leu 965 970 975Phe Leu His
Leu Phe Ala Leu Met Arg Ala Ala Gly Leu Pro Glu Leu 980 985 990Ser
Cys Ser Lys Asp Ile Gln Tyr Leu Lys Asp Ser Leu Ala Leu Gly 995
1000 1005Lys Thr Glu Glu Glu Ala Leu Lys His Phe Arg Val Lys Phe
Asn 1010 1015 1020Glu Ala Leu Arg Glu Ser Trp Lys Thr Lys Val Asn
Trp Leu Ala 1025 1030 1035His Asn Val Ser Lys Asp Asn Arg Gln 1040
1045313144DNAMus musculus 31atgccccctg gggtggactg ccccatggag
ttctggacca aagaggagag ccagagcgtg 60gttgttgact tcttgctgcc cacaggggtc
tacttgaact tccccgtgtc ccgcaatgcc 120aacctcagca ccatcaagca
ggtgctgtgg caccgtgcac agtatgagcc actcttccac 180atgctcagtg
accccgaggc ctatgtgttc acctgtgtga accagacggc ggagcagcag
240gagttggagg atgagcagcg gaggctgtgc gacatccagc ccttcctgcc
cgtgctgcgc 300ctcgtggccc gagaggggga ccgcgtgaag aagctcatta
actcccagat cagcctcctc 360attggcaaag gtctccatga gtttgattcc
ctgcgggacc cggaagtaaa cgacttccgc 420actaagatgc gccagttttg
tgaagaggct gctgctcacc gccagcagct gggctgggtg 480gaatggctgc
agtacagctt ccccctgcag ctggagccct cagcaagggg ttggcgggcc
540ggcttattgc gtgtcagcaa ccgagccctg ctggtcaacg tgaagttcga
gggcagtgag 600gagagcttca ccttccaggt atccaccaag gacatgcccc
tggcactgat ggcctgtgcc 660ctccgaaaaa aggccacagt gttccggcag
cctctggtgg agcagcctga ggaatatgcc 720ctgcaggtga acgggaggca
cgaatacctc tacggcaact acccgctctg ccactttcag 780tacatctgca
gctgcctaca cagcgggctg acccctcatc tgaccatggt ccactcctcc
840tccatccttg ctatgcggga tgagcagagc aatcctgccc cccaagtaca
gaaaccacgt 900gccaaacctc ccccgatccc tgccaagaag ccctcctctg
tgtccctgtg gtccctggaa 960cagccattct ccattgagct gatcgagggc
cgaaaagtga atgctgacga gcggatgaag 1020ctggttgttc aggccgggct
cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc 1080tcggaggtga
atgtatgctc agagcccgtg tggaagcagc gactggagtt cgatatcagc
1140gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc tctatgccgt
cgtggagaag 1200gctaagaagg cacgctccac aaagaagaag tctaagaagg
cggactgccc catcgcttgg 1260gccaacctca tgctattcga ctacaaagat
cagctcaaga cgggggagcg ctgcctctac 1320atgtggccct ctgtcccaga
tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1380gggaacccca
acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc
1440caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca
cggggagcgt 1500gggcgcatca cggaggagga gcagctgcag ctgcgggaga
tcctggaacg gcggggatcc 1560ggggaactgt acgaacatga gaaggacctg
gtgtggaaga tgcgccacga agtccaggag 1620catttcccag aggcgctggc
ccgcctgctg ctggtcacca agtggaataa acacgaggat 1680gtggcccagc
tgtcccagat gctctatttg ctgtgctcct ggcccgagct gcctgtgctg
1740agcgccctgg aacttctgga ctttagcttt cccgactgct acgtgggctc
cttcgccatc 1800aagtcccttc ggaagctgac ggacgatgag ctcttccagt
accttctgca gctggtgcaa 1860gtgctcaaat atgagtccta cctggactgc
gagctgacca aattcttgct gggccgagcc 1920ctggctaacc gcaagatcgg
acacttcctg ttctggcacc tccgctctga gatgcacgta 1980ccatcagtgg
ctctgcggtt tggtctcatc atggaagcct actgcagagg cagcacccac
2040cacatgaagg tgctgatgaa gcagggggaa gcactgagca agcttaaggc
actgaatgac 2100tttgtgaagg tgagttccca gaagaccacc aagccccaaa
ccaaggagat gatgcatatg 2160tgcatgcgcc aggagaccta catggaggcc
ctgtcccacc tgcagtctcc actcgacccc 2220agcaccctgc tggaggaagt
ctgtgtggag cagtgcacct tcatggactc caaaatgaag 2280cccctgtgga
tcatgtacag cagcgaggag gcgggcagtg ctggcaacgt gggcatcatc
2340tttaagaacg gggatgacct ccgccaggac atgctgactc tgcagatgat
ccagctcatg 2400gacgtcctgt ggaagcagga gggcctggac ctgaggatga
cgccctacgg ctgcctcccc 2460accggggacc gcacaggtct catcgaggtg
gtcctccact cggacaccat cgccaacatc 2520cagctgaaca aaagcaacat
ggcggccaca gctgccttca acaaggacgc cctgctcaac 2580tggctcaagt
ccaagaaccc tggggaggcc ctggatcggg ccattgagga attcaccctc
2640tcctgtgctg gctactgtgt ggccacatat gttctgggca tcggtgaccg
gcacagcgac 2700aacatcatga tcagagagag tgggcagctc ttccacattg
attttggcca ctttctgggg 2760aacttcaaga ccaagtttgg aatcaaccga
gagcgcgtcc ccttcattct cacctacgac 2820tttgtccacg tgatccagca
ggggaagact aacaacagtg agaagtttga aaggttccgc 2880ggctactgtg
aacgagccta taccatcctg cggcgccacg ggctgctttt cctccatctc
2940ttcgccctga tgcgggccgc aggtctgcct gagcttagct gctccaaaga
tatccagtat 3000ctcaaggact ctctggcact ggggaagacg gaggaagagg
cgctaaagca cttccgggtg 3060aagttcaacg aagctctccg agaaagctgg
aaaaccaaag tcaactggct ggcgcacaat 3120gtgtccaagg ataaccgaca gtag
3144321047PRTMus musculus 32Met Pro Pro Gly Val Asp Cys Pro Met Glu
Phe Trp Thr Lys Glu Glu1 5 10 15Ser Gln Ser Val Val Val Asp Phe Leu
Leu Pro Thr Gly Val Tyr Leu 20 25 30Asn Phe Pro Val Ser Arg Asn Ala
Asn Leu Ser Thr Ile Lys Gln Val 35 40 45Leu Trp His Arg Ala Gln Tyr
Glu Pro Leu Phe His Met Leu Ser Asp 50 55 60Pro Glu Ala Tyr Val Phe
Thr Cys Val Asn Gln Thr Ala Glu Gln Gln65 70 75 80Glu Leu Glu Asp
Glu Gln Arg Arg Leu Cys Asp Ile Gln Pro Phe Leu 85 90 95Pro Val Leu
Arg Leu Val Ala Arg Glu Gly Asp Arg Val Lys Lys Leu 100 105 110Ile
Asn Ser Gln Ile Ser Leu Leu Ile Gly Lys Gly Leu His Glu Phe 115 120
125Asp Ser Leu Arg Asp Pro Glu Val Asn Asp Phe Arg Thr Lys Met Arg
130 135 140Gln Phe Cys Glu Glu Ala Ala Ala His Arg Gln Gln Leu Gly
Trp Val145 150 155 160Glu Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu
Glu Pro Ser Ala Arg 165 170 175Gly Trp Arg Ala Gly Leu Leu Arg Val
Ser Asn Arg Ala Leu Leu Val 180 185 190Asn Val Lys Phe Glu Gly Ser
Glu Glu Ser Phe Thr Phe Gln Val Ser 195 200 205Thr Lys Asp Met Pro
Leu Ala Leu Met Ala Cys Ala Leu Arg Lys Lys 210 215 220Ala Thr Val
Phe Arg Gln Pro Leu Val Glu Gln Pro Glu Glu Tyr Ala225 230 235
240Leu Gln Val Asn Gly Arg His Glu Tyr Leu Tyr Gly Asn Tyr Pro Leu
245 250 255Cys His Phe Gln Tyr Ile Cys Ser Cys Leu His Ser Gly Leu
Thr Pro 260 265 270His Leu Thr Met Val His Ser Ser Ser Ile Leu Ala
Met Arg Asp Glu 275 280 285Gln Ser Asn Pro Ala Pro Gln Val Gln Lys
Pro Arg Ala Lys Pro Pro 290 295 300Pro Ile Pro Ala Lys Lys Pro Ser
Ser Val Ser Leu Trp Ser Leu Glu305 310 315 320Gln Pro Phe Ser Ile
Glu Leu Ile Glu Gly Arg Lys Val Asn Ala Asp 325 330 335Glu Arg Met
Lys Leu Val Val Gln Ala Gly Leu Phe His Gly Asn Glu 340 345 350Met
Leu Cys Lys Thr Val Ser Ser Ser Glu Val Asn Val Cys Ser Glu 355 360
365Pro Val Trp Lys Gln Arg Leu Glu Phe Asp Ile Ser Val Cys Asp Leu
370 375 380Pro Arg Met Ala Arg Leu Cys Phe Ala Leu Tyr Ala Val Val
Glu Lys385 390 395 400Ala Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser
Lys Lys Ala Asp Cys 405 410 415Pro Ile Ala Trp Ala Asn Leu Met Leu
Phe Asp Tyr Lys Asp Gln Leu 420 425 430Lys Thr Gly Glu Arg Cys Leu
Tyr Met Trp Pro Ser Val Pro Asp Glu 435 440 445Lys Gly Glu Leu Leu
Asn Pro Ala Gly Thr Val Arg Gly Asn Pro Asn 450 455 460Thr Glu Ser
Ala Ala Ala Leu Val Ile Tyr Leu Pro Glu Val Ala Pro465 470 475
480His Pro Val Tyr Phe Pro Ala Leu Glu Lys Ile Leu Glu Leu Gly Arg
485 490 495His Gly Glu Arg Gly Arg Ile Thr Glu Glu Glu Gln Leu Gln
Leu Arg 500 505 510Glu Ile Leu Glu Arg Arg Gly Ser Gly Glu Leu Tyr
Glu His Glu Lys 515 520 525Asp Leu Val Trp Lys Met Arg His Glu Val
Gln Glu His Phe Pro Glu 530 535 540Ala Leu Ala Arg Leu Leu Leu Val
Thr Lys Trp Asn Lys His Glu Asp545 550 555 560Val Ala Gln Leu Ser
Gln Met Leu Tyr Leu Leu Cys Ser Trp Pro Glu 565 570 575Leu Pro Val
Leu Ser Ala Leu Glu Leu Leu Asp Phe Ser Phe Pro Asp 580 585 590Cys
Tyr Val Gly Ser Phe Ala Ile Lys Ser Leu Arg Lys Leu Thr Asp 595
600 605Asp Glu Leu Phe Gln Tyr Leu Leu Gln Leu Val Gln Val Leu Lys
Tyr 610 615 620Glu Ser Tyr Leu Asp Cys Glu Leu Thr Lys Phe Leu Leu
Gly Arg Ala625 630 635 640Leu Ala Asn Arg Lys Ile Gly His Phe Leu
Phe Trp His Leu Arg Ser 645 650 655Glu Met His Val Pro Ser Val Ala
Leu Arg Phe Gly Leu Ile Met Glu 660 665 670Ala Tyr Cys Arg Gly Ser
Thr His His Met Lys Val Leu Met Lys Gln 675 680 685Gly Glu Ala Leu
Ser Lys Leu Lys Ala Leu Asn Asp Phe Val Lys Val 690 695 700Ser Ser
Gln Lys Thr Thr Lys Pro Gln Thr Lys Glu Met Met His Met705 710 715
720Cys Met Arg Gln Glu Thr Tyr Met Glu Ala Leu Ser His Leu Gln Ser
725 730 735Pro Leu Asp Pro Ser Thr Leu Leu Glu Glu Val Cys Val Glu
Gln Cys 740 745 750Thr Phe Met Asp Ser Lys Met Lys Pro Leu Trp Ile
Met Tyr Ser Ser 755 760 765Glu Glu Ala Gly Ser Ala Gly Asn Val Gly
Ile Ile Phe Lys Asn Gly 770 775 780Asp Asp Leu Arg Gln Asp Met Leu
Thr Leu Gln Met Ile Gln Leu Met785 790 795 800Asp Val Leu Trp Lys
Gln Glu Gly Leu Asp Leu Arg Met Thr Pro Tyr 805 810 815Gly Cys Leu
Pro Thr Gly Asp Arg Thr Gly Leu Ile Glu Val Val Leu 820 825 830His
Ser Asp Thr Ile Ala Asn Ile Gln Leu Asn Lys Ser Asn Met Ala 835 840
845Ala Thr Ala Ala Phe Asn Lys Asp Ala Leu Leu Asn Trp Leu Lys Ser
850 855 860Lys Asn Pro Gly Glu Ala Leu Asp Arg Ala Ile Glu Glu Phe
Thr Leu865 870 875 880Ser Cys Ala Gly Tyr Cys Val Ala Thr Tyr Val
Leu Gly Ile Gly Asp 885 890 895Arg His Ser Asp Asn Ile Met Ile Arg
Glu Ser Gly Gln Leu Phe His 900 905 910Ile Asp Phe Gly His Phe Leu
Gly Asn Phe Lys Thr Lys Phe Gly Ile 915 920 925Asn Arg Glu Arg Val
Pro Phe Ile Leu Thr Tyr Asp Phe Val His Val 930 935 940Ile Gln Gln
Gly Lys Thr Asn Asn Ser Glu Lys Phe Glu Arg Phe Arg945 950 955
960Gly Tyr Cys Glu Arg Ala Tyr Thr Ile Leu Arg Arg His Gly Leu Leu
965 970 975Phe Leu His Leu Phe Ala Leu Met Arg Ala Ala Gly Leu Pro
Glu Leu 980 985 990Ser Cys Ser Lys Asp Ile Gln Tyr Leu Lys Asp Ser
Leu Ala Leu Gly 995 1000 1005Lys Thr Glu Glu Glu Ala Leu Lys His
Phe Arg Val Lys Phe Asn 1010 1015 1020Glu Ala Leu Arg Glu Ser Trp
Lys Thr Lys Val Asn Trp Leu Ala 1025 1030 1035His Asn Val Ser Lys
Asp Asn Arg Gln 1040 1045333135DNAMus musculus 33atgccccctg
gggtggactg ccccatggag ttctggacca aagaggagag ccagagcgtg 60gttgttgact
tcttgctgcc cacaggggtc tacttgaact tccccgtgtc ccgcaatgcc
120aacctcagca ccatcaagca ggtgctgtgg caccgtgcac agtatgagcc
actcttccac 180atgctcagtg accccgaggc ctatgtgttc acctgtgtga
accagacggc ggagcagcag 240gagttggagg atgagcagcg gaggctgtgc
gacatccagc ccttcctgcc cgtgctgcgc 300ctcgtggccc gagaggggga
ccgcgtgaag aagctcatta actcccagat cagcctcctc 360attggcaaag
gtctccatga gtttgattcc ctgcgggacc cggaagtaaa cgacttccgc
420actaagatgc gccagttttg tgaagaggct gctgctcacc gccagcagct
gggctgggtg 480gaatggctgc agtacagctt ccccctgcag ctggagccct
cagcaagggg ttggcgggcc 540ggcttattgc gtgtcagcaa ccgagccctg
ctggtcaacg tgaagttcga gggcagtgag 600gagagcttca ccttccaggt
atccaccaag gacatgcccc tggcactgat ggcctgtgcc 660ctccgaaaaa
aggccacagt gttccggcag cctctggtgg agcagcctga ggaatatgcc
720ctgcaggtga acgggaggca cgaatacctc tacggcaact acccgctctg
ccactttcag 780tacatctgca gctgcctaca cagcgggctg acccctcatc
tgaccatggt ccactcctcc 840tccatccttg ctatgcggga tgagcagagc
aatcctgccc cccaagtaca gaaaccacgt 900gccaaacctc ccccgatccc
tgccaagaag ccctcctctg tgtccctgtg gtccctggaa 960cagccattct
ccattgagct gatcgagggc cgaaaagtga atgctgacga gcggatgaag
1020ctggttgttc aggccgggct cttccatggc aatgagatgc tgtgcaagac
tgtgtcaagc 1080tcggaggtga atgtatgctc agagcccgtg tggaagcagc
gactggagtt cgatatcagc 1140gtctgtgacc tcccgcgcat ggctcgactc
tgttttgctc tctatgccgt cgtggagaag 1200gctaagaagg cacgctccac
aaagaagaag tctaagaagg cggactgccc catcgcttgg 1260gccaacctca
tgctattcga ctacaaagat cagctcaaga cgggggagcg ctgcctctac
1320atgtggccct ctgtcccaga tgagaaggga gagctgctga atcctgcggg
tacagtgcgc 1380gggaacccca acacggagag tgccgctgcc ctggtcatct
acctgcctga ggtggccccc 1440caccctgtgt acttccccgc tctggagaag
atcctggagc tggggcgtca cggggagcgt 1500gggcgcatca cggaggagga
gcagctgcag ctgcgggaga tcctggaacg gcggggatcc 1560ggggaactgt
acgaacatga gaaggacctg gtgtggaaga tgcgccacga agtccaggag
1620catttcccag aggcgctggc ccgcctgctg ctggtcacca agtggaataa
acacgaggat 1680gtggcccaga tgctctattt gctgtgctcc tggcccgagc
tgcctgtgct gagcgccctg 1740gaacttctgg actttagctt tcccgactgc
tacgtgggct ccttcgccat caagtccctt 1800cggaagctga cggacgatga
gctcttccag taccttctgc agctggtgca agtgctcaaa 1860tatgagtcct
acctggactg cgagctgacc aaattcttgc tgggccgagc cctggctaac
1920cgcaagatcg gacacttcct gttctggcac ctccgctctg agatgcacgt
accatcagtg 1980gctctgcggt ttggtctcat catggaagcc tactgcagag
gcagcaccca ccacatgaag 2040gtgctgatga agcaggggga agcactgagc
aagcttaagg cactgaatga ctttgtgaag 2100gtgagttccc agaagaccac
caagccccaa accaaggaga tgatgcatat gtgcatgcgc 2160caggagacct
acatggaggc cctgtcccac ctgcagtctc cactcgaccc cagcaccctg
2220ctggaggaag tctgtgtgga gcagtgcacc ttcatggact ccaaaatgaa
gcccctgtgg 2280atcatgtaca gcagcgagga ggcgggcagt gctggcaacg
tgggcatcat ctttaagaac 2340ggggatgacc tccgccagga catgctgact
ctgcagatga tccagctcat ggacgtcctg 2400tggaagcagg agggcctgga
cctgaggatg acgccctacg gctgcctccc caccggggac 2460cgcacaggtc
tcatcgaggt ggtcctccac tcggacacca tcgccaacat ccagctgaac
2520aaaagcaaca tggcggccac agctgccttc aacaaggacg ccctgctcaa
ctggctcaag 2580tccaagaacc ctggggaggc cctggatcgg gccattgagg
aattcaccct ctcctgtgct 2640ggctactgtg tggccacata tgttctgggc
atcggtgacc ggcacagcga caacatcatg 2700atcagagaga gtgggcagct
cttccacatt gattttggcc actttctggg gaacttcaag 2760accaagtttg
gaatcaaccg agagcgcgtc cccttcattc tcacctacga ctttgtccac
2820gtgatccagc aggggaagac taacaacagt gagaagtttg aaaggttccg
cggctactgt 2880gaacgagcct ataccatcct gcggcgccac gggctgcttt
tcctccatct cttcgccctg 2940atgcgggccg caggtctgcc tgagcttagc
tgctccaaag atatccagta tctcaaggac 3000tctctggcac tggggaagac
ggaggaagag gcgctaaagc acttccgggt gaagttcaac 3060gaagctctcc
gagaaagctg gaaaaccaaa gtcaactggc tggcgcacaa tgtgtccaag
3120gataaccgac agtag 3135341044PRTMus musculus 34Met Pro Pro Gly
Val Asp Cys Pro Met Glu Phe Trp Thr Lys Glu Glu1 5 10 15Ser Gln Ser
Val Val Val Asp Phe Leu Leu Pro Thr Gly Val Tyr Leu 20 25 30Asn Phe
Pro Val Ser Arg Asn Ala Asn Leu Ser Thr Ile Lys Gln Val 35 40 45Leu
Trp His Arg Ala Gln Tyr Glu Pro Leu Phe His Met Leu Ser Asp 50 55
60Pro Glu Ala Tyr Val Phe Thr Cys Val Asn Gln Thr Ala Glu Gln Gln65
70 75 80Glu Leu Glu Asp Glu Gln Arg Arg Leu Cys Asp Ile Gln Pro Phe
Leu 85 90 95Pro Val Leu Arg Leu Val Ala Arg Glu Gly Asp Arg Val Lys
Lys Leu 100 105 110Ile Asn Ser Gln Ile Ser Leu Leu Ile Gly Lys Gly
Leu His Glu Phe 115 120 125Asp Ser Leu Arg Asp Pro Glu Val Asn Asp
Phe Arg Thr Lys Met Arg 130 135 140Gln Phe Cys Glu Glu Ala Ala Ala
His Arg Gln Gln Leu Gly Trp Val145 150 155 160Glu Trp Leu Gln Tyr
Ser Phe Pro Leu Gln Leu Glu Pro Ser Ala Arg 165 170 175Gly Trp Arg
Ala Gly Leu Leu Arg Val Ser Asn Arg Ala Leu Leu Val 180 185 190Asn
Val Lys Phe Glu Gly Ser Glu Glu Ser Phe Thr Phe Gln Val Ser 195 200
205Thr Lys Asp Met Pro Leu Ala Leu Met Ala Cys Ala Leu Arg Lys Lys
210 215 220Ala Thr Val Phe Arg Gln Pro Leu Val Glu Gln Pro Glu Glu
Tyr Ala225 230 235 240Leu Gln Val Asn Gly Arg His Glu Tyr Leu Tyr
Gly Asn Tyr Pro Leu 245 250 255Cys His Phe Gln Tyr Ile Cys Ser Cys
Leu His Ser Gly Leu Thr Pro 260 265 270His Leu Thr Met Val His Ser
Ser Ser Ile Leu Ala Met Arg Asp Glu 275 280 285Gln Ser Asn Pro Ala
Pro Gln Val Gln Lys Pro Arg Ala Lys Pro Pro 290 295 300Pro Ile Pro
Ala Lys Lys Pro Ser Ser Val Ser Leu Trp Ser Leu Glu305 310 315
320Gln Pro Phe Ser Ile Glu Leu Ile Glu Gly Arg Lys Val Asn Ala Asp
325 330 335Glu Arg Met Lys Leu Val Val Gln Ala Gly Leu Phe His Gly
Asn Glu 340 345 350Met Leu Cys Lys Thr Val Ser Ser Ser Glu Val Asn
Val Cys Ser Glu 355 360 365Pro Val Trp Lys Gln Arg Leu Glu Phe Asp
Ile Ser Val Cys Asp Leu 370 375 380Pro Arg Met Ala Arg Leu Cys Phe
Ala Leu Tyr Ala Val Val Glu Lys385 390 395 400Ala Lys Lys Ala Arg
Ser Thr Lys Lys Lys Ser Lys Lys Ala Asp Cys 405 410 415Pro Ile Ala
Trp Ala Asn Leu Met Leu Phe Asp Tyr Lys Asp Gln Leu 420 425 430Lys
Thr Gly Glu Arg Cys Leu Tyr Met Trp Pro Ser Val Pro Asp Glu 435 440
445Lys Gly Glu Leu Leu Asn Pro Ala Gly Thr Val Arg Gly Asn Pro Asn
450 455 460Thr Glu Ser Ala Ala Ala Leu Val Ile Tyr Leu Pro Glu Val
Ala Pro465 470 475 480His Pro Val Tyr Phe Pro Ala Leu Glu Lys Ile
Leu Glu Leu Gly Arg 485 490 495His Gly Glu Arg Gly Arg Ile Thr Glu
Glu Glu Gln Leu Gln Leu Arg 500 505 510Glu Ile Leu Glu Arg Arg Gly
Ser Gly Glu Leu Tyr Glu His Glu Lys 515 520 525Asp Leu Val Trp Lys
Met Arg His Glu Val Gln Glu His Phe Pro Glu 530 535 540Ala Leu Ala
Arg Leu Leu Leu Val Thr Lys Trp Asn Lys His Glu Asp545 550 555
560Val Ala Gln Met Leu Tyr Leu Leu Cys Ser Trp Pro Glu Leu Pro Val
565 570 575Leu Ser Ala Leu Glu Leu Leu Asp Phe Ser Phe Pro Asp Cys
Tyr Val 580 585 590Gly Ser Phe Ala Ile Lys Ser Leu Arg Lys Leu Thr
Asp Asp Glu Leu 595 600 605Phe Gln Tyr Leu Leu Gln Leu Val Gln Val
Leu Lys Tyr Glu Ser Tyr 610 615 620Leu Asp Cys Glu Leu Thr Lys Phe
Leu Leu Gly Arg Ala Leu Ala Asn625 630 635 640Arg Lys Ile Gly His
Phe Leu Phe Trp His Leu Arg Ser Glu Met His 645 650 655Val Pro Ser
Val Ala Leu Arg Phe Gly Leu Ile Met Glu Ala Tyr Cys 660 665 670Arg
Gly Ser Thr His His Met Lys Val Leu Met Lys Gln Gly Glu Ala 675 680
685Leu Ser Lys Leu Lys Ala Leu Asn Asp Phe Val Lys Val Ser Ser Gln
690 695 700Lys Thr Thr Lys Pro Gln Thr Lys Glu Met Met His Met Cys
Met Arg705 710 715 720Gln Glu Thr Tyr Met Glu Ala Leu Ser His Leu
Gln Ser Pro Leu Asp 725 730 735Pro Ser Thr Leu Leu Glu Glu Val Cys
Val Glu Gln Cys Thr Phe Met 740 745 750Asp Ser Lys Met Lys Pro Leu
Trp Ile Met Tyr Ser Ser Glu Glu Ala 755 760 765Gly Ser Ala Gly Asn
Val Gly Ile Ile Phe Lys Asn Gly Asp Asp Leu 770 775 780Arg Gln Asp
Met Leu Thr Leu Gln Met Ile Gln Leu Met Asp Val Leu785 790 795
800Trp Lys Gln Glu Gly Leu Asp Leu Arg Met Thr Pro Tyr Gly Cys Leu
805 810 815Pro Thr Gly Asp Arg Thr Gly Leu Ile Glu Val Val Leu His
Ser Asp 820 825 830Thr Ile Ala Asn Ile Gln Leu Asn Lys Ser Asn Met
Ala Ala Thr Ala 835 840 845Ala Phe Asn Lys Asp Ala Leu Leu Asn Trp
Leu Lys Ser Lys Asn Pro 850 855 860Gly Glu Ala Leu Asp Arg Ala Ile
Glu Glu Phe Thr Leu Ser Cys Ala865 870 875 880Gly Tyr Cys Val Ala
Thr Tyr Val Leu Gly Ile Gly Asp Arg His Ser 885 890 895Asp Asn Ile
Met Ile Arg Glu Ser Gly Gln Leu Phe His Ile Asp Phe 900 905 910Gly
His Phe Leu Gly Asn Phe Lys Thr Lys Phe Gly Ile Asn Arg Glu 915 920
925Arg Val Pro Phe Ile Leu Thr Tyr Asp Phe Val His Val Ile Gln Gln
930 935 940Gly Lys Thr Asn Asn Ser Glu Lys Phe Glu Arg Phe Arg Gly
Tyr Cys945 950 955 960Glu Arg Ala Tyr Thr Ile Leu Arg Arg His Gly
Leu Leu Phe Leu His 965 970 975Leu Phe Ala Leu Met Arg Ala Ala Gly
Leu Pro Glu Leu Ser Cys Ser 980 985 990Lys Asp Ile Gln Tyr Leu Lys
Asp Ser Leu Ala Leu Gly Lys Thr Glu 995 1000 1005Glu Glu Ala Leu
Lys His Phe Arg Val Lys Phe Asn Glu Ala Leu 1010 1015 1020Arg Glu
Ser Trp Lys Thr Lys Val Asn Trp Leu Ala His Asn Val 1025 1030
1035Ser Lys Asp Asn Arg Gln 1040353138DNAMus musculus 35atgccccctg
gggtggactg ccccatggag ttctggacca aagaggagag ccagagcgtg 60gttgttgact
tcttgctgcc cacaggggtc tacttgaact tccccgtgtc ccgcaatgcc
120aacctcagca ccatcaagca ggtgctgtgg caccgtgcac agtatgagcc
actcttccac 180atgctcagtg accccgaggc ctatgtgttc acctgtgtga
accagacggc ggagcagcag 240gagttggagg atgagcagcg gaggctgtgc
gacatccagc ccttcctgcc cgtgctgcgc 300ctcgtggccc gagaggggga
ccgcgtgaag aagctcatta actcccagat cagcctcctc 360attggcaaag
gtctccatga gtttgattcc ctgcgggacc cggaagtaaa cgacttccgc
420actaagatgc gccagttttg tgaagaggct gctgctcacc gccagcagct
gggctgggtg 480gaatggctgc agtacagctt ccccctgcag ctggagccct
cagcaagggg ttggcgggcc 540ggcttattgc gtgtcagcaa ccgagccctg
ctggtcaacg tgaagttcga gggcagtgag 600gagagcttca ccttccaggt
atccaccaag gacatgcccc tggcactgat ggcctgtgcc 660ctccgaaaaa
aggccacagt gttccggcag cctctggtgg agcagcctga ggaatatgcc
720ctgcaggtga acgggaggca cgaatacctc tacggcaact acccgctctg
ccactttcag 780tacatctgca gctgcctaca cagcgggctg acccctcatc
tgaccatggt ccactcctcc 840tccatccttg ctatgcggga tgagcagagc
aatcctgccc cccaagtaca gaaaccacgt 900gccaaacctc ccccgatccc
tgccaagaag ccctcctctg tgtccctgtg gtccctggaa 960cagccattct
ccattgagct gatcgagggc cgaaaagtga atgctgacga gcggatgaag
1020ctggttgttc aggccgggct cttccatggc aatgagatgc tgtgcaagac
tgtgtcaagc 1080tcggaggtga atgtatgctc agagcccgtg tggaagcagc
gactggagtt cgatatcagc 1140gtctgtgacc tcccgcgcat ggctcgactc
tgttttgctc tctatgccgt cgtggagaag 1200gctaagaagg cacgctccac
aaagaagaag tctaagaagg cggactgccc catcgcttgg 1260gccaacctca
tgctattcga ctacaaagat cagctcaaga cgggggagcg ctgcctctac
1320atgtggccct ctgtcccaga tgagaaggga gagctgctga atcctgcggg
tacagtgcgc 1380gggaacccca acacggagag tgccgctgcc ctggtcatct
acctgcctga ggtggccccc 1440caccctgtgt acttccccgc tctggagaag
atcctggagc tggggcgtca cggggagcgt 1500gggcgcatca cggaggagga
gcagctgcag ctgcgggaga tcctggaacg gcggggatcc 1560ggggaactgt
acgaacatga gaaggacctg gtgtggaaga tgcgccacga agtccaggag
1620catttcccag aggcgctggc ccgcctgctg ctggtcacca agtggaataa
acacgaggat 1680gtggcccaga tgctctattt gctgtgctcc tggcccgagc
tgcctgtgct gagcgccctg 1740gaacttctgg actttagctt tcccgactgc
tacgtgggct ccttcgccat caagtccctt 1800cggaagctga cggacgatga
gctcttccag taccttctgc agctggtgca agtgctcaaa 1860tatgagtcct
acctggactg cgagctgacc aaattcttgc tgggccgagc cctggctaac
1920cgcaagatcg gacacttcct gttctggcac ctccgctctg agatgcacgt
accatcagtg 1980gctctgcggt ttggtctcat catggaagcc tactgcagag
gcagcaccca ccacatgaag 2040gtgctgatga agcaggggga agcactgagc
aagcttaagg cactgaatga ctttgtgaag 2100gtgagttccc agaagaccac
caagccccaa accaaggaga tgatgcatat gtgcatgcgc 2160caggagacct
acatggaggc cctgtcccac ctgcagtctc cactcgaccc cagcaccctg
2220ctggaggaag tctgcagtgt ggagcagtgc accttcatgg actccaaaat
gaagcccctg 2280tggatcatgt acagcagcga ggaggcgggc agtgctggca
acgtgggcat catctttaag 2340aacggggatg acctccgcca ggacatgctg
actctgcaga tgatccagct catggacgtc 2400ctgtggaagc aggagggcct
ggacctgagg atgacgccct acggctgcct ccccaccggg 2460gaccgcacag
gtctcatcga ggtggtcctc cactcggaca ccatcgccaa catccagctg
2520aacaaaagca acatggcggc cacagctgcc ttcaacaagg acgccctgct
caactggctc 2580aagtccaaga accctgggga ggccctggat cgggccattg
aggaattcac cctctcctgt 2640gctggctact gtgtggccac atatgttctg
ggcatcggtg accggcacag cgacaacatc 2700atgatcagag agagtgggca
gctcttccac attgattttg gccactttct ggggaacttc
2760aagaccaagt ttggaatcaa ccgagagcgc gtccccttca ttctcaccta
cgactttgtc 2820cacgtgatcc agcaggggaa gactaacaac agtgagaagt
ttgaaaggtt ccgcggctac 2880tgtgaacgag cctataccat cctgcggcgc
cacgggctgc ttttcctcca tctcttcgcc 2940ctgatgcggg ccgcaggtct
gcctgagctt agctgctcca aagatatcca gtatctcaag 3000gactctctgg
cactggggaa gacggaggaa gaggcgctaa agcacttccg ggtgaagttc
3060aacgaagctc tccgagaaag ctggaaaacc aaagtcaact ggctggcgca
caatgtgtcc 3120aaggataacc gacagtag 3138361045PRTMus musculus 36Met
Pro Pro Gly Val Asp Cys Pro Met Glu Phe Trp Thr Lys Glu Glu1 5 10
15Ser Gln Ser Val Val Val Asp Phe Leu Leu Pro Thr Gly Val Tyr Leu
20 25 30Asn Phe Pro Val Ser Arg Asn Ala Asn Leu Ser Thr Ile Lys Gln
Val 35 40 45Leu Trp His Arg Ala Gln Tyr Glu Pro Leu Phe His Met Leu
Ser Asp 50 55 60Pro Glu Ala Tyr Val Phe Thr Cys Val Asn Gln Thr Ala
Glu Gln Gln65 70 75 80Glu Leu Glu Asp Glu Gln Arg Arg Leu Cys Asp
Ile Gln Pro Phe Leu 85 90 95Pro Val Leu Arg Leu Val Ala Arg Glu Gly
Asp Arg Val Lys Lys Leu 100 105 110Ile Asn Ser Gln Ile Ser Leu Leu
Ile Gly Lys Gly Leu His Glu Phe 115 120 125Asp Ser Leu Arg Asp Pro
Glu Val Asn Asp Phe Arg Thr Lys Met Arg 130 135 140Gln Phe Cys Glu
Glu Ala Ala Ala His Arg Gln Gln Leu Gly Trp Val145 150 155 160Glu
Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu Glu Pro Ser Ala Arg 165 170
175Gly Trp Arg Ala Gly Leu Leu Arg Val Ser Asn Arg Ala Leu Leu Val
180 185 190Asn Val Lys Phe Glu Gly Ser Glu Glu Ser Phe Thr Phe Gln
Val Ser 195 200 205Thr Lys Asp Met Pro Leu Ala Leu Met Ala Cys Ala
Leu Arg Lys Lys 210 215 220Ala Thr Val Phe Arg Gln Pro Leu Val Glu
Gln Pro Glu Glu Tyr Ala225 230 235 240Leu Gln Val Asn Gly Arg His
Glu Tyr Leu Tyr Gly Asn Tyr Pro Leu 245 250 255Cys His Phe Gln Tyr
Ile Cys Ser Cys Leu His Ser Gly Leu Thr Pro 260 265 270His Leu Thr
Met Val His Ser Ser Ser Ile Leu Ala Met Arg Asp Glu 275 280 285Gln
Ser Asn Pro Ala Pro Gln Val Gln Lys Pro Arg Ala Lys Pro Pro 290 295
300Pro Ile Pro Ala Lys Lys Pro Ser Ser Val Ser Leu Trp Ser Leu
Glu305 310 315 320Gln Pro Phe Ser Ile Glu Leu Ile Glu Gly Arg Lys
Val Asn Ala Asp 325 330 335Glu Arg Met Lys Leu Val Val Gln Ala Gly
Leu Phe His Gly Asn Glu 340 345 350Met Leu Cys Lys Thr Val Ser Ser
Ser Glu Val Asn Val Cys Ser Glu 355 360 365Pro Val Trp Lys Gln Arg
Leu Glu Phe Asp Ile Ser Val Cys Asp Leu 370 375 380Pro Arg Met Ala
Arg Leu Cys Phe Ala Leu Tyr Ala Val Val Glu Lys385 390 395 400Ala
Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser Lys Lys Ala Asp Cys 405 410
415Pro Ile Ala Trp Ala Asn Leu Met Leu Phe Asp Tyr Lys Asp Gln Leu
420 425 430Lys Thr Gly Glu Arg Cys Leu Tyr Met Trp Pro Ser Val Pro
Asp Glu 435 440 445Lys Gly Glu Leu Leu Asn Pro Ala Gly Thr Val Arg
Gly Asn Pro Asn 450 455 460Thr Glu Ser Ala Ala Ala Leu Val Ile Tyr
Leu Pro Glu Val Ala Pro465 470 475 480His Pro Val Tyr Phe Pro Ala
Leu Glu Lys Ile Leu Glu Leu Gly Arg 485 490 495His Gly Glu Arg Gly
Arg Ile Thr Glu Glu Glu Gln Leu Gln Leu Arg 500 505 510Glu Ile Leu
Glu Arg Arg Gly Ser Gly Glu Leu Tyr Glu His Glu Lys 515 520 525Asp
Leu Val Trp Lys Met Arg His Glu Val Gln Glu His Phe Pro Glu 530 535
540Ala Leu Ala Arg Leu Leu Leu Val Thr Lys Trp Asn Lys His Glu
Asp545 550 555 560Val Ala Gln Met Leu Tyr Leu Leu Cys Ser Trp Pro
Glu Leu Pro Val 565 570 575Leu Ser Ala Leu Glu Leu Leu Asp Phe Ser
Phe Pro Asp Cys Tyr Val 580 585 590Gly Ser Phe Ala Ile Lys Ser Leu
Arg Lys Leu Thr Asp Asp Glu Leu 595 600 605Phe Gln Tyr Leu Leu Gln
Leu Val Gln Val Leu Lys Tyr Glu Ser Tyr 610 615 620Leu Asp Cys Glu
Leu Thr Lys Phe Leu Leu Gly Arg Ala Leu Ala Asn625 630 635 640Arg
Lys Ile Gly His Phe Leu Phe Trp His Leu Arg Ser Glu Met His 645 650
655Val Pro Ser Val Ala Leu Arg Phe Gly Leu Ile Met Glu Ala Tyr Cys
660 665 670Arg Gly Ser Thr His His Met Lys Val Leu Met Lys Gln Gly
Glu Ala 675 680 685Leu Ser Lys Leu Lys Ala Leu Asn Asp Phe Val Lys
Val Ser Ser Gln 690 695 700Lys Thr Thr Lys Pro Gln Thr Lys Glu Met
Met His Met Cys Met Arg705 710 715 720Gln Glu Thr Tyr Met Glu Ala
Leu Ser His Leu Gln Ser Pro Leu Asp 725 730 735Pro Ser Thr Leu Leu
Glu Glu Val Cys Ser Val Glu Gln Cys Thr Phe 740 745 750Met Asp Ser
Lys Met Lys Pro Leu Trp Ile Met Tyr Ser Ser Glu Glu 755 760 765Ala
Gly Ser Ala Gly Asn Val Gly Ile Ile Phe Lys Asn Gly Asp Asp 770 775
780Leu Arg Gln Asp Met Leu Thr Leu Gln Met Ile Gln Leu Met Asp
Val785 790 795 800Leu Trp Lys Gln Glu Gly Leu Asp Leu Arg Met Thr
Pro Tyr Gly Cys 805 810 815Leu Pro Thr Gly Asp Arg Thr Gly Leu Ile
Glu Val Val Leu His Ser 820 825 830Asp Thr Ile Ala Asn Ile Gln Leu
Asn Lys Ser Asn Met Ala Ala Thr 835 840 845Ala Ala Phe Asn Lys Asp
Ala Leu Leu Asn Trp Leu Lys Ser Lys Asn 850 855 860Pro Gly Glu Ala
Leu Asp Arg Ala Ile Glu Glu Phe Thr Leu Ser Cys865 870 875 880Ala
Gly Tyr Cys Val Ala Thr Tyr Val Leu Gly Ile Gly Asp Arg His 885 890
895Ser Asp Asn Ile Met Ile Arg Glu Ser Gly Gln Leu Phe His Ile Asp
900 905 910Phe Gly His Phe Leu Gly Asn Phe Lys Thr Lys Phe Gly Ile
Asn Arg 915 920 925Glu Arg Val Pro Phe Ile Leu Thr Tyr Asp Phe Val
His Val Ile Gln 930 935 940Gln Gly Lys Thr Asn Asn Ser Glu Lys Phe
Glu Arg Phe Arg Gly Tyr945 950 955 960Cys Glu Arg Ala Tyr Thr Ile
Leu Arg Arg His Gly Leu Leu Phe Leu 965 970 975His Leu Phe Ala Leu
Met Arg Ala Ala Gly Leu Pro Glu Leu Ser Cys 980 985 990Ser Lys Asp
Ile Gln Tyr Leu Lys Asp Ser Leu Ala Leu Gly Lys Thr 995 1000
1005Glu Glu Glu Ala Leu Lys His Phe Arg Val Lys Phe Asn Glu Ala
1010 1015 1020Leu Arg Glu Ser Trp Lys Thr Lys Val Asn Trp Leu Ala
His Asn 1025 1030 1035Val Ser Lys Asp Asn Arg Gln 1040
1045373141DNAMus musculus 37atgccccctg gggtggactg ccccatggag
ttctggacca aagaggagag ccagagcgtg 60gttgttgact tcttgctgcc cacaggggtc
tacttgaact tccccgtgtc ccgcaatgcc 120aacctcagca ccatcaagca
ggtgctgtgg caccgtgcac agtatgagcc actcttccac 180atgctcagtg
accccgaggc ctatgtgttc acctgtgtga accagacggc ggagcagcag
240gagttggagg atgagcagcg gaggctgtgc gacatccagc ccttcctgcc
cgtgctgcgc 300ctcgtggccc gagaggggga ccgcgtgaag aagctcatta
actcccagat cagcctcctc 360attggcaaag gtctccatga gtttgattcc
ctgcgggacc cggaagtaaa cgacttccgc 420actaagatgc gccagttttg
tgaagaggct gctgctcacc gccagcagct gggctgggtg 480gaatggctgc
agtacagctt ccccctgcag ctggagccct cagcaagggg ttggcgggcc
540ggcttattgc gtgtcagcaa ccgagccctg ctggtcaacg tgaagttcga
gggcagtgag 600gagagcttca ccttccaggt atccaccaag gacatgcccc
tggcactgat ggcctgtgcc 660ctccgaaaaa aggccacagt gttccggcag
cctctggtgg agcagcctga ggaatatgcc 720ctgcaggtga acgggaggca
cgaatacctc tacggcaact acccgctctg ccactttcag 780tacatctgca
gctgcctaca cagcgggctg acccctcatc tgaccatggt ccactcctcc
840tccatccttg ctatgcggga tgagcagagc aatcctgccc cccaagtaca
gaaaccacgt 900gccaaacctc ccccgatccc tgccaagaag ccctcctctg
tgtccctgtg gtccctggaa 960cagccattct ccattgagct gatcgagggc
cgaaaagtga atgctgacga gcggatgaag 1020ctggttgttc aggccgggct
cttccatggc aatgagatgc tgtgcaagac tgtgtcaagc 1080tcggaggtga
atgtatgctc agagcccgtg tggaagcagc gactggagtt cgatatcagc
1140gtctgtgacc tcccgcgcat ggctcgactc tgttttgctc tctatgccgt
cgtggagaag 1200gctaagaagg cacgctccac aaagaagaag tctaagaagg
cggactgccc catcgcttgg 1260gccaacctca tgctattcga ctacaaagat
cagctcaaga cgggggagcg ctgcctctac 1320atgtggccct ctgtcccaga
tgagaaggga gagctgctga atcctgcggg tacagtgcgc 1380gggaacccca
acacggagag tgccgctgcc ctggtcatct acctgcctga ggtggccccc
1440caccctgtgt acttccccgc tctggagaag atcctggagc tggggcgtca
cggggagcgt 1500gggcgcatca cggaggagga gctgcagctg cgggagatcc
tggaacggcg gggatccggg 1560gaactgtacg aacatgagaa ggacctggtg
tggaagatgc gccacgaagt ccaggagcat 1620ttcccagagg cgctggcccg
cctgctgctg gtcaccaagt ggaataaaca cgaggatgtg 1680gcccagctgt
cccagatgct ctatttgctg tgctcctggc ccgagctgcc tgtgctgagc
1740gccctggaac ttctggactt tagctttccc gactgctacg tgggctcctt
cgccatcaag 1800tcccttcgga agctgacgga cgatgagctc ttccagtacc
ttctgcagct ggtgcaagtg 1860ctcaaatatg agtcctacct ggactgcgag
ctgaccaaat tcttgctggg ccgagccctg 1920gctaaccgca agatcggaca
cttcctgttc tggcacctcc gctctgagat gcacgtacca 1980tcagtggctc
tgcggtttgg tctcatcatg gaagcctact gcagaggcag cacccaccac
2040atgaaggtgc tgatgaagca gggggaagca ctgagcaagc ttaaggcact
gaatgacttt 2100gtgaaggtga gttcccagaa gaccaccaag ccccaaacca
aggagatgat gcatatgtgc 2160atgcgccagg agacctacat ggaggccctg
tcccacctgc agtctccact cgaccccagc 2220accctgctgg aggaagtctg
tgtggagcag tgcaccttca tggactccaa aatgaagccc 2280ctgtggatca
tgtacagcag cgaggaggcg ggcagtgctg gcaacgtggg catcatcttt
2340aagaacgggg atgacctccg ccaggacatg ctgactctgc agatgatcca
gctcatggac 2400gtcctgtgga agcaggaggg cctggacctg aggatgacgc
cctacggctg cctccccacc 2460ggggaccgca caggtctcat cgaggtggtc
ctccactcgg acaccatcgc caacatccag 2520ctgaacaaaa gcaacatggc
ggccacagct gccttcaaca aggacgccct gctcaactgg 2580ctcaagtcca
agaaccctgg ggaggccctg gatcgggcca ttgaggaatt caccctctcc
2640tgtgctggct actgtgtggc cacatatgtt ctgggcatcg gtgaccggca
cagcgacaac 2700atcatgatca gagagagtgg gcagctcttc cacattgatt
ttggccactt tctggggaac 2760ttcaagacca agtttggaat caaccgagag
cgcgtcccct tcattctcac ctacgacttt 2820gtccacgtga tccagcaggg
gaagactaac aacagtgaga agtttgaaag gttccgcggc 2880tactgtgaac
gagcctatac catcctgcgg cgccacgggc tgcttttcct ccatctcttc
2940gccctgatgc gggccgcagg tctgcctgag cttagctgct ccaaagatat
ccagtatctc 3000aaggactctc tggcactggg gaagacggag gaagaggcgc
taaagcactt ccgggtgaag 3060ttcaacgaag ctctccgaga aagctggaaa
accaaagtca actggctggc gcacaatgtg 3120tccaaggata accgacagta g
3141381046PRTMus musculus 38Met Pro Pro Gly Val Asp Cys Pro Met Glu
Phe Trp Thr Lys Glu Glu1 5 10 15Ser Gln Ser Val Val Val Asp Phe Leu
Leu Pro Thr Gly Val Tyr Leu 20 25 30Asn Phe Pro Val Ser Arg Asn Ala
Asn Leu Ser Thr Ile Lys Gln Val 35 40 45Leu Trp His Arg Ala Gln Tyr
Glu Pro Leu Phe His Met Leu Ser Asp 50 55 60Pro Glu Ala Tyr Val Phe
Thr Cys Val Asn Gln Thr Ala Glu Gln Gln65 70 75 80Glu Leu Glu Asp
Glu Gln Arg Arg Leu Cys Asp Ile Gln Pro Phe Leu 85 90 95Pro Val Leu
Arg Leu Val Ala Arg Glu Gly Asp Arg Val Lys Lys Leu 100 105 110Ile
Asn Ser Gln Ile Ser Leu Leu Ile Gly Lys Gly Leu His Glu Phe 115 120
125Asp Ser Leu Arg Asp Pro Glu Val Asn Asp Phe Arg Thr Lys Met Arg
130 135 140Gln Phe Cys Glu Glu Ala Ala Ala His Arg Gln Gln Leu Gly
Trp Val145 150 155 160Glu Trp Leu Gln Tyr Ser Phe Pro Leu Gln Leu
Glu Pro Ser Ala Arg 165 170 175Gly Trp Arg Ala Gly Leu Leu Arg Val
Ser Asn Arg Ala Leu Leu Val 180 185 190Asn Val Lys Phe Glu Gly Ser
Glu Glu Ser Phe Thr Phe Gln Val Ser 195 200 205Thr Lys Asp Met Pro
Leu Ala Leu Met Ala Cys Ala Leu Arg Lys Lys 210 215 220Ala Thr Val
Phe Arg Gln Pro Leu Val Glu Gln Pro Glu Glu Tyr Ala225 230 235
240Leu Gln Val Asn Gly Arg His Glu Tyr Leu Tyr Gly Asn Tyr Pro Leu
245 250 255Cys His Phe Gln Tyr Ile Cys Ser Cys Leu His Ser Gly Leu
Thr Pro 260 265 270His Leu Thr Met Val His Ser Ser Ser Ile Leu Ala
Met Arg Asp Glu 275 280 285Gln Ser Asn Pro Ala Pro Gln Val Gln Lys
Pro Arg Ala Lys Pro Pro 290 295 300Pro Ile Pro Ala Lys Lys Pro Ser
Ser Val Ser Leu Trp Ser Leu Glu305 310 315 320Gln Pro Phe Ser Ile
Glu Leu Ile Glu Gly Arg Lys Val Asn Ala Asp 325 330 335Glu Arg Met
Lys Leu Val Val Gln Ala Gly Leu Phe His Gly Asn Glu 340 345 350Met
Leu Cys Lys Thr Val Ser Ser Ser Glu Val Asn Val Cys Ser Glu 355 360
365Pro Val Trp Lys Gln Arg Leu Glu Phe Asp Ile Ser Val Cys Asp Leu
370 375 380Pro Arg Met Ala Arg Leu Cys Phe Ala Leu Tyr Ala Val Val
Glu Lys385 390 395 400Ala Lys Lys Ala Arg Ser Thr Lys Lys Lys Ser
Lys Lys Ala Asp Cys 405 410 415Pro Ile Ala Trp Ala Asn Leu Met Leu
Phe Asp Tyr Lys Asp Gln Leu 420 425 430Lys Thr Gly Glu Arg Cys Leu
Tyr Met Trp Pro Ser Val Pro Asp Glu 435 440 445Lys Gly Glu Leu Leu
Asn Pro Ala Gly Thr Val Arg Gly Asn Pro Asn 450 455 460Thr Glu Ser
Ala Ala Ala Leu Val Ile Tyr Leu Pro Glu Val Ala Pro465 470 475
480His Pro Val Tyr Phe Pro Ala Leu Glu Lys Ile Leu Glu Leu Gly Arg
485 490 495His Gly Glu Arg Gly Arg Ile Thr Glu Glu Glu Leu Gln Leu
Arg Glu 500 505 510Ile Leu Glu Arg Arg Gly Ser Gly Glu Leu Tyr Glu
His Glu Lys Asp 515 520 525Leu Val Trp Lys Met Arg His Glu Val Gln
Glu His Phe Pro Glu Ala 530 535 540Leu Ala Arg Leu Leu Leu Val Thr
Lys Trp Asn Lys His Glu Asp Val545 550 555 560Ala Gln Leu Ser Gln
Met Leu Tyr Leu Leu Cys Ser Trp Pro Glu Leu 565 570 575Pro Val Leu
Ser Ala Leu Glu Leu Leu Asp Phe Ser Phe Pro Asp Cys 580 585 590Tyr
Val Gly Ser Phe Ala Ile Lys Ser Leu Arg Lys Leu Thr Asp Asp 595 600
605Glu Leu Phe Gln Tyr Leu Leu Gln Leu Val Gln Val Leu Lys Tyr Glu
610 615 620Ser Tyr Leu Asp Cys Glu Leu Thr Lys Phe Leu Leu Gly Arg
Ala Leu625 630 635 640Ala Asn Arg Lys Ile Gly His Phe Leu Phe Trp
His Leu Arg Ser Glu 645 650 655Met His Val Pro Ser Val Ala Leu Arg
Phe Gly Leu Ile Met Glu Ala 660 665 670Tyr Cys Arg Gly Ser Thr His
His Met Lys Val Leu Met Lys Gln Gly 675 680 685Glu Ala Leu Ser Lys
Leu Lys Ala Leu Asn Asp Phe Val Lys Val Ser 690 695 700Ser Gln Lys
Thr Thr Lys Pro Gln Thr Lys Glu Met Met His Met Cys705 710 715
720Met Arg Gln Glu Thr Tyr Met Glu Ala Leu Ser His Leu Gln Ser Pro
725 730 735Leu Asp Pro Ser Thr Leu Leu Glu Glu Val Cys Val Glu Gln
Cys Thr 740 745 750Phe Met Asp Ser Lys Met Lys Pro Leu Trp Ile Met
Tyr Ser Ser Glu 755 760 765Glu Ala Gly Ser Ala Gly Asn Val Gly Ile
Ile Phe Lys Asn Gly Asp 770 775 780Asp Leu Arg Gln Asp Met Leu Thr
Leu Gln Met Ile Gln Leu Met Asp785 790 795 800Val Leu Trp Lys Gln
Glu Gly Leu Asp Leu Arg Met Thr Pro Tyr Gly 805 810 815Cys Leu Pro
Thr Gly Asp Arg Thr Gly Leu Ile Glu Val Val Leu His
820 825 830Ser Asp Thr Ile Ala Asn Ile Gln Leu Asn Lys Ser Asn Met
Ala Ala 835 840 845Thr Ala Ala Phe Asn Lys Asp Ala Leu Leu Asn Trp
Leu Lys Ser Lys 850 855 860Asn Pro Gly Glu Ala Leu Asp Arg Ala Ile
Glu Glu Phe Thr Leu Ser865 870 875 880Cys Ala Gly Tyr Cys Val Ala
Thr Tyr Val Leu Gly Ile Gly Asp Arg 885 890 895His Ser Asp Asn Ile
Met Ile Arg Glu Ser Gly Gln Leu Phe His Ile 900 905 910Asp Phe Gly
His Phe Leu Gly Asn Phe Lys Thr Lys Phe Gly Ile Asn 915 920 925Arg
Glu Arg Val Pro Phe Ile Leu Thr Tyr Asp Phe Val His Val Ile 930 935
940Gln Gln Gly Lys Thr Asn Asn Ser Glu Lys Phe Glu Arg Phe Arg
Gly945 950 955 960Tyr Cys Glu Arg Ala Tyr Thr Ile Leu Arg Arg His
Gly Leu Leu Phe 965 970 975Leu His Leu Phe Ala Leu Met Arg Ala Ala
Gly Leu Pro Glu Leu Ser 980 985 990Cys Ser Lys Asp Ile Gln Tyr Leu
Lys Asp Ser Leu Ala Leu Gly Lys 995 1000 1005Thr Glu Glu Glu Ala
Leu Lys His Phe Arg Val Lys Phe Asn Glu 1010 1015 1020Ala Leu Arg
Glu Ser Trp Lys Thr Lys Val Asn Trp Leu Ala His 1025 1030 1035Asn
Val Ser Lys Asp Asn Arg Gln 1040 104539921DNAHomo
sapiensCDS(25)..(888) 39cactctggtg gggctgctcc aggc atg cag atc cca
cag gcg ccc tgg cca 51 Met Gln Ile Pro Gln Ala Pro Trp Pro 1 5gtc
gtc tgg gcg gtg cta caa ctg ggc tgg cgg cca gga tgg ttc tta 99Val
Val Trp Ala Val Leu Gln Leu Gly Trp Arg Pro Gly Trp Phe Leu10 15 20
25gac tcc cca gac agg ccc tgg aac ccc ccc acc ttc tcc cca gcc ctg
147Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu
30 35 40ctc gtg gtg acc gaa ggg gac aac gcc acc ttc acc tgc agc ttc
tcc 195Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe
Ser 45 50 55aac aca tcg gag agc ttc gtg cta aac tgg tac cgc atg agc
ccc agc 243Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser
Pro Ser 60 65 70aac cag acg gac aag ctg gcc gcc ttc ccc gag gac cgc
agc cag ccc 291Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg
Ser Gln Pro 75 80 85ggc cag gac tgc cgc ttc cgt gtc aca caa ctg ccc
aac ggg cgt gac 339Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro
Asn Gly Arg Asp90 95 100 105ttc cac atg agc gtg gtc agg gcc cgg cgc
aat gac agc ggc acc tac 387Phe His Met Ser Val Val Arg Ala Arg Arg
Asn Asp Ser Gly Thr Tyr 110 115 120ctc tgt ggg gcc atc tcc ctg gcc
ccc aag gcg cag atc aaa gag agc 435Leu Cys Gly Ala Ile Ser Leu Ala
Pro Lys Ala Gln Ile Lys Glu Ser 125 130 135ctg cgg gca gag ctc agg
gtg aca gag aga agg gca gaa gtg ccc aca 483Leu Arg Ala Glu Leu Arg
Val Thr Glu Arg Arg Ala Glu Val Pro Thr 140 145 150gcc cac ccc agc
ccc tca ccc agg tca gcc ggc cag ttc caa acc ctg 531Ala His Pro Ser
Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln Thr Leu 155 160 165gtg gtt
ggt gtc gtg ggc ggc ctg ctg ggc agc ctg gtg ctg cta gtc 579Val Val
Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val170 175 180
185tgg gtc ctg gcc gtc atc tgc tcc cgg gcc gca cga ggg aca ata gga
627Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly
190 195 200gcc agg cgc acc ggc cag ccc ctg aag gag gac ccc tca gcc
gtg cct 675Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala
Val Pro 205 210 215gtg ttc tct gtg gac tat ggg gag ctg gat ttc cag
tgg cga gag aag 723Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln
Trp Arg Glu Lys 220 225 230acc ccg gag ccc ccc gtg ccc tgt gtc cct
gag cag acg gag tat gcc 771Thr Pro Glu Pro Pro Val Pro Cys Val Pro
Glu Gln Thr Glu Tyr Ala 235 240 245acc att gtc ttt cct agc gga atg
ggc acc tca tcc ccc gcc cgc agg 819Thr Ile Val Phe Pro Ser Gly Met
Gly Thr Ser Ser Pro Ala Arg Arg250 255 260 265ggc tca gct gac ggc
cct cgg agt gcc cag cca ctg agg cct gag gat 867Gly Ser Ala Asp Gly
Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp 270 275 280gga cac tgc
tct tgg ccc ctc tgaccggctt ccttggccac cagtgttctg cag 921Gly His Cys
Ser Trp Pro Leu 28540288PRTHomo sapiens 40Met Gln Ile Pro Gln Ala
Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro
Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr
Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr
Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn
Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Ser Ala Gly Gln Phe
Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser
Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190Ser Arg
Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200
205Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro
Val Pro225 230 235 240Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile
Val Phe Pro Ser Gly 245 250 255Met Gly Thr Ser Ser Pro Ala Arg Arg
Gly Ser Ala Asp Gly Pro Arg 260 265 270Ser Ala Gln Pro Leu Arg Pro
Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 28541968DNAHomo
sapiensCDS(59)..(793) 41gcttcccgag gctccgcacc agccgcgctt ctgtccgcct
gcagggcatt ccagaaag 58atg agg ata ttt gct gtc ttt ata ttc atg acc
tac tgg cat ttg ctg 106Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr
Tyr Trp His Leu Leu1 5 10 15aac gca ttt act gtc acg gtt ccc aag gac
cta tat gtg gta gag tat 154Asn Ala Phe Thr Val Thr Val Pro Lys Asp
Leu Tyr Val Val Glu Tyr 20 25 30ggt agc aat atg aca att gaa tgc aaa
ttc cca gta gaa aaa caa tta 202Gly Ser Asn Met Thr Ile Glu Cys Lys
Phe Pro Val Glu Lys Gln Leu 35 40 45gac ctg gct gca cta att gtc tat
tgg gaa atg gag gat aag aac att 250Asp Leu Ala Ala Leu Ile Val Tyr
Trp Glu Met Glu Asp Lys Asn Ile 50 55 60att caa ttt gtg cat gga gag
gaa gac ctg aag gtt cag cat agt agc 298Ile Gln Phe Val His Gly Glu
Glu Asp Leu Lys Val Gln His Ser Ser65 70 75 80tac aga cag agg gcc
cgg ctg ttg aag gac cag ctc tcc ctg gga aat 346Tyr Arg Gln Arg Ala
Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95gct gca ctt cag
atc aca gat gtg aaa ttg cag gat gca ggg gtg tac 394Ala Ala Leu Gln
Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110cgc tgc
atg atc agc tat ggt ggt gcc gac tac aag cga att act gtg 442Arg Cys
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120
125aaa gtc aat gcc cca tac aac aaa atc aac caa aga att ttg gtt gtg
490Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140gat cca gtc acc tct gaa cat gaa ctg aca tgt cag gct gag
ggc tac 538Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu
Gly Tyr145 150 155 160ccc aag gcc gaa gtc atc tgg aca agc agt gac
cat caa gtc ctg agt 586Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln Val Leu Ser 165 170 175ggt aag acc acc acc acc aat tcc aag
aga gag gag aag ctt ttc aat 634Gly Lys Thr Thr Thr Thr Asn Ser Lys
Arg Glu Glu Lys Leu Phe Asn 180 185 190gtg acc agc aca ctg aga atc
aac aca aca act aat gag att ttc tac 682Val Thr Ser Thr Leu Arg Ile
Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205tgc act ttt agg aga
tta gat cct gag gaa aac cat aca gct gaa ttg 730Cys Thr Phe Arg Arg
Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220gtc atc cca
ggt aat att ctg aat gtg tcc att aaa ata tgt cta aca 778Val Ile Pro
Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu Thr225 230 235
240ctg tcc cct agc acc tagcatgatg tctgcctatc atagtcattc agtgattgtt
833Leu Ser Pro Ser Thr 245gaataaatga atgaatgaat aacactatgt
ttacaaaata tatcctaatt cctcacctcc 893attcatccaa accatattgt
tacttaataa acattcagca gatatttatg gaataaaaaa 953aaaaaaaaaa aaaaa
96842245PRTHomo sapiens 42Met Arg Ile Phe Ala Val Phe Ile Phe Met
Thr Tyr Trp His Leu Leu1 5 10 15Asn Ala Phe Thr Val Thr Val Pro Lys
Asp Leu Tyr Val Val Glu Tyr 20 25 30Gly Ser Asn Met Thr Ile Glu Cys
Lys Phe Pro Val Glu Lys Gln Leu 35 40 45Asp Leu Ala Ala Leu Ile Val
Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60Ile Gln Phe Val His Gly
Glu Glu Asp Leu Lys Val Gln His Ser Ser65 70 75 80Tyr Arg Gln Arg
Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95Ala Ala Leu
Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Arg
Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120
125Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu
Gly Tyr145 150 155 160Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln Val Leu Ser 165 170 175Gly Lys Thr Thr Thr Thr Asn Ser Lys
Arg Glu Glu Lys Leu Phe Asn 180 185 190Val Thr Ser Thr Leu Arg Ile
Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205Cys Thr Phe Arg Arg
Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220Val Ile Pro
Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu Thr225 230 235
240Leu Ser Pro Ser Thr 245431553DNAHomo sapiensCDS(53)..(922)
43cgaggctccg caccagccgc gcttctgtcc gcctgcaggg cattccagaa ag atg agg
58 Met Arg 1ata ttt gct gtc ttt ata ttc atg acc tac tgg cat ttg ctg
aac gca 106Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
Asn Ala 5 10 15ttt act gtc acg gtt ccc aag gac cta tat gtg gta gag
tat ggt agc 154Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu
Tyr Gly Ser 20 25 30aat atg aca att gaa tgc aaa ttc cca gta gaa aaa
caa tta gac ctg 202Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys
Gln Leu Asp Leu35 40 45 50gct gca cta att gtc tat tgg gaa atg gag
gat aag aac att att caa 250Ala Ala Leu Ile Val Tyr Trp Glu Met Glu
Asp Lys Asn Ile Ile Gln 55 60 65ttt gtg cat gga gag gaa gac ctg aag
gtt cag cat agt agc tac aga 298Phe Val His Gly Glu Glu Asp Leu Lys
Val Gln His Ser Ser Tyr Arg 70 75 80cag agg gcc cgg ctg ttg aag gac
cag ctc tcc ctg gga aat gct gca 346Gln Arg Ala Arg Leu Leu Lys Asp
Gln Leu Ser Leu Gly Asn Ala Ala 85 90 95ctt cag atc aca gat gtg aaa
ttg cag gat gca ggg gtg tac cgc tgc 394Leu Gln Ile Thr Asp Val Lys
Leu Gln Asp Ala Gly Val Tyr Arg Cys 100 105 110atg atc agc tat ggt
ggt gcc gac tac aag cga att act gtg aaa gtc 442Met Ile Ser Tyr Gly
Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val115 120 125 130aat gcc
cca tac aac aaa atc aac caa aga att ttg gtt gtg gat cca 490Asn Ala
Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 135 140
145gtc acc tct gaa cat gaa ctg aca tgt cag gct gag ggc tac ccc aag
538Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
150 155 160gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt
ggt aag 586Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
Gly Lys 165 170 175acc acc acc acc aat tcc aag aga gag gag aag ctt
ttc aat gtg acc 634Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu
Phe Asn Val Thr 180 185 190agc aca ctg aga atc aac aca aca act aat
gag att ttc tac tgc act 682Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn
Glu Ile Phe Tyr Cys Thr195 200 205 210ttt agg aga tta gat cct gag
gaa aac cat aca gct gaa ttg gtc atc 730Phe Arg Arg Leu Asp Pro Glu
Glu Asn His Thr Ala Glu Leu Val Ile 215 220 225cca gaa cta cct ctg
gca cat cct cca aat gaa agg act cac ttg gta 778Pro Glu Leu Pro Leu
Ala His Pro Pro Asn Glu Arg Thr His Leu Val 230 235 240att ctg gga
gcc atc tta tta tgc ctt ggt gta gca ctg aca ttc atc 826Ile Leu Gly
Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr Phe Ile 245 250 255ttc
cgt tta aga aaa ggg aga atg atg gat gtg aaa aaa tgt ggc atc 874Phe
Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys Gly Ile 260 265
270caa gat aca aac tca aag aag caa agt gat aca cat ttg gag gag acg
922Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu Glu
Thr275 280 285 290taatccagca ttggaacttc tgatcttcaa gcagggattc
tcaacctgtg gtttaggggt 982tcatcggggc tgagcgtgac aagaggaagg
aatgggcccg tgggatgcag gcaatgtggg 1042acttaaaagg cccaagcact
gaaaatggaa cctggcgaaa gcagaggagg agaatgaaga 1102aagatggagt
caaacaggga gcctggaggg agaccttgat actttcaaat gcctgagggg
1162ctcatcgacg cctgtgacag ggagaaagga tacttctgaa caaggagcct
ccaagcaaat 1222catccattgc tcatcctagg aagacgggtt gagaatccct
aatttgaggg tcagttcctg 1282cagaagtgcc ctttgcctcc actcaatgcc
tcaatttgtt ttctgcatga ctgagagtct 1342cagtgttgga acgggacagt
atttatgtat gagtttttcc tatttatttt gagtctgtga 1402ggtcttcttg
tcatgtgagt gtggttgtga atgatttctt ttgaagatat attgtagtag
1462atgttacaat tttgtcgcca aactaaactt gctgcttaat gatttgctca
catctagtaa 1522aacatggagt atttgtaaaa aaaaaaaaaa a 155344290PRTHomo
sapiens 44Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His
Leu Leu1 5 10 15Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val
Val Glu Tyr 20 25 30Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val
Glu Lys Gln Leu 35 40 45Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met
Glu Asp Lys Asn Ile 50 55 60Ile Gln Phe Val His Gly Glu Glu Asp Leu
Lys Val Gln His Ser Ser65 70 75 80Tyr Arg Gln Arg Ala Arg Leu Leu
Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95Ala Ala Leu Gln Ile Thr Asp
Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Arg Cys Met Ile Ser
Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125Lys Val Asn
Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140Asp
Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr145 150
155 160Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu
Ser 165 170
175Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile
Phe Tyr 195 200 205Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His
Thr Ala Glu Leu 210 215 220Val Ile Pro Glu Leu Pro Leu Ala His Pro
Pro Asn Glu Arg Thr His225 230 235 240Leu Val Ile Leu Gly Ala Ile
Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255Phe Ile Phe Arg Leu
Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270Gly Ile Gln
Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285Glu
Thr 29045819DNAHomo sapiensCDS(1)..(819) 45atg atc ttc ctc ctg cta
atg ttg agc ctg gaa ttg cag ctt cac cag 48Met Ile Phe Leu Leu Leu
Met Leu Ser Leu Glu Leu Gln Leu His Gln1 5 10 15ata gca gct tta ttc
aca gtg aca gtc cct aag gaa ctg tac ata ata 96Ile Ala Ala Leu Phe
Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30gag cat ggc agc
aat gtg acc ctg gaa tgc aac ttt gac act gga agt 144Glu His Gly Ser
Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45cat gtg aac
ctt gga gca ata aca gcc agt ttg caa aag gtg gaa aat 192His Val Asn
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60gat aca
tcc cca cac cgt gaa aga gcc act ttg ctg gag gag cag ctg 240Asp Thr
Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu65 70 75
80ccc cta ggg aag gcc tcg ttc cac ata cct caa gtc caa gtg agg gac
288Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp
85 90 95gaa gga cag tac caa tgc ata atc atc tat ggg gtc gcc tgg gac
tac 336Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp
Tyr 100 105 110aag tac ctg act ctg aaa gtc aaa gct tcc tac agg aaa
ata aac act 384Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys
Ile Asn Thr 115 120 125cac atc cta aag gtt cca gaa aca gat gag gta
gag ctc acc tgc cag 432His Ile Leu Lys Val Pro Glu Thr Asp Glu Val
Glu Leu Thr Cys Gln 130 135 140gct aca ggt tat cct ctg gca gaa gta
tcc tgg cca aac gtc agc gtt 480Ala Thr Gly Tyr Pro Leu Ala Glu Val
Ser Trp Pro Asn Val Ser Val145 150 155 160cct gcc aac acc agc cac
tcc agg acc cct gaa ggc ctc tac cag gtc 528Pro Ala Asn Thr Ser His
Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175acc agt gtt ctg
cgc cta aag cca ccc cct ggc aga aac ttc agc tgt 576Thr Ser Val Leu
Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190gtg ttc
tgg aat act cac gtg agg gaa ctt act ttg gcc agc att gac 624Val Phe
Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200
205ctt caa agt cag atg gaa ccc agg acc cat cca act tgg ctg ctt cac
672Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His
210 215 220att ttc atc ccc tcc tgc atc att gct ttc att ttc ata gcc
aca gtg 720Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala
Thr Val225 230 235 240ata gcc cta aga aaa caa ctc tgt caa aag ctg
tat tct tca aaa gac 768Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu
Tyr Ser Ser Lys Asp 245 250 255aca aca aaa aga cct gtc acc aca aca
aag agg gaa gtg aac agt gct 816Thr Thr Lys Arg Pro Val Thr Thr Thr
Lys Arg Glu Val Asn Ser Ala 260 265 270atc 819Ile46273PRTHomo
sapiens 46Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu
His Gln1 5 10 15Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu
Tyr Ile Ile 20 25 30Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe
Asp Thr Gly Ser 35 40 45His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu
Gln Lys Val Glu Asn 50 55 60Asp Thr Ser Pro His Arg Glu Arg Ala Thr
Leu Leu Glu Glu Gln Leu65 70 75 80Pro Leu Gly Lys Ala Ser Phe His
Ile Pro Gln Val Gln Val Arg Asp 85 90 95Glu Gly Gln Tyr Gln Cys Ile
Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105 110Lys Tyr Leu Thr Leu
Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr 115 120 125His Ile Leu
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln 130 135 140Ala
Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val145 150
155 160Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln
Val 165 170 175Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn
Phe Ser Cys 180 185 190Val Phe Trp Asn Thr His Val Arg Glu Leu Thr
Leu Ala Ser Ile Asp 195 200 205Leu Gln Ser Gln Met Glu Pro Arg Thr
His Pro Thr Trp Leu Leu His 210 215 220Ile Phe Ile Pro Ser Cys Ile
Ile Ala Phe Ile Phe Ile Ala Thr Val225 230 235 240Ile Ala Leu Arg
Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255Thr Thr
Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265
270Ile47906DNAHomo sapiens 47atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc
ggtcagaatg cctatctgcc ctgcttctac 120accccagccg ccccagggaa
cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg
gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
240agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 300actctagcag acagtgggat ctactgctgc cggatccaaa
tcccaggcat aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactcgg 420cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 480gagacacaga
cactggggag cctccctgat ataaatctaa cacaaatatc cacattggcc
540aatgagttac gggactctag attggccaat gacttacggg actctggagc
aaccatcaga 600ataggcatct acatcggagc agggatctgt gctgggctgg
ctctggctct tatcttcggc 660gctttaattt tcaaatggta ttctcatagc
aaagagaaga tacagaattt aagcctcatc 720tctttggcca acctccctcc
ctcaggattg gcaaatgcag tagcagaggg aattcgctca 780gaagaaaaca
tctataccat tgaagagaac gtatatgaag tggaggagcc caatgagtat
840tattgctatg tcagcagcag gcagcaaccc tcacaacctt tgggttgtcg
ctttgcaatg 900ccatag 90648301PRTHomo sapiens 48Met Phe Ser His Leu
Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Thr Arg
Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn Ala Tyr
Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40 45Val Pro
Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly 50 55 60Asn
Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser65 70 75
80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr
85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg
Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu
Lys Leu Val 115 120 125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr
Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr
Thr Arg Gly His Gly Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly
Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala
Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu 180 185 190Arg Asp
Ser Gly Ala Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly 195 200
205Ile Cys Ala Gly Leu Ala Leu Ala Leu Ile Phe Gly Ala Leu Ile Phe
210 215 220Lys Trp Tyr Ser His Ser Lys Glu Lys Ile Gln Asn Leu Ser
Leu Ile225 230 235 240Ser Leu Ala Asn Leu Pro Pro Ser Gly Leu Ala
Asn Ala Val Ala Glu 245 250 255Gly Ile Arg Ser Glu Glu Asn Ile Tyr
Thr Ile Glu Glu Asn Val Tyr 260 265 270Glu Val Glu Glu Pro Asn Glu
Tyr Tyr Cys Tyr Val Ser Ser Arg Gln 275 280 285Gln Pro Ser Gln Pro
Leu Gly Cys Arg Phe Ala Met Pro 290 295 30049846DNAMus musculus
49atgttttcag gtcttaccct caactgtgtc ctgctgctgc tgcaactact acttgcaagg
60tcattggaaa atgcttatgt gtttgaggtt ggtaagaatg cctatctgcc ctgcagttac
120actctatcta cacctggggc acttgtgcct atgtgctggg gcaagggatt
ctgtccttgg 180tcacagtgta ccaacgagtt gctcagaact gatgaaagaa
atgtgacata tcagaaatcc 240agcagatacc agctaaaggg cgatctcaac
aaaggagacg tgtctctgat cataaagaat 300gtgactctgg atgaccatgg
gacctactgc tgcaggatac agttccctgg tcttatgaat 360gataaaaaat
tagaactgaa attagacatc aaagcagcca aggtcactcc agctcagact
420gcccatgggg actctactac agcttctcca agaaccctaa ccacggagag
aaatggttca 480gagacacaga cactggtgac cctccataat aacaatggaa
caaaaatttc cacatgggct 540gatgaaatta aggactctgg agaaacgatc
agaactgcta tccacattgg agtgggagtc 600tctgctgggt tgaccctggc
acttatcatt ggtgtcttaa tccttaaatg gtattcctgt 660aagaaaaaga
agttatcgag tttgagcctt attacactgg ccaacttgcc tccaggaggg
720ttggcaaatg caggagcagt caggattcgc tctgaggaaa atatctacac
catcgaggag 780aacgtatatg aagtggagaa ttcaaatgag tactactgct
acgtcaacag ccagcagcca 840tcctga 84650281PRTMus musculus 50Met Phe
Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu
Leu Ala Arg Ser Leu Glu Asn Ala Tyr Val Phe Glu Val Gly Lys 20 25
30Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Ser Thr Pro Gly Ala Leu
35 40 45Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys
Thr 50 55 60Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln
Lys Ser65 70 75 80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly
Asp Val Ser Leu 85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly
Thr Tyr Cys Cys Arg 100 105 110Ile Gln Phe Pro Gly Leu Met Asn Asp
Lys Lys Leu Glu Leu Lys Leu 115 120 125Asp Ile Lys Ala Ala Lys Val
Thr Pro Ala Gln Thr Ala His Gly Asp 130 135 140Ser Thr Thr Ala Ser
Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser145 150 155 160Glu Thr
Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile 165 170
175Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr
180 185 190Ala Ile His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu
Ala Leu 195 200 205Ile Ile Gly Val Leu Ile Leu Lys Trp Tyr Ser Cys
Lys Lys Lys Lys 210 215 220Leu Ser Ser Leu Ser Leu Ile Thr Leu Ala
Asn Leu Pro Pro Gly Gly225 230 235 240Leu Ala Asn Ala Gly Ala Val
Arg Ile Arg Ser Glu Glu Asn Ile Tyr 245 250 255Thr Ile Glu Glu Asn
Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Tyr 260 265 270Cys Tyr Val
Asn Ser Gln Gln Pro Ser 275 280511578DNAHomo sapiens 51atgtgggagg
ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60aagcctctcc
agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc
120cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg
aagagcaggg 180gtcacttggc agcatcagcc agacagtggc ccgcccgctg
ccgcccccgg ccatcccctg 240gcccccggcc ctcacccggc ggcgccctcc
tcctgggggc ccaggccccg ccgctacacg 300gtgctgagcg tgggtcccgg
aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360cagctggatg
agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg
420cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc
cctctcctgc 480cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca
gccccccagg atctctcaga 540gcctccgact gggtcatttt gaactgctcc
ttcagccgcc ctgaccgccc agcctctgtg 600cattggttcc ggaaccgggg
ccagggccga gtccctgtcc gggagtcccc ccatcaccac 660ttagcggaaa
gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc
720tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct
cactgttctg 780ggtctggagc ccccaactcc cttgacagtg tacgctggag
caggttccag ggtggggctg 840ccctgccgcc tgcctgctgg tgtggggacc
cggtctttcc tcactgccaa gtggactcct 900cctgggggag gccctgacct
cctggtgact ggagacaatg gcgactttac ccttcgacta 960gaggatgtga
gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag
1020cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc
ctttgggtca 1080cctggatccc tggggaagct gctttgtgag gtgactccag
tatctggaca agaacgcttt 1140gtgtggagct ctctggacac cccatcccag
aggagtttct caggaccttg gctggaggca 1200caggaggccc agctcctttc
ccagccttgg caatgccagc tgtaccaggg ggagaggctt 1260cttggagcag
cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga
1320gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcatccttgg
tgtcctttct 1380ctgctccttt tggtgactgg agcctttggc tttcaccttt
ggagaagaca gtggcgacca 1440agacgatttt ctgccttaga gcaagggatt
caccctccgc aggctcagag caagatagag 1500gagctggagc aagaaccgga
gccggagccg gagccggaac cggagcccga gcccgagccc 1560gagccggagc agctctga
157852525PRTHomo sapiens 52Met Trp Glu Ala Gln Phe Leu Gly Leu Leu
Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val Lys Pro Leu Gln Pro
Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Ala
Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu Gln Asp Leu Ser Leu
Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His Gln Pro Asp Ser Gly
Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75 80Ala Pro Gly Pro
His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95Arg Arg Tyr
Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110Arg
Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120
125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala
130 135 140Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu
Ser Cys145 150 155 160Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met
Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg Ala Ser Asp Trp Val
Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro Asp Arg Pro Ala Ser
Val His Trp Phe Arg Asn Arg Gly Gln 195 200 205Gly Arg Val Pro Val
Arg Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220Phe Leu Phe
Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly225 230 235
240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn
245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val
Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu
Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp
Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu Leu Val Thr Gly Asp
Asn Gly Asp Phe Thr Leu Arg Leu305 310 315 320Glu Asp Val Ser Gln
Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335Leu Gln Glu
Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350Val
Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360
365Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser
370 375 380Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu
Glu Ala385 390 395 400Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln
Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg Leu Leu Gly Ala Ala Val
Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro Gly Ala Gln Arg Ser Gly
Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440 445His Leu Leu Leu Phe
Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu 450 455 460Val Thr
Gly
Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro465 470 475
480Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln
485 490 495Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro
Glu Pro 500 505 510Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln
Leu 515 520 525531566DNAMus musculus 53atgagggagg acctgctcct
tggctttttg cttctgggac tgctttggga agctccagtt 60gtgtcttcag ggcctgggaa
agagctcccc gtggtgtggg cccaggaggg agctcccgtc 120catcttccct
gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg
180gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc
ccttgacctt 240caccagggga tgccctcgcc tagacaaccc gcacccggtc
gctacacggt gctgagcgtg 300gctccaggag gcctgcgcag cgggaggcag
cccctgcatc cccacgtgca gctggaggag 360cgcggcctcc agcgcgggga
cttctctctg tggttgcgcc cagctctgcg caccgatgcg 420ggcgagtacc
acgccaccgt gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg
480cgcgtcggcc aggcctcgat gattgctagt ccctcaggag tcctcaagct
gtctgattgg 540gtccttttga actgctcctt cagccgtcct gaccgcccag
tctctgtgca ctggttccag 600ggccagaacc gagtgcctgt ctacaactca
ccgcgtcatt ttttagctga aactttcctg 660ttactgcccc aagtcagccc
cctggactct gggacctggg gctgtgtcct cacctacaga 720gatggcttca
atgtctccat cacgtacaac ctcaaggttc tgggtctgga gcccgtagcc
780cctctgacag tgtacgctgc tgaaggttct agggtggagc tgccctgtca
tttgccccca 840ggagtgggga ccccttcttt gctcattgcc aagtggactc
ctcctggagg aggtcctgag 900ctccccgtgg ctggaaagag tggcaatttt
acccttcacc ttgaggctgt gggtctggca 960caggctggga cctacacctg
tagcatccat ctgcagggac agcagctcaa tgccactgtc 1020acgttggcgg
tcatcacagt gactcccaaa tccttcgggt tacctggctc ccgggggaag
1080ctgttgtgtg aggtaacccc ggcatctgga aaggaaagat ttgtgtggcg
tcccctgaac 1140aatctgtcca ggagttgccc gggccctgtg ctggagattc
aggaggccag gctccttgct 1200gagcgatggc agtgtcagct gtacgagggc
cagaggcttc ttggagcgac agtgtacgcc 1260gcagagtcta gctcaggcgc
ccacagtgct aggagaatct caggtgacct taaaggaggc 1320catctcgttc
tcgttctcat ccttggtgcc ctctccctgt tccttttggt ggccggggcc
1380tttggctttc actggtggag aaaacagttg ctactgagaa gattttctgc
cttagaacat 1440gggattcagc catttccggc tcagaggaag atagaggagc
tggagcgaga actggagacg 1500gagatgggac aggagccgga gcccgagccg
gagccacagc tggagccaga gcccaggcag 1560ctctga 156654521PRTMus
musculus 54Met Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu
Leu Trp1 5 10 15Glu Ala Pro Val Val Ser Ser Gly Pro Gly Lys Glu Leu
Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys
Ser Leu Lys Ser 35 40 45Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly
Gly Val Ile Trp Gln 50 55 60His Gln Pro Asp Ser Gly Gln Pro Thr Pro
Ile Pro Ala Leu Asp Leu65 70 75 80His Gln Gly Met Pro Ser Pro Arg
Gln Pro Ala Pro Gly Arg Tyr Thr 85 90 95Val Leu Ser Val Ala Pro Gly
Gly Leu Arg Ser Gly Arg Gln Pro Leu 100 105 110His Pro His Val Gln
Leu Glu Glu Arg Gly Leu Gln Arg Gly Asp Phe 115 120 125Ser Leu Trp
Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His 130 135 140Ala
Thr Val Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu145 150
155 160Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu
Lys 165 170 175Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg
Pro Asp Arg 180 185 190Pro Val Ser Val His Trp Phe Gln Gly Gln Asn
Arg Val Pro Val Tyr 195 200 205Asn Ser Pro Arg His Phe Leu Ala Glu
Thr Phe Leu Leu Leu Pro Gln 210 215 220Val Ser Pro Leu Asp Ser Gly
Thr Trp Gly Cys Val Leu Thr Tyr Arg225 230 235 240Asp Gly Phe Asn
Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu 245 250 255Glu Pro
Val Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val 260 265
270Glu Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu
275 280 285Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro
Val Ala 290 295 300Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala
Val Gly Leu Ala305 310 315 320Gln Ala Gly Thr Tyr Thr Cys Ser Ile
His Leu Gln Gly Gln Gln Leu 325 330 335Asn Ala Thr Val Thr Leu Ala
Val Ile Thr Val Thr Pro Lys Ser Phe 340 345 350Gly Leu Pro Gly Ser
Arg Gly Lys Leu Leu Cys Glu Val Thr Pro Ala 355 360 365Ser Gly Lys
Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg 370 375 380Ser
Cys Pro Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala385 390
395 400Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly
Ala 405 410 415Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser
Ala Arg Arg 420 425 430Ile Ser Gly Asp Leu Lys Gly Gly His Leu Val
Leu Val Leu Ile Leu 435 440 445Gly Ala Leu Ser Leu Phe Leu Leu Val
Ala Gly Ala Phe Gly Phe His 450 455 460Trp Trp Arg Lys Gln Leu Leu
Leu Arg Arg Phe Ser Ala Leu Glu His465 470 475 480Gly Ile Gln Pro
Phe Pro Ala Gln Arg Lys Ile Glu Glu Leu Glu Arg 485 490 495Glu Leu
Glu Thr Glu Met Gly Gln Glu Pro Glu Pro Glu Pro Glu Pro 500 505
510Gln Leu Glu Pro Glu Pro Arg Gln Leu 515 520
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References