U.S. patent application number 16/513118 was filed with the patent office on 2020-06-04 for raf1 fusions.
This patent application is currently assigned to BLUEPRINT MEDICINES CORPORATION. The applicant listed for this patent is BLUEPRINT MEDICINES CORPORATION. Invention is credited to Joseph L. Kim, Nicolas Stransky.
Application Number | 20200172981 16/513118 |
Document ID | / |
Family ID | 54545424 |
Filed Date | 2020-06-04 |
View All Diagrams
United States Patent
Application |
20200172981 |
Kind Code |
A1 |
Stransky; Nicolas ; et
al. |
June 4, 2020 |
RAF1 FUSIONS
Abstract
The invention provides to RAF1 gene fusions, RAF1 fusion
proteins, and fragments of those genes and polypeptides. The
invention further provides methods of diagnosing and treating
diseases or disorders associated with RAF1 fusions, such as
conditions mediated by aberrant RAF1 expression or activity, or
overexpression of RAF1.
Inventors: |
Stransky; Nicolas;
(Charlestown, MA) ; Kim; Joseph L.; (Wayland,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BLUEPRINT MEDICINES CORPORATION |
Cambridge |
MA |
US |
|
|
Assignee: |
BLUEPRINT MEDICINES
CORPORATION
Cambridge
MA
|
Family ID: |
54545424 |
Appl. No.: |
16/513118 |
Filed: |
July 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15317685 |
Dec 9, 2016 |
10378063 |
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PCT/US2015/035019 |
Jun 10, 2015 |
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16513118 |
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62010242 |
Jun 10, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/34 20130101;
C12Q 1/6886 20130101; C12Q 2600/106 20130101; G01N 33/5748
20130101; C12Q 2600/156 20130101; C07K 16/32 20130101; C12Q
2600/118 20130101; G01N 2333/91205 20130101; C07K 2317/33
20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574; C07K 16/32 20060101
C07K016/32 |
Claims
1-76. (canceled)
77. A method for detecting in a patient a RAF1 fusion protein or a
RAF1 gene fusion that results in aberrant activity or expression of
RAF1 or overexpression of RAF1, wherein the RAF1 fusion protein is
an AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or a
TRAK1:RAF1 fusion protein, and the RAF1 gene fusion is an
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or a TRAK1:RAF1 gene
fusion, said method comprising: a) contacting a biological sample
from the patient with a reagent selected from i) an antibody that
specifically binds to the RAF1 fusion protein, but does not
specifically bind to wild-type RAF1, AGGF1, LMNA, MPRIP, PAPD7,
CLCN6, or TRAK1; or ii) an oligonucleotide that hybridizes to the
fusion junction of the RAF1 gene fusion; and b) detecting binding
between the RAF1 fusion protein or the RAF1 gene fusion and the
reagent.
78. The method of claim 77, wherein: a) the RAF1 gene fusion
comprises: i) SEQ ID NO:3, 5, 7, 9, or 11, or ii) a portion of SEQ
ID NO:3, 5, 7, 9, or 11, wherein the portion comprises a fusion
junction between RAF1 and its fusion partner and the gene fusion
encodes a polypeptide having RAF1 kinase activity, or b) the RAF1
fusion protein comprises: i) SEQ ID NO:2, 4, 6, 8, 10, or 12, or
ii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein the
portion comprises a fusion junction between RAF1 and its fusion
partner and the fusion protein has RAF1 kinase activity.
79. The method of claim 77, wherein the reagent is an
oligonucleotide that hybridizes under stringent conditions to: a) a
fragment of SEQ ID NO:3 comprising nucleotides 1693-1702 of SEQ ID
NO:3; b) a fragment of SEQ ID NO:5 comprising nucleotides 3041-3050
of SEQ ID NO:5; c) a fragment of SEQ ID NO:7 comprising nucleotides
1265-1274 of SEQ ID NO:7; d) a fragment of SEQ ID NO:9 comprising
nucleotides 143-152 of SEQ ID NO:9; e) a fragment of SEQ ID NO:11
comprising nucleotides 971-980 of SEQ ID NO:11; or f) a
complementary oligonucleotide of any one of a)-e).
80. The method of claim 77, wherein the reagent is an antibody that
specifically binds to: a) a fragment of SEQ ID NO:2 comprising
amino acids 286-295 of SEQ ID NO:2; b) a fragment of SEQ ID NO:4
comprising amino acids 562-572 of SEQ ID NO:4; c) a fragment of SEQ
ID NO:6 comprising amino acids 1011-1020 of SEQ ID NO:6; or d) a
fragment of SEQ ID NO:8 comprising amino acids 419-428 of SEQ ID
NO:8; e) a fragment of SEQ ID NO:10 comprising amino acids 45-54 of
SEQ ID NO:10; or f) a fragment of SEQ ID NO:12 comprising amino
acids 321-330 of SEQ ID NO:12.
81. The method of claim 77, wherein the patient is suffering from
or susceptible to a cancer.
82. The method of claim 81, wherein the cancer is prostate
adenocarcinoma or melanoma.
83. A method of treating a patient in which a RAF1 fusion protein
selected from AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,
CLCN6:RAF1, and TRAK1:RAF1 or a RAF1 gene fusion selected from
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, and TRAK1:RAF1 has
been detected, said method comprising administering to the patient
a therapeutically effective amount of a compound that inhibits RAF1
activity or the activity or expression of the RAF1 fusion protein
or RAF1 gene fusion, wherein the RAF1 fusion protein or RAF1 gene
fusion has been detected in the patient by a method comprising: a)
contacting a biological sample from the patient with a reagent
selected from: i) an antibody that specifically binds to a RAF1
fusion protein, but does not specifically bind to wild-type RAF1,
AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1; or ii) an
oligonucleotide that hybridizes to the fusion junction of a RAF1
gene fusion; and b) detecting binding between the RAF1 fusion
protein or the RAF1 gene fusion and the reagent.
84. The method of claim 83, wherein: a) the RAF1 gene fusion
comprises: i) SEQ ID NO:3, 5, 7, 9, or 11, or ii) a portion of SEQ
ID NO:3, 5, 7, 9, or 11, wherein the portion comprises a fusion
junction between RAF1 and its fusion partner and the gene fusion
encodes a polypeptide having RAF1 kinase activity, or b) the RAF1
fusion protein comprises: i) SEQ ID NO:2, 4, 6, 8, 10, or 12, or
ii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein the
portion comprises a fusion junction between RAF1 and its fusion
partner and the fusion protein has RAF1 kinase activity.
85. The method of claim 83, wherein the reagent is an
oligonucleotide that hybridizes under stringent conditions to: a) a
fragment of SEQ ID NO:3 comprising nucleotides 1693-1702 of SEQ ID
NO:3; b) a fragment of SEQ ID NO:5 comprising nucleotides 3041-3050
of SEQ ID NO:5; c) a fragment of SEQ ID NO:7 comprising nucleotides
1265-1274 of SEQ ID NO:7; d) a fragment of SEQ ID NO:9 comprising
nucleotides 143-152 of SEQ ID NO:9; e) a fragment of SEQ ID NO:11
comprising nucleotides 971-980 of SEQ ID NO:11; or f) a
complementary oligonucleotide of any one of a)-e).
86. The method of claim 83, wherein the reagent is an antibody that
specifically binds to: a) a fragment of SEQ ID NO:2 comprising
amino acids 286-295 of SEQ ID NO:2; b) a fragment of SEQ ID NO:4
comprising amino acids 562-572 of SEQ ID NO:4; c) a fragment of SEQ
ID NO:6 comprising amino acids 1011-1020 of SEQ ID NO:6; or d) a
fragment of SEQ ID NO:8 comprising amino acids 419-428 of SEQ ID
NO:8; e) a fragment of SEQ ID NO:10 comprising amino acids 45-54 of
SEQ ID NO:10; or f) a fragment of SEQ ID NO:12 comprising amino
acids 321-330 of SEQ ID NO:12.
87. The method of claim 83, wherein the patient is suffering from
or susceptible to a cancer.
88. The method of claim 87, wherein the cancer is prostate
adenocarcinoma or melanoma.
89. A molecule capable of specifically binding to a RAF1 fusion
protein or a RAF1 gene fusion that results in aberrant activity or
expression of RAF1 or overexpression RAF1, wherein the molecule is
selected from: a) an antibody that specifically binds to the RAF1
fusion protein selected from AGGF1:RAF1, LMNA: RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, and TRAK1:RAF1, but does not specifically
bind to wild-type RAF1, AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1;
or b) an oligonucleotide that hybridizes to the fusion junction of
the RAF1 gene fusion selected from AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, and TRAK1:RAF1.
90. The molecule of claim 89, wherein: a) the RAF1 gene fusion that
the molecule specifically binds to comprises: i) SEQ ID NO:1, 3, 5,
7, 9, or 11, or ii) a portion of SEQ ID NO:1, 3, 5, 7, 9, or 11,
wherein the portion comprises a fusion junction between RAF1 and
its fusion partner and the gene fusion encodes a polypeptide having
RAF1 kinase activity, or b) the RAF1 fusion protein that the
molecule specifically binds to comprises: i) SEQ ID NO:2, 4, 6, 8,
10, or 12, or ii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12,
wherein the portion comprises a fusion junction between RAF1 and
its fusion partner and the fusion protein has RAF1 kinase
activity.
91. The molecule of claim 89, wherein the molecule is an
oligonucleotide that hybridizes under stringent conditions to: a) a
fragment of SEQ ID NO:1 comprising nucleotides 866-875 of SEQ ID
NO:1; b) a fragment of SEQ ID NO:3 comprising nucleotides 1693-1702
of SEQ ID NO:3; c) a fragment of SEQ ID NO:5 comprising nucleotides
3041-3050 of SEQ ID NO:5; d) a fragment of SEQ ID NO:7 comprising
nucleotides 1265-1274 of SEQ ID NO:7; e) a fragment of SEQ ID NO:9
comprising nucleotides 143-152 of SEQ ID NO:9; f) a fragment of SEQ
ID NO:11 comprising nucleotides 971-980 of SEQ ID NO:11; or g) a
complementary oligonucleotide of any one of a)-f).
92. The molecule of claim 89, wherein the molecule is an antibody
that specifically binds to: a) a fragment of SEQ ID NO:2 comprising
amino acids 286-295 of SEQ ID NO:2; b) a fragment of SEQ ID NO:4
comprising amino acids 562-572 of SEQ ID NO:4; c) a fragment of SEQ
ID NO:6 comprising amino acids 1011-1020 of SEQ ID NO:6; or d) a
fragment of SEQ ID NO:8 comprising amino acids 419-428 of SEQ ID
NO:8; e) a fragment of SEQ ID NO:10 comprising amino acids 45-54 of
SEQ ID NO:10; or f) a fragment of SEQ ID NO:12 comprising amino
acids 321-330 of SEQ ID NO:12.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/010,242, filed Jun. 10, 2014, which is
incorporated here by reference in its entirety to provide
continuity of disclosure.
[0002] This invention relates to RAF1 (RAF proto-oncogene
serine/threonine-protein kinase, also known as proto-oncogene
c-RAF, or c-Raf) gene fusions and RAF1 fusion proteins. The
invention further relates to methods of diagnosing and treating
diseases or disorders associated with RAF1 fusions, such as
conditions mediated by aberrant RAF1 expression or activity, or
conditions associated with overexpression of RAF1.
[0003] Many forms of cancer are caused by genetic lesions that give
rise to tumor initiation and growth. Genetic lesions may include
chromosomal aberrations, such as translocations, inversions,
deletions, copy number changes, gene expression level changes, and
somatic and germline mutations. Indeed, the presence of such
genomic aberrations is a hallmark feature of many cancers,
including, for example, B cell cancer, lung cancer, breast cancer,
ovarian cancer, pancreatic cancer, and colon cancer. In some
models, cancer represents the phenotypic end-point of multiple
genetic lesions that endow cells with a full range of biological
properties required for tumorigenesis.
[0004] Recent efforts by The Cancer Genome Atlas (TCGA), the
International Cancer Genome Consortium (ICGC), and dozens of other
large-scale profiling efforts have generated an enormous amount of
new sequencing data for dozens of cancer types--this includes
whole-genome DNA, whole-exome DNA, and full-transcriptome RNA
sequencing. These efforts have led to the identification of new
driver genes and fusion genes within multiple cancer types.
Fusions, particularly fusions involving kinases, are of particular
interest, as such fusions have been shown to be oncogenic, and have
been successfully targeted by new therapeutics. For example,
anaplastic lymphoma kinase (ALK), one of the receptor tyrosine
kinases, is known to become oncogenic when fused with various
genes. See, e.g., M. Soda et al, "Identification of the
transforming EML4-ALK fusion gene in non-small-cell lung cancer,"
Nature 444:561-566 (2007).
[0005] A need exists for identifying novel genetic lesions
associated with cancer. For example, the presence of fusions
involving a kinase in samples collected from more than one source
can indicate that the kinase is an oncogenic driver. The
identification of such fusions can be an effective approach to
diagnosis of cancers and development of compounds, compositions,
methods, and assays for evaluating and treating cancer
patients.
[0006] In one aspect, the invention provides methods for detecting
the presence of a RAF1 fusion in a biological sample; the methods
include the steps of: (a) obtaining a biological sample from a
mammal; and (b) contacting the sample with a reagent that detects a
RAF1 fusion, to determine whether a RAF1 fusion is present in the
biological sample. In some embodiments, the sample can be from,
e.g., a cancer patient. In some embodiments, the cancer is prostate
adenocarcinoma. In some embodiments, the cancer is melanoma. In
some embodiments, the fusion can be, e.g., an AGGF1:RAF1 fusion, an
LMNA:RAF1 fusion, an MPRIP:RAF1 fusion, a PAPD7:RAF1 fusion, a
CLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion. In some embodiments, the
AGGF1:RAF1 fusion has all or a part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:1 and SEQ ID NO:2, respectively. In some embodiments, the
LMNA:RAF1 fusion has all or part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, the
MPRIP:RAF1 fusion has all or part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, the
PAPD7:RAF1 fusion has all or part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:7 and SEQ ID NO:8, respectively. In some embodiments, the
CLCN6:RAF1 fusion has all or part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:9 and SEQ ID NO:10, respectively. In some embodiments, the
TRAK1:RAF1 fusion has all or part of the nucleotide and/or amino
acid sequence (such as, e.g., the fusion junction) set forth in SEQ
ID NO:11 and SEQ ID NO:12, respectively,
[0007] In another aspect, the invention provides methods of
diagnosing a pattern having a disease or disorder associated with
aberrant RAF1 expression or activity, or overexpression of RAF1;
the methods include: (a) obtaining a biological sample from the
patient; and (b) contacting the sample with a reagent that detects
a RAF1 fusion to determine whether a RAF1 fusion is present in the
biological sample, wherein the detection of the RAF1 fusion
indicates the presence of a disorder associated with aberrant RAF1
expression or activity, or overexpression of RAF1.
[0008] The invention also includes methods of determining a
therapeutic regimen for treating a cancer in a human subject;
methods of identifying a patient likely to respond to treatment
with a RAF1 inhibitor or a RAF1 fusion inhibitor; methods of
stratifying a patient population by detecting a RAF1 fusion;
methods of treating a patient; methods of inhibiting the
proliferation of cells containing a RAF1 fusion; methods of
reducing an activity of a RAF1 fusion; methods of treating a
condition mediated by aberrant RAF1 expression or activity; methods
of treating a condition characterized by overexpression of RAF1;
methods of identifying an agent that modulates the activity of a
RAF1 fusion; and methods of monitoring disease burden in a patient
having a condition mediated by RAF1.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 depicts the nucleotide sequence of an AGGF1:RAF1 gene
fusion (SEQ ID NO:1) comprising a portion of the AGGF1 gene
(NM_018146) up to and including exon 5 (amino acid 290) and a
portion of the RAF1 gene (NM_002880) starting at exon 8 (amino acid
279). The underlined codons at nucleotides 868-870 and 871-873
encode the last amino acid of AGGF1 and the first amino acid of
RAF1, respective. The slash after nucleotide 870 indicates the
breakpoint (fusion junction) where translocation and in-frame
fusion has occurred.
[0010] FIG. 2 depicts the amino acid sequence of an AGGF1:RAF1
fusion protein (SEQ ID NO:2). The slash between amino acids 290 and
291 indicates the breakpoint or fusion junction between the AGGF1
and RAF1 proteins. Amino acids 290-291 correspond to nucleotides
868-870 and 871-873 in SEQ ID NO:1.
[0011] FIG. 3 depicts the nucleotide sequence of an LMNA:RAF1 gene
fusion (SEQ ID NO:3) comprising a portion of the LMNA gene
(NM_170707) up to and including exon 10 (amino acid 566) and a
portion of the RAF1 gene (NM_002880) starting at exon 8 (amino acid
279). The underlined codons at nucleotides 1696-1698 and 1699-1701
encode the last amino acid of LMNA and the first amino acid of
RAF1, respectively. The slash after nucleotide 1698 indicates the
breakpoint (fusion junction) where translocation and in-frame
fusion has occurred.
[0012] FIG. 4 depicts the amino acid sequence of an LMNA:RAF1
fusion protein (SEQ ID NO:4). The slash between amino acids 566 and
567 indicates the breakpoint or fusion junction between the LMNA
and RAF1 proteins. Amino acids 566-567 correspond to nucleotides
1696-1698 and 1699-1671 in SEQ ID NO:3.
[0013] FIGS. 5A & 5B depict the nucleotide sequence of an
MPRIP:RAF1 gene fusion (SEQ ID NO:5) comprising an MPRIP gene
(NM_201274) up to and including exon 22 (amino acid 1015) and a
portion of the RAF1 gene (NM_002880) starting at exon 8 (amino acid
279). The underlined codons at nucleotides 3043-3045 and 3046-3048
encode the last amino acid of MPRIP and the first amino acid of
RAF1, respectively. The slash between nucleotides 3045 and 3046
indicates the breakpoint or fusion junction where translocation and
in-frame fusion has occurred.
[0014] FIG. 6 depicts the amino acid sequence of an MPRIP:RAF1
fusion protein (SEQ ID NO:6). The slash between amino acids 1015
and 1016 represents the location where the two proteins are fused
and corresponds to nucleotides 3043-3045 and 3046-3048 of SEQ ID
NO:5.
[0015] FIG. 7 depicts the nucleotide sequence of a PAPD7:RAF1 gene
fusion (SEQ ID NO:7) comprising a PAPD7 gene (NM_006999) up to and
including exon 11 (amino acid 423) and a portion of the RAF1 gene
(NM_002880) starting at exon 10 (amino acid 331). The underlined
codons at nucleotides 1267-1269 and 1270-1272 encode the last amino
acid of PAPD7 and the first amino acid of RAF1, respectively. The
slash between nucleotides 1269 and 1270 indicates the breakpoint or
fusion junction where translocation and in-frame fusion has
occurred.
[0016] FIG. 8 depicts the amino acid sequence of a PAPD7:RAF1
fusion protein (SEQ ID NO:8). The slash between amino acids 423 and
424 represents the location where the two proteins are fused and
corresponds to nucleotides 1267-1269 and 1270-1272 of SEQ ID
NO:7.
[0017] FIG. 9 depicts the nucleotide sequence of a CLCN6:RAF1 gene
fusion (SEQ ID NO:9) comprising a CLCN6 gene (NM_001286) up to and
including exon 2 (amino acid 49) and a portion of the RAF1 gene
(NM_002880) starting at exon 8 (amino acid 279). The underlined
codons at nucleotides 145-147 and 148-150 encode the last amino
acid of CLCN6 and the first amino acid of RAF1, respectively. The
slash between nucleotides 147 and 148 indicates the breakpoint or
fusion junction where translocation and in-frame fusion has
occurred.
[0018] FIG. 10 depicts the amino acid sequence of a CLCN6:RAF1
fusion protein (SEQ ID NO:10). The slash between amino acids 49 and
50 represents the location where the two proteins are fused and
corresponds to nucleotides 145-147 and 148-150 of SEQ ID NO:9.
[0019] FIG. 11 depicts the nucleotide sequence of a TRAK1:RAF1 gene
fusion (SEQ ID NO:11) comprising a TRAK1 gene (NM_001042646) up to
and including exon 9 (amino acid 325) and a portion of the RAF1
gene (NM_002880) starting at exon 8 (amino acid 279). The
underlined codons at nucleotides 973-975 and 976-978 encode the
last amino acid of TRAK1 and the first amino acid of RAF1,
respectively. The slash between nucleotides 975 and 976 indicates
the breakpoint or fusion junction where translocation and in-frame
fusion has occurred.
[0020] FIG. 12 depicts the amino acid sequence of an TRAK1:RAF1
fusion protein (SEQ ID NO:12). The slash between amino acids 325
and 326 represents the location where the two proteins are fused
and corresponds to nucleotides 973-975 and 976-978 of SEQ ID
NO:11.
EXEMPLARY EMBODIMENTS OF THE INVENTION
[0021] The invention is based, at least in part, on the discovery
of novel recombination or translocation events in cancer patients
that result in at least a fragment of a RAF1 gene linked to a
non-homologous promoter via a recombination or translocation event
that may result in aberrant expression (e.g., in a location where
the kinase is not typically expressed) or overexpression of the
kinase domain of the RAF1 gene and thus, an increase in kinase
activity. Thus, a new patient population is identified, which is
characterized by the presence of a RAF1 fusion, e.g., a RAF1 gene
fusion or fusion protein. This new patient population suffers from
or is susceptible to disorders mediated by aberrant RAF1 expression
or activity, or overexpression of RAF1, such as, e.g., a cancer. In
another aspect of the invention, a new subtype of cancer is
identified, which is characterized by the presence of the RAF1
fusions described herein. In some embodiments, the new patient
population suffers from or is susceptible to prostate
adenocarcinoma or melanoma characterized by the presence of a RAF1
fusion. New methods of diagnosing and treating the patient
population and the RAF1 fusion cancer subtype are also
provided.
[0022] The term "RAF1 fusion" is used generically herein, and
includes any fusion molecule (e.g., gene, gene product (e.g., cDNA,
mRNA, or protein), and variants thereof) that includes a fragment
of RAF1, particularly the coding region for the kinase domain of
RAF1, and the coding region of a second, non-homologous gene and a
promoter sequence from the non-homologous gene, such that the
coding sequence for the kinase domain of RAF1 is under control of
the promoter of the non-homologous gene. A RAF1 fusion protein
generally includes the kinase domain of RAF1.
RAF1 Gene Fusions and Fusion Proteins
[0023] RAF1 is a member of the Raf kinase family of
serine/threonine-specific protein kinases, from the TKL
(Tyrosine-kinase-like) group of kinases. RAF1 is a MAP kinase
(MAP3K) that can initiate the entire kinase cascade. Normal,
cellular Raf genes have been shown to imitate to become oncogenes,
by over stimulation of MEK1/2 and ERK1/2 activity.
[0024] RAF1 gene fusions are generated by a fusion between at least
a part of the RAF1 gene and a part of another gene as a result of a
translocation (including inversion) within a chromosome or between
chromosomes. As a result of a translocation, the RAF1 gene maybe
placed under the transcriptional control of the partner gene
promoter, resulting in aberrant RAF1 expression or activity, or
overexpression of RAF1. The overexpression can lead to certain
cancers. Alternatively or additionally, the partner gene may
include a dimerization domain that causes RAF1 to become
constitutively activated, or the fusion event may delete an
autoregulatory region of RAF1 leading to a constitutively activated
kinase. As used herein, the 5'-region is upstream of, and the
3'-region is downstream of, a fusion junction or breakpoint in one
of the component genes. RAF1 and the gene or protein that it is
fused to is referred to as "fusion partners." Alternatively, they
may be identified as a "RAF1 gene fusion" or a "RAF1 fusion
protein" which are collectively termed "RAF1 fusions." The RAF1
fusions disclosed herein have a kinase activity. The phrase "having
a kinase activity" as used in this application means having an
activity as an enzyme phosphorylating the side chain of an amino
acid, such as serine or threonine.
[0025] In some exemplary embodiments, the fusion partner is all or
a portion of AGGF1 (Angiogenic factor with G patch and FHA domains
1). In other exemplary embodiments, the fusion partner is all or a
portion of LMNA (Lamin A/C). In other exemplary embodiments, the
fusion partner is all or a portion of MPRIP (Myosin Phosphatase Rho
Interacting Protein). In certain exemplary embodiments, the fusion
partner is all or a portion of PAPD7 (PAP Associated Domain
Containing 7). In other exemplary embodiments, the fusion partner
is all or a portion of CLCN6 (chloride transport protein 6). In yet
other exemplary embodiments, the fusion partner is all or a portion
of TRAK1 (trafficking protein, kinesin binding 1).
[0026] Reference to "all or a portion" or "all or part" of a RAF1
gene fusion or SEQ NO:1, 3, 5, 7, 9, 11, or 13, means that the
nucleotide sequence comprises the entire RAF1 gene fusion
nucleotide sequence or a fragment of that sequence that comprises
the fusion junction or breakpoint between RAF1 and its fusion
partner (such as, e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or
TRAK1). The fragment may comprise 7, 8, 9, 10, 12, 14, 16, 18, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 120, 150, 175, 200, 250, 300, or more nucleotides spanning
the fusion junction of the RAF1 gene fusion. Reference to "all or a
portion" or "all or part" of a RAF1 fusion protein or SEQ ID NO:2,
4, 6, 8, 10, 12, or 14, means an amino acid sequence that comprises
the entire RAF1 fusion protein amino acid sequence or a fragment of
that sequence that comprises the fusion junction or breakpoint
between RAF1 and its fusion partner (such as, e.g., AGGF1, LMNA,
MPRIP, PAPD7, CLCN6, or TRAK1). The fragment may comprise 8, 10,
12, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 75, or more amino acids spanning the fusion
junction.
[0027] In certain embodiments of the invention, a fusion includes
an in-frame fusion of all or a portion of the AGGF1 gene (e.g., an
AGGF1 promotor or a functional fragment thereof, and one or more
exons encoding AGGF1 or a fragment thereof) and an exon of the RAF1
gene (e.g., one or more exons encoding a RAF1 kinase domain or a
functional fragment thereof). Such a fusion can be referred to as
an AGGF1:RAF1 fusion. In one embodiment, the AGGF1:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0028] In some embodiments, the invention provides an AGGF1:RAF1
gene fusion comprising the nucleotide sequence depicted in FIG. 1
(SEQ ID NO:1), or a fragment thereof that includes the fusion
junction. SEQ ID NO:1 comprises AGGF1 (NM_018046) up to oxen 5
(amino acid number 290) fused to RAF1 (NM_002880), beginning at
exon 8 (amino acid number 279). In some embodiments the AGGF1:RAF1
gene fusion comprises a nucleotide sequence that is at least 85%,
at least 90%, at least 95%, at least 97%, at least 98%, or at least
99% identical to all or part of SEQ ID NO:1. In some embodiments,
the AGGF1:RAF1 gene fusion encodes a protein having the sequence
depicted in FIG. 2 (SEQ ID NO:2) or a sequence that is at least
85%, at least 90%, at least 95%, at least 97%, at least 98%, or at
least 99% identical to all or part of SEQ ID NO:2.
[0029] In other embodiments of the invention, a fusion includes an
in-frame fusion of all or a portion of the LMNA gene (e.g., an LMNA
promoter or a functional fragment thereof, and one or more axons
encoding an LMNA or a fragment thereof) and an exon of the RAF1
gene (e.g., one or more exons encoding a RAF1 kinase domain or a
functional fragment thereof). Such a fusion can be referred to as
an LMNA:RAF1 fusion. In one embodiment, the LMNA:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0030] In some embodiments, the invention provides an LMNA:RAF1
gene fusion comprising the nucleotide sequence depicted in FIG. 3
(SEQ ID NO:3), or a fragment thereof that includes the fusion
junction. SEQ ID NO:3 comprises LMNA (NM_170707) up to exon 10
(amino acid number 566) fused to RAF1 (NM_002880), beginning at
exon 8 (amino acid number 279). In some embodiments the LMNA:RAF1
gene fusion comprises a nucleotide sequence that is at least 85%,
at least 90%, at least 95%, at least 97%, at least 98%, or at least
99% identical to all or part of SEQ ID NO:3. In some embodiments,
the LMNA:RAF1 gene fusion encodes a protein having the sequence
depicted in FIG. 4 (SEQ ID NO:4) or a sequence that is at least
85%, at least 90%, at least 95%, at least 97%, at least 98%, or at
least 99% identical to all or part of SEQ ID NO:4.
[0031] In certain embodiments of the invention, a fusion includes
an in-frame fusion of all or a portion of the MPRIP gene (e.g., an
MPRIP promotor or a functional fragment thereof, and one or more
exons encoding an MPRIP or a fragment thereof) and an exon of the
RAF1 gene (e.g., one or more exons encoding a RAF1 kinase domain or
a functional fragment thereof). Such a fusion can be referred to as
an MPRIP:RAF1 fusion. In some embodiments, the MPRIP:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0032] In other embodiments, the MPRIP:RAF1 fusion has the
nucleotide sequence depicted in FIG. 5 (SEQ ID NO:5), or a fragment
thereof that includes the fusion junction. SEQ ID NO:5 comprises
MPRIP (NM_201274) up to and including exon 22 (amino acid number
1015) fused to RAF1 (NM_002880), beginning at exon 8 (amino acid
279). In some embodiments the MPRIP:RAF1 gene fusion comprises a
nucleotide sequence that is at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identical to all
or part of SEQ ID NO:5. In sonic embodiments, the MPRIP:RAF1 fusion
encodes a protein having the sequence depicted in FIG. 6 (SEQ ID
NO:6) or a sequence that is at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identical to all
or part of SEQ ID NO:6.
[0033] In certain embodiments of the invention, a fusion includes
an in-frame fusion of all or a portion of the PAPD7 gene (e.g., a
PAPD7 promotor or a functional fragment thereof, and one or more
exons encoding a PAPD7 or a fragment thereof) and an exon of the
RAF1 gene (e.g., one or more exons encoding a RAF1 kinase domain or
a functional fragment thereof). Such a fusion can be referred to as
a PAPD7:RAF1 fusion. In one embodiment, the PAPD7:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0034] In some embodiments, the invention provides a PAPD7:RAF1
gene fusion comprising the nucleotide sequence depicted in FIG. 7
(SEQ ID NO:'7), or a fragment of thereof that includes the fusion
junction. SEQ ID NO:7 comprises PAPD7 (NM_006999) up to exon 11
(amino acid number 423) fused to RAF1 (NM_002880), beginning at
exon 10 (amino acid number 331). In some embodiments the PAPD7:RAF1
gene fusion comprises a nucleotide sequence that is at least 85%,
at least 90%, at least 95%, at least 97%, at least 98%, or at least
99% identical to all or part of SEQ ID NO:7. In some embodiments,
the PAPD7:RAF1 gene fusion encodes a protein having the sequence
depicted in FIG. 8 (SEQ ID NO:8) or a sequence that is at least
85%, at least 90%, at least 95%, at least 97%, at least 98%, or at
least 99% identical to all or part of SEQ ID NO:8.
[0035] In certain embodiments of the invention, a fusion includes
an in-frame fusion of all or a portion of the CLCN6 gene (e.g., a
CLCN6 promotor or a functional fragment thereof, and one or more
exons encoding a CLCN6 or a fragment thereof) and an exon of the
RAF1 gene (e.g., one or more exons encoding a RAF1 kinase domain or
a functional fragment thereof). Such a fusion can be referred to as
a CLCN6:RAF1 fusion. In one embodiment, the CLCN6:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0036] In some embodiments, the invention provides a CLCN6:RAF1
gene fusion comprising the nucleotide sequence depicted in FIG. 9
(SEQ ID NO:9), or a fragment of thereof that includes the fusion
junction. SEQ ID NO:9 comprises the CLCN6 gene (NM_001286) up to
and including exon 2 fused to RAF1 (NM_002880), beginning at exon
8. In some embodiments the CLCN6:RAF1 gene fusion comprises a
nucleotide sequence that is at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identical to all
or part of SEQ ID NO:9. In some embodiments, the CLCN6:RAF1 gene
fusion encodes a protein having the sequence depicted in FIG. 10
(SEQ ID NO:10) or a sequence that is at least 85%, at least 90%, at
least 95%, at least 97%, at least 98%, or at least 99% identical to
all or part of SEQ ID NO:10.
[0037] In certain embodiments of the invention, a fusion includes
an in-frame fusion of all or a portion of the TRAK1 gene (e.g., a
TRAK1 promotor or a functional fragment thereof, and one or more
exons encoding a TRAK1 or a fragment thereof) and an exon of the
RAF1 gene (e.g., one or more exons encoding a RAF1 kinase domain or
a functional fragment thereof). Such a fusion can be referred to as
a TRAK1:RAF1 fusion. In one embodiment, the TRAK1:RAF1 fusion
comprises sufficient RAF1 sequence to drive expression of a fusion
protein that has kinase activity, e.g., has elevated activity as
compared with wild type RAF1 in the same tissue or cell.
[0038] In some, embodiments, the invention provides a TRAK1:RAF1
gene fusion comprising the nucleotide sequence depicted in FIG. 11
(SEQ NO:11), or a fragment of thereof that includes the fusion
junction. SEQ ID NO:11 comprises the TRAK1 gene (NM_001042646) up
to and including exon 9 fused to RAF1 (NM_002880), beginning at
exon 8. In some embodiments the TRAK1:RAF1 gene fusion comprises a
nucleotide sequence that is at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identical to SEQ
ID NO:11. In some embodiments, the TRAK1:RAF1 gene fusion encodes a
protein having the sequence depicted in FIG. 12 (SEQ ID NO:12) or a
sequence that is at least 85%, at least 90%, at least 95%, at least
97%, at least 98%, or at least 99% identical to SEQ ID NO:12.
[0039] The nucleic acid sequences of RAF1 gene fusions may be used
as probes, primers, or bait to identify nucleotides from a
biological sample that include, flank, or hybridize to, RAF1
fusions, such as AGGF1:RAF1 (e.g., all or part of SEQ ID NO: 1),
LMNA:RAF1 (e.g., all or part of SEQ ID NO:3), MPRIP:RAF1 (e.g., all
or part of SEQ ID NO:5), CLCN6:RAF1 (e.g., all or part of SEQ ID
NO:9), or TRAK1:RAF1 (e.g., all or part of SEQ ID NO:11) at, e.g.,
the fusion junctions. In certain embodiments, the probe, primer, or
bait molecule is an oligonucleotide that allows capture, detection,
and/or isolation of a RAF1 gene fusion in a biological sample. In
certain embodiments, the probes or primers derived from the nucleic
acid sequences of RAF1 gene fusions (e.g., from the fusion
junctions) may be used, for example, for polymerase chain reaction
(PCR) amplification. The oligonucleotide can comprise a nucleotide
sequence substantially complementary to a fragment of the RAF1 gene
fusion nucleic acid molecules described herein. The sequence
identity between the nucleic acid fragment, e.g., the
oligonucleotide and the target RAF1 gene fusion sequence, need not
be exact, so long as the sequences are sufficiently complementary
to allow the capture, detection, and/or isolation of the target
sequence. In one embodiment, the nucleic acid fragment is a probe
or primer that includes an oligonucleotide between about 5 and 25,
e.g., between 10 and 20, or 10 and 15 nucleotides in length that
includes the fusion junction of a RAF1 fusion, such as e.g.,
AGGF1:RAF1 (e.g., all or part of SEQ ID NO: 1), LMNA:RAF1 (e.g. all
or part of SEQ ID NO:3), MPRIP:RAF1 (e.g., all or part of SEQ ID
NO:5), PAPD7:RAF1 (e.g., all or part of SEQ ID NO:7, CLCN6:RAF1
(e.g., all or part of SEQ ID NO:9), or TRAK1:RAF1 (e.g., all or
part of SEQ ID NO:11). In other embodiments, the nucleic acid
fragment is a bait that includes an oligonucleotide between about
100 to 300 nucleotides, 130 and 230 nucleotides, or 150 and 200
nucleotides in length that includes the fusion junction of a RAF1
fusion, such as e.g., AGGF1:RAF1 (e.g., all or part of SEQ ID NO:
1), LMNA:RAF1 (e.g., all or part of SEQ ID NO:3), MPRIP:RAF1 (e.g.,
all or part of SEQ ID NO:5), PAPD7:RAF1 (e.g., all or part of SEQ
ID NO:7), CLCN6:RAF1 (e.g., all or part of SEQ ID NO:9), or
TRAK1:RAF1 (e.g., all or part of SEQ ID NO:11).
[0040] In certain embodiments, the nucleic acid fragments hybridize
to a nucleotide sequence that includes a breakpoint or fusion
junction, e.g., a breakpoint or fusion junction as identified by a
slash ("/") in FIGS. 1, 3, 5, 7, 9, or 11. For example, the nucleic
acid fragment can hybridize to a nucleotide sequence that includes
the fusion junction between the AGGF1 transcript and the RAF1
transcript (e.g., nucleotides 868-873 of SEQ ID NO:1), between the
MPRIP transcript and the RAF1 transcript (e.g., nucleotides
1696-1701 of SEQ ID NO:3), between the LMNA transcript and the RAF1
transcript (e.g., nucleotides 3043-3048 of SEQ ID NO:5), between
the PAPD7 transcript and the RAF1 transcript (e.g., nucleotides
1267-1272 of SEQ ID NO:7), between the CLCN6 transcript and the
RAF1 transcript (e.g., nucleotides 145-150 of SEQ ID NO:9), or
between the TRAK1 transcript and the RAF1 transcript (e.g.,
nucleotides 973-978 of SEQ ID NO:11), i.e., a nucleotide sequence
that includes a portion of SEQ ID NO: 1, 3, 5, 7, 9, or 11.
Examples include a nucleotide sequence within exons 1 to 5 of an
AGGF1 gene and exons 8-17 of a RAF1 gene (e.g., a portion of SEQ ID
NO:1 comprising nucleotides 868-873, 866-875, 861-880, 846-895, or
821-920); a nucleotide sequence within exons 1-10 of an LMNA gene
and exons 8 to 17 of a RAF1 gene (e.g., a portion of SEQ ID NO:3
comprising nucleotides 1696-1701, 1693-1702, 1688-1707, 1673-1722,
or 1648-1747); a nucleotide sequence within exons 1-22 of an MPRIP
gene and exons 8 to 17 of a RAF1 gene (e.g., a portion of SEQ ID
NO:5 comprising nucleotides 3043-3048, 3041-3050, 3036-3055,
3021-3070, or 2996-3095); a nucleotide sequence within exons 1-11
of a PAPD7 gene and exons 10 to 17 of a RAF1 gene (e.g., a portion
of SEQ ID NO:7 comprising nucleotides 1267-1272, 1265-1274,
1260-1279, 1245-1294); a nucleotide sequence within exons 1-2 of a
CLCN6 gene and exons 8 to 17 of a RAF1 gene (e.g., a portion of SEQ
ID NO:9 comprising nucleotides 145-150, 143-152, 138-157, 123-172,
or 98-197); or a nucleotide sequence within exons 1-9 of a TRAK1
gene and exons 8 to 17 of a RAF1 gene (e.g., a portion of SEQ ID
NO:11 comprising nucleotides 973-978, 971-980, 966-975, 951-990, or
926-1015).
[0041] In other embodiments, the nucleic acid fragment includes a
bait that comprises a nucleotide sequence that hybridizes to a RAF1
gene fusion nucleic acid molecule described herein, and thereby
allows the detection, capture, and/or isolation of the nucleic acid
molecule. In one embodiment, a bait is suitable fur solution phase
hybridization. In other embodiments, a bait includes a binding
entity or detection entity, e.g., an affinity tag or fluorescent
label, that allows detection, capture, and/or separation, e.g., by
binding to a binding entity, of a hybrid formed by a bait and a
nucleic acid hybridized to the bait.
[0042] In exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequence that includes a fusion junction
between the AGGF1 transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:1 comprising nucleotides
868-873 (such as, e.g., a sequence comprising nucleotides 866-875,
861-880, 846-895, or 821-920 of SEQ ID NO:1).
[0043] In other exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequence that includes a fusion junction
between the LMNA transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:3 comprising nucleotides
1696-1701 (such as, e.g., a sequence comprising nucleotides
1693-1702, 1688-1707, 1673-1722, or 1648-1747 of SEQ ID NO:3).
[0044] In other exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequence that includes a fusion junction
between the MPRIP transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:5 comprising nucleotides
3043-3048 (such as, e.g., a sequence comprising nucleotides
3041-3050, 3036-3055, 3021-3070, or 2996-3095 of SEQ ID NO:5).
[0045] In other exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequences that includes a fusion junction
between the PAPD7 transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:7 comprising nucleotides
1267-1272 (such as, e.g., a sequence comprising nucleotides
1265-1274, 1260-1279, 1245-1294, or 1219-1318 of SEQ ID NO:7).
[0046] In some exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequence that includes a fusion junction
between the CLCN6 transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:9 comprising nucleotides
145-150 (such as, e.g., a sequence comprising nucleotides 143-152,
138-157, 123-172, or 98-197 of SEQ ID NO:9).
[0047] In other exemplary embodiments, the nucleic acid fragments
hybridize to a nucleotide sequence that includes a fusion junction
between the TRAK1 transcript and the RAF1 transcript, e.g., a
nucleotide sequence within SEQ ID NO:11 comprising nucleotides
973-978 (such as, e.g., a sequence comprising nucleotides 971-980,
966-975, 951-990, or 926-1015 of SEQ ID NO:11).
[0048] Another aspect of the invention provides RAF1 fusion
proteins (such as, e.g., a purified or isolated AGGF1:RAF1,
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1 fusion
protein), biologically active or antigenic fragments thereof, and
use of those polypeptides for detecting and/or modulating the
biological activity (such as tumorigenic activity) of a RAF1 fusion
protein. Exemplary embodiments of the RAF1 fusion proteins comprise
the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, or
12, and fragments of those sequences.
[0049] In some embodiments, the RAF1 fusion protein of the
invention can include a fragment of an AGGF1 protein, an LMNA
protein, an MPRIP protein, a PAPD7 protein, a CLCN6 protein, or a
TRAK1 protein, and a fragment of a RAF1 protein. In one embodiment,
the RAF1 fusion protein is AGGF1:RAF1 fusion protein having the
amino acid sequence of SEQ ID NO:2 or a fragment thereof, such as,
e.g., amino acids 286-295, 281-300, or 266-315 of SEQ ID NO:2. In
other embodiments, the RAF1 fusion protein is an LMNA:RAF1 fusion
protein having the amino acid sequence of SEQ ID NO:4 or a fragment
thereof, such as, e.g., amino acids 562-572, 556-575, or 542-591 of
SEQ ID NO:4. In some embodiments, the RAF1 fusion protein is an
MPRIP:RAF1 fusion protein having the amino acid sequence of SEQ ID
NO:6 or a fragment thereof, such as, e.g., amino acids 1011-1020,
1006-1025, or 991-1040 of SEQ ID NO:6. In other embodiments, the
RAF1 fusion protein is a PAPD7:RAF1 fusion protein having the amino
acid sequence of SEQ ID NO:8, or a fragment thereof, such as, e.g.,
amino acids 419-428, 414-433, or 399-448 of SEQ ID NO:8. In some
embodiments, the RAF1 fusion protein is a CLCN6:RAF1 fusion protein
having the amino acid sequence of SEQ ID NO:10 or a fragment
thereof, such as, e.g., amino acids 45-54, 40-59, or 25-74 of SEQ
ID NO:10. In other embodiments, the RAF1 fusion protein is a
TRAK1:RAF1 fusion protein having the amino acid sequence of SEQ ID
NO:12 or a fragment thereof, such as, e.g., amino acids 321-330,
316-335, or 301-350 of SEQ ID NO:12.
[0050] In some embodiments, the RAF1 fusion protein is an
AGGF1:RAF1 fusion protein comprising an amino acid sequence that is
at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, or at least 99% identical to SEQ ID NO:2 or a fragment thereof
(e.g., amino acids 286-295, 281-300, or 266-315 of SEQ ID NO:2). In
other embodiments, the RAF1 fusion protein is an LMNA:RAF1 fusion
protein comprising au amino acid sequence that is at least 85%, at
least 90%, at least 95%, at least 97%, at least 98%, or at least
99% identical to SEQ ID NO:4 or a fragment thereof (e.g., amino
acids 562-572, 556-575, or 542-591 of SEQ ID NO:4). In yet other
embodiments, the RAF1 fusion protein is an MPRIP:RAF1 fusion
protein comprising an amino acid sequence that is at least 85%, at
least 90%, at least 95%, at least 97%, at least 98%, or at least
99% identical to SEQ ID NO:6 or a fragment thereof (e.g., amino
acids 1011-1020, 1006-1025, or 991-1040 of SEQ ID NO:6). In some
embodiments the RAF1 fusion protein is a PAPD7:RAF1 fusion protein
comprising an amino acid sequence that is at least 85%, at least
90%, at least 95%, at least 97%, at least 98%, or at least 99%
identical to SEQ ID NO:8 or a fragment thereof (e.g., amino acids
419-428, 414-433, or 399-448 of SEQ ID NO:8). In some embodiments,
the RAF1 fusion protein is a CLCN6:RAF1 fusion protein comprising
an amino acid sequence that is at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identical to SEQ
ID NO:10 or a fragment thereof (e.g., amino acids 45-54, 40-59, or
25-74 of SEQ ID NO:10). In other embodiments, the RAF1 fusion
protein is a TRAK1:RAF1 fusion protein comprising an amino acid
sequence that is at least 85%, at least 90%, at least 95%, at least
97%, at least 98%, or at least 99% identical to SEQ ID NO:12 or a
fragment thereof (e.g., amino acids 321-330, 316-335, or 301-350 of
SEQ ID NO:12).
[0051] In certain embodiments, the RAF1 fusion protein includes a
functional kinase domain. In some embodiments, the RAF1 fusion
protein comprises elevated RAF1 activity as compared with wild type
RAF1 activity (e.g., in a cancer cell, a non-cancer cell adjacent
to the cancer cell, or a non-cancer cell from a control sample,
such as a cancer free subject). In one exemplary embodiment, the
RAF1 fusion protein is an AGGF1:RAF1 fusion and includes a RAF1
serine/threonine kinase domain or a functional fragment thereof. In
other exemplary embodiments, the RAF1 fusion protein is an
LMNA:RAF1 fusion and includes a RAF1 serine/threonine kinase domain
or a functional fragment thereof. In some exemplary embodiments,
the RAF1 fusion protein is an MPRIP:RAF1 fusion and includes a RAF1
serine/threonine kinase domain or a functional fragment thereof. In
yet other exemplary embodiments, the RAF1 fusion protein is a
PAPD7:RAF1 fusion and includes a RAF1 serine/threonine kinase
domain or a functional fragment thereof. In some exemplary
embodiments, the RAF1 fusion protein is a CLCN6:RAF1 fusion and
includes a RAF1 serine/threonine kinase domain or a functional
fragment thereof. In other exemplary embodiments, the RAF1 fusion
protein is a TRAK1:RAF1 fusion and includes a RAF1 serine/threonine
kinase domain or a functional fragment thereof.
[0052] In another embodiment, the RAF1 fusion protein or fragment
is a peptide, e.g., an immunogenic peptide or protein, that
contains a fusion junction with a heterologous protein as described
herein. Such immunogenic peptides or proteins can be used for
vaccine preparation for use in the treatment or prevention of
cancers caused by or exacerbated by RAF1 gene fusions and RAF1
fusion proteins. In other embodiments, such immunogenic peptides or
proteins can be used to raise antibodies specific to the fusion
protein. In some embodiments, the RAF1 fusion protein is present in
combination with or is further conjugated to one or more
adjuvant(s) or immunogen(s), e.g., a protein capable of enhancing
an immune response to the RAF1 fusion protein (e.g., a hapten, a
toxoid, etc.). In some embodiments, the RAF1 fusion protein is an
AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or
TRAK1:RAF1 fusion protein. In some embodiments, the RAF1 fusion
protein comprises the fusion junction of SEQ ID NO:2, 4, 6, 8, 10,
or 12.
[0053] Thus, another aspect of the invention provides an antibody
that binds to a RAF1 fusion protein (such as, e.g., an AGGF1:RAF1,
an LMNA:RAF1, an MPRIP:RAF1, a PAPD7:RAF1, a CLCN6:RAF1, or a
TRAK1:RAF1 fusion protein) or a fragment thereof. In certain
embodiments, the antibody recognizes a RAF1 fusion protein but does
not recognize wild type RAF1 or the wild type fusion partner (such
as, e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1). In some
embodiments, the antibody binds to an epitope comprising the
junction between RAF1 and the fusion partner (e.g., the junction of
AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or
TRAK1:RAF1). In one embodiment, the antibody binds to an AGGF1:RAF1
fusion protein having the amino acid sequence of SEQ ID NO:2 or a
fragment thereof, such as, e.g., amino acids 286-295, 281-300, or
266-315 of SEQ ID NO:2. In other embodiments, the the antibody
binds to an LMNA:RAF1 fusion protein having the amino acid sequence
of SEQ ID NO:4 or a fragment thereof, such as, e.g., amino acids
562-572, 556-575, or 542-591 of SEQ ID NO:4. In some embodiments,
the the antibody binds to an MPRIP:RAF1 fusion protein having the
amino acid sequence of SEQ ID NO:6 or a fragment thereof, such as,
e.g., amino acids 1011-1020, 1006-1025, or 991-1040 of SEQ ID NO:6.
In other embodiments, the the antibody binds to a PAPD7:RAF1 fusion
protein having the amino acid sequence of SEQ ID NO:8 or a fragment
thereof, such as, e.g., amino acids 419-428, 414-433, or 399-448 of
SEQ ID NO:8. In other embodiments, the the antibody binds to a
CLCN6:RAF1 fusion protein having the amino acid sequence of SEQ ID
NO:10 or a fragment thereof, such as, e.g., amino acids 45-54,
40-59, or 25-74 of SEQ ID NO:10. In yet other embodiments, the the
antibody binds to a TRAK1:RAF1 fusion protein having the amino acid
sequence of SEQ ID NO:12 or a fragment thereof, such as, e.g.,
amino acids 321-330, 316-335, or 301-350 of SEQ ID NO:12.
[0054] In certain embodiments, the antibodies of the invention
inhibit and/or neutralize the biological activity of the RAF1
fusion protein, and more specifically, in some embodiments, the
kinase activity of the RAF1 fusion protein. In other embodiments,
the antibodies may be used to detect a RAF1 fusion protein or to
diagnose a patient suffering from a disease or disorder associated
with the expression of a RAF1 fusion protein.
Detection and Diagnostic Methods
[0055] In another aspect, the invention provides a method of
determining the presence of a RAF1 gene fusion or fusion protein,
such as, e.g., an AGGF1:RAF1, an LMNA:RAF1, an MPRIP:RAF1, a
PAPD7:RAF1, a CLCN6:RAF1, or a TRAK1:RAF1 fusion as described
herein. The presence of a RAF1 fusion can indicate that the mammal
providing the biological sample suffers from or is at risk of
developing a disorder mediated by aberrant RAF1 expression or
activity, or overexpression of RAF1, such as, e.g., a cancer. The
presence of a RAF1 gene fusion may also indicate that the cancer is
treatable with a RAF1 inhibitor (such as, e.g., a kinase inhibitor
or an antibody specific to RAF1) or a RAF1 fusion inhibitor. In
some embodiments, the RAF1 fusion present in the sample is
AGGF1:RAF1 and the cancer to be treated is prostate,
adenocarcinoma. In other embodiments, the RAF1 fusion present in
the sample is MPRIP:RAF1, LMNA:RAF1, CLCN6:RAF1, or TRAK1:RAF1 and
the cancer to be treated is melanoma. In other embodiments, the
cancer is a different cancer associated with aberrant expression or
activity of RAF1 or overexpression of RAF1.
[0056] In one embodiment, the RAF1 fusion detected is a nucleic
acid molecule or a polypeptide. The method includes detecting
whether a RAF1 fusion nucleic acid molecule or polypeptide is
present in a cell (e.g., a circulating cell or a cancer cell), a
tissue (e.g., a tumor), or a sample, e.g., a tumor sample, from a
subject. In one embodiment, the sample is a nucleic acid sample. In
one embodiment, the nucleic acid sample comprises DNA, e.g.,
genomic DNA or cDNA, or RNA, e.g., mRNA. In other embodiments, the
sample is a protein sample.
[0057] The sample can be chosen from one or more sample types, such
as, for example, tissue, e.g., cancerous tissue (e.g., a tissue
biopsy), whole blood, serum, plasma, buccal serape, sputum, saliva,
cerebrospinal fluid, urine, stool, circulating tumor cells,
circulating nucleic acids, or bone marrow.
I. Methods for Detecting Gene Fusions
[0058] In some embodiments, the RAF1 fusion is detected in a
nucleic acid molecule by one or more methods chosen from: nucleic
acid hybridization assays (e.g. in situ hybridization, comparative
genomic hybridization, microarray, Southern blot, northern blot),
amplification-based assays (e.g., PCR, PCR-RFLP assay, or real-time
PCR), sequencing and genotyping (e.g. sequence-specific primers,
high-performance liquid chromatography, or mass-spectrometric
genotyping), and screening analysis (including metaphase
cytogenetic analysis by karyotype methods).
(1) Hybridization Methods
[0059] In some embodiments, the reagent hybridizes to a RAF1 gene
fusion, such as, e.g., nucleotides 868-873, 866-875, 861-880,
846-895, or 821-920 of SEQ ID NO:1. In alternate embodiments, the
reagent detects the presence of nucleotides 1696-1701, 1693-1702,
1688-1707, 1673-1722, or 1648-1747of SEQ NO:3, nucleotides
3043-3048, 3041-3050, 3036-3055, 3021-3070, or 2996-3095 of SEQ ID
NO:5, nucleotides 1267-1272, 1265-1274, 1260-1279, 1245-1294, or
1219-1318 of SEQ ID NO:7, nucleotides 145-150, 143-152, 138-157,
123-172, or 98-197 of SEQ ID NO:9, or nucleotides 973-978, 971-980,
966-975, 951-990, or 926-1015 of SEQ ID NO:11.
[0060] In one embodiment, the method includes: contacting a nucleic
acid sample, e.g., a genomic DNA sample (e.g., a chromosomal sample
or a fractionated, enriched or otherwise pre-treated sample) or a
gene product (mRNA, or cDNA), obtained from the subject, with a
nucleic acid fragment e.g., a probe or primer as described herein
(e.g., an exon-specific or a breakpoint-specific probe or primer)
under conditions suitable for hybridization, and determining the
presence or absence of the RAF1 gene fusion, such as, e.g.
AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or
TRAK1:RAF1, as described herein.
[0061] In an alternate embodiment, the method includes the steps of
obtaining a sample; exposing the sample to a nucleic acid probe
which hybridizes to an mRNA or cDNA encoding a RAF1 fusion protein
that comprises amino acids 286-295, 281-300, or 266-315 of SEQ ID
NO:2, amino acids 562-572, 556-575, or 542-591 of SEQ ID NO:4,
amino acids 1011-1020, 1006-1025, or 991-1040 of SEQ ID NO:6, amino
acids 419-428, 414-433, or 399-448 of SEQ ID NO:8, amino acids
45-54, 40-59, or 25-74 of SEQ ID NO:10, or amino acids 321-330,
316-335, or 301-350 of SEQ ID NO:12.
[0062] Hybridization, as described throughout the specification,
may be carried out under stringent conditions, e.g., medium or high
stringency. See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Pr; 2nd edition (1989); T. Brown, Hybridization Analysis
of DNA Blots. Current Protocols in Molecular Biology at
21:2.10.1-2.10.16 (2001). High stringency conditions for
hybridization refer to conditions under which two nucleic acids
must possess a high degree of base pair homology to each other in
order to hybridize. Examples of highly stringent conditions for
hybridization include hybridization in 4.times.sodium
chloride/sodium citrate (SSC), at 65 or 70.degree. C., or
hybridization in 4.times.SSC plus 50% formamide at about 42 or
50.degree. C., followed by at least one, at least two, or at least
three washes in 1.times.SSC, at 65 or 70.degree. C. Another example
of highly stringent conditions includes hybridization in
2.times.SSC; 10.times.Denhardt solution (Fikoll 400+PEG+BSA; ratio
1:1:1); 0.1% SDS; 5 mM EDTA; 50 nM Na.sub.2HPO.sub.4; 250 .mu.g/ml
of herring sperm DNA; 50 .mu.g/ml of tRNA; or 0.25 M of sodium
phosphate buffer, pH 7.2; 1 mM EDTA7% SDS at 60.degree. C.;
followed by washing 2.times.SSC, 0.1% SDS at 60.degree. C.
[0063] The nucleic acid fragments can be detectably labeled with,
e.g., a radiolabel, a fluorescent label, a bioluminescent label, a
chemiluminescent label, an enzyme label, a binding pair label
(e.g., biotin/streptavidin), an antigen lable, or can include an
affinity tag, or identifier (e.g., an adaptor, barcode or other
sequence identifier). Labeled or unlabeled nucleic acids and/or
nucleic acid fragments may be used in reagents for detecting,
capturing, or isolating RAF1 gene fusions. Labeled or unlabeled
nucleic acids and/or nucleic acid fragments may be used in reagents
for detecting, capturing, and/or isolating RAF1 gene fusions, such
as, e.g., AGGF1:RAF1 (e.g., all or part of SEQ ID NO: 1), LMNA:RAF1
(e.g., all or part of SEQ ID NO:3), MPRIP:RAF1 (e.g., all or part
of SEQ ID NO:5), PAPD7:RAF1 (e.g., all or pall of SEQ ID NO:7),
CLCN6:RAF1 (e.g., all or part of SEQ ID NO:9), or TRAK1:RAF1 (e.g.,
all or part of SEQ ID NO:11). In some embodiments, the labeled
reagent can be detected using, e.g., autoradiography, microscopy
(e.g., brightfield, fluorescence, or electron microscopy),
enzyme-linked immunosorbent assay (ELISA), or
immunohistochemistry.
[0064] In some embodiments, the method comprises performing
chromosome in situ hybridization with chromosomal DNA from a
biological sample to detect the presence of a RAF1 gene fusion
(such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,
CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein). In some
embodiments, the chromosome in situ hybridization comprises the
steps of: providing a chromosome (e.g., interphase or metaphase
chromosome) preparation (e.g., by attaching the chromosomes to a
substrate (e.g., glass)); denaturing the chromosomal DNA (e.g., by
exposure to formamide) to separate the double strands of the
polynucleotides from each other; exposing the nucleic acid probe to
the chromosomes under conditions to allow hybridization of the
probe to the target DNA; removing unhybridized or non-specifically
hybridized probes by washing; and detecting the hybridization of
the probe with the target DNA. In some embodiments, the chromosome
in situ hybridization is fluorescence in situ hybridization (FISH).
In some embodiments, the probe is labeled directly by a fluorescent
label, or indirectly by incorporation of a nucleotide containing a
tag or reporter molecule (e.g., biotin, digoxigenin, or hapten)
which after hybridization to the target DNA is then bound by
fluorescently labeled affinity molecule (e.g., an antibody or
streptavidin). In sonic embodiments, the hybridization of the probe
with the target DNA in FISH can be visualized using a fluorescence
microscope.
[0065] In other embodiments, the method comprises performing
Southern blot with DNA polynucleotides from a biological sample to
detect the presence of a RAF1 gene fusion (such as, e.g.,
AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or
TRAK1:RAF1, as disclosed herein). In some embodiments, the Southern
blot comprises the steps of: optionally fragmenting the
polynucleotides into smaller sizes by restriction endonucleases;
separating the polynucleotides by gel electrophoresis; denaturing
the polynucleotides (e.g., by heat or alkali treatment) to separate
the double strands of the polynucleotides from each other;
transferring the polynucleotides from the gel to a membrane (e.g.,
a nylon or nitrocellulose membrane); immobilizing the
polynucleotides to the membrane (e.g., by UV light or heat);
exposing the nucleic acid probe to the polynucleotides under
conditions to allow hybridization of the probe to the target DNA;
removing unhybridized or non-specifically hybridized probes by
washing; and detecting the hybridization of the probe with the
target DNA.
(2) Amplification-Based Assays
[0066] In certain embodiments, the method of determining the
presence of a RAF1 gene fusion, comprises (a) performing a PCR
amplification reaction with polynucleotides from a biological
sample, wherein the amplification reaction utilizes a pair of
primers which will amplify at least a fragment of the RAF1 gene
fusion, wherein the fragment comprises the fusion junction, wherein
the first primer is in sense orientation and the second primer is
in antisense orientation; and (b) detecting an amplification
product, wherein the presence of the amplification product is
indicative of the presence of a RAF1 fusion polynucleotide in the
sample. In specific exemplary embodiments, the RAF1 gene fusion is
AGGF1:RAF1, such as, e.g., the gene fusion of SEQ ID NO: 1 or a
fragment thereof comprising nucleotides 868-873, 866-875, 861-880,
846-895, or 821-920 of SEQ ID NO:1 In other exemplary embodiments,
the gene fission is LMNA:RAF1 such as, e.g. the gene fusion of SEQ
ID NO:3 or a fragment thereof comprising nucleotides 1696-1701,
1693-1702, 1688-1707, 1673-1722, or 1648-1747 of SEQ ID NO:3. In
other exemplary embodiments, the gene fusion is MPRIP:RAF1 such as,
e.g. the gene fusion of SEQ ID NO:5 or a fragment thereof
comprising nucleotides 3043-3048, 3041-3050, 3036-3055, 3021-3070,
or 2996-3095 of SEQ ID NO:5. In certain exemplary embodiments, the
gene fusion is PAPD7:RAF1 such as, e.g. the gene fusion of SEQ ID
NO:7 or a fragment thereof comprising nucleotides 1267-1272,
1265-1274, 1260-1279, 1245-1294, or 1219-1318 of SEQ ID NO:7. In
some exemplary embodiments, the gene fusion is CLCN6:RAF1 such as,
e.g. the gene fusion of SEQ ID NO:9 or a fragment thereof
comprising nucleotides 145-150, 143-152, 138-157, 123-172, or
98-197 of SEQ ID NO:9. In other exemplary embodiments, the gene
fusion is TRAK1:RAF1 such as, e.g. the gene fusion of SEQ ID NO:11
or a fragment thereof comprising nucleotides 973-978, 971-980,
966-975, 951-990, or 926-1015 of SEQ ID NO:11.
[0067] In some embodiments, step (a) of performing a PCR
amplification reaction comprises: (i) providing a reaction mixture
comprising the polynucleotides (e.g., DNA or cDNA) from the
biological sample, the pair of primers which will amplify at least
a fragment of the RAF1 gene fusion wherein the first primer is
complementary to a sequence on the first strand of the
polynucleotides and the second printer is complementary to a
sequence on the second strand of the polynucleotides, a DNA
polymerase, and a plurality of free nucleotides comprising adenine,
thymine cytosine, and guanine (dNTPs); (ii) heating the reaction
mixture to a first predetermined temperature for a first
predetermined time to separate the double strands of the
polynucleotides from each other; (iii) cooling the reaction mixture
to a second predetermined temperature for a second predetermined
time under conditions to allow the first and second primers to
hybridize with their complementary sequences on the first and
second strands of the polynucleotides, and to allow the DNA
polymerase to extend the primers; and (iv) repeating steps (ii) and
(iii) for a predetermined number of cycles (e.g., 10, 15, 20, 25,
30, 35, 40, 45, or 50 cycles).
[0068] In some embodiments, the polynucleotides from the biological
sample comprise RNA, and the method further comprises performing a
RT-PCR amplification reaction with the RNA to synthesize cDNA as
the template for subsequent or simultaneous PCR reactions. In some
embodiments, the RT-PCR amplification reaction comprises providing
a reaction mixture comprising the RNA, a primer which will amplify
the RNA (e.g., a sequence-specific primer, a random primer, or
oligo(dT)s), a reverse transcriptase, and dNTPs, and heating the
reaction mixture to a third predetermined temperature for a third
predetermined time under conditions to allow the reverse
transcriptase to extend the primer.
(3) Sequencing and Genotyping
[0069] Another method for determining the presence of a RAF1 gene
fusion molecule (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)
includes: sequencing a portion of the nucleic acid molecule (e.g.,
sequencing the portion of the nucleic acid molecule that comprises
the fusion junction of a RAF1 gene fusion), thereby determining
that the RAF1 gene fusion is present in the nucleic acid molecule.
Optionally, the sequence acquired is compared to a reference
sequence, or a wild type reference sequence. In one embodiment, the
sequence is determined by a next generation sequencing method. In
some embodiments, the sequencing is automated and/or
high-throughput sequencing. The method can further include
acquiring, e.g., directly or indirectly acquiring, a sample, e.g.,
a tumor or cancer sample, from a patient.
[0070] In some embodiments, the sequencing comprises chain
terminator sequencing (Sanger sequencing) comprising: providing a
reaction mixture comprising a nucleic acid molecule from a
biological sample, a primer complementary to a region of the
template nucleic acid molecule, a DNA polymerase, a plurality of
free nucleotides comprising adenine, thymine, cytosine, and guanine
(dNTPs), and at least one chain terminating nucleotide (e.g., at
least one di-deoxynucleotide (ddNTPs) chosen from ddATP, ddTTP,
ddCTP, and ddGTP), wherein the at least one chain terminating
nucleotide is present in a low concentration so that chain
termination occurs randomly at any one of the positions containing
the corresponding base on the DNA strand; annealing the primer to a
single strand of the nucleic acid molecule; extending the primer to
allow incorporation of the chain terminating nucleotide by the DNA
polymerase to produce a series of DNA fragments that are terminated
at positions where that particular nucleotide is used; separating
the polynucleotides by electrophoresis (e.g., gel or capillary
electrophoresis); and determining the nucleotide order of the
template nucleic acid molecule based on the positions of chain
termination on the DNA fragments. In some embodiments, the
sequencing is carried out with four separate base-specific
reactions, wherein the primer or the chain terminating nucleotide
in each reaction is labeled with a separate fluorescent label. In
other embodiments, the sequencing is carried out in a single
reaction, wherein the four chain terminating nucleotides mixed in
the single reaction are each labeled with a separate fluorescent
label.
[0071] In some embodiments, the sequencing comprises pyrosequencing
(sequencing by synthesis), comprising: (i) providing a reaction
mixture comprising, a nucleic acid molecule from a biological
sample, a primer complementary to a region of the template nucleic
acid molecule, a DNA polymerase, a first enzyme capable of
converting pyrophosphate into ATP, and a second enzyme capable
using ATP to generates a detectable signal (e.g., a
chemiluminescent signal, such as light) in an amount that is
proportional to the amount of ATP; (ii) annealing the primer to a
single strand of the nucleic acid molecule; (iii) adding one of the
four free nucleotides (dNTPs) to allow incorporation of the
correct, complementary dNTP onto the template by the DNA polymerase
and release of pyrophosphate stoichiometrically; (iv) converting
the released pyrophosphate to ATP by the first enzyme; (v)
generating a detectable signal by the second enzyme using the ATP;
(vi) detecting the generated signal and analyzing the amount of
signal generated in a program; (vii) removing the unincorporated
nucleotides; and (viii) repeating steps (iii) to (vii). The method
allows sequencing of a single strand of DNA, one base pair at a
time, and detecting which base was actually added at each step. The
solutions of each type of nucleotides are sequentially added and
removed from the reaction. Light is produced only when the
nucleotide solution complements the first unpaired base of die
template. The order of solutions which produce detectable signals
allows the determination of the sequence of the template.
[0072] In some embodiments, the method of determining the presence
of a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)
comprises analyzing a nucleic acid sample (e.g., DNA, cDNA, or RNA,
or an amplification product thereof) by HPLC. The method may
comprise: passing a pressurized liquid solution containing the
sample through a column filled with a sorbent, wherein the nucleic
acid or protein components in the sample interact differently with
the sorbent, causing different flow rates for the different
components; separating the components as they flow out the column
at different flow rates. In some embodiments, the HPLC is chosen
from, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange
HPLC, and bioaffinity HPLC.
[0073] In sonic embodiments, the method of determining the presence
of a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)
comprises analyzing a nucleic acid sample (e.g., DNA, eDNA, or RNA,
or an amplification product thereof) by mass spectrometry. The
method may comprise: ionizing the components in the sample (e.g.,
by chemical or electron ionization); accelerating and subjecting
the ionized components to an electric or magnetic field; separating
the ionized components based on their mass-to-charge ratios; and
detecting the separated components by a detector capable of
detecting charged particles (e.g., by an electron multiplier).
II. Methods for Detecting Fusion Proteins
[0074] Another aspect of the invention provides a method of
determining the presence of a RAF1 fusion protein in a mammal. The
method comprises the steps of obtaining a biological sample of a
mammal (such as, e.g., a human cancer), and exposing that sample to
at least one reagent that detects a RAF1 fusion protein (e.g., an
antibody that recognizes the RAF1 fusion but does not recognize the
wild type RAF1 or the wild type fusion partner) to determine
whether a RAF1 fusion protein is present in the biological sample.
The detection of a RAF1 fusion protein indicates the presence of a
mutant RAF1 in the mammal (such as, e.g., in the human cancer). In
some embodiments, the RAF1 fusion protein comprises an amino acid
sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% identity
with an amino acid sequence of all or part of SEQ ID NO:2, 4, 6, 8,
10, or 12. In some embodiments the cancer is melanoma. In some
embodiments, the cancer is prostate adenocarcinoma.
[0075] In some embodiments, the reagent that detects a RAF1 fusion
protein can be detectably labeled with, e.g., a radiolabel, a
fluorescent label, a bioluminescent label, a chemiluminescent
label, an enzyme label, a binding pair label (e.g.,
biotin/streptavidin), an antigen label, or can include an affinity
tag or identifier (e.g., an adaptor, barcode or other sequence
identifier). In some embodiments, the labeled reagent can be
detected using, e.g., autoradiography, microscopy (e.g.,
brightfield, fluorescence, or electron microscopy), ELISA, or
immunohistochemistry. In some embodiments, the RAF1 fusion protein
is detected in a biological sample by a method chosen from one or
more of: antibody-based detection (e.g., western blot, ELISA,
immunohistochemistry), size-based detection methods (e.g., HPLC or
mass spectrometry), or protein sequencing.
(1) Antibody-Based Detection
[0076] In some embodiments, the method comprises performing a
western blot with polypeptides from a biological sample to detect
the presence of a RAF1 fusion protein (such as, e.g., AGGF1:RAF1,
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as
disclosed herein). In some embodiments, the western blot comprises
the steps of: separating the polypeptides by gel electrophoresis;
transferring the polypeptides from the gel to a membrane (e.g., a
nitrocellulose or polyvinylidene difluoride (PVDF) membrane);
blocking the membrane to prevent nonspecific binding by incubating
the membrane in a dilute solution of protein (e.g., 3-5% bovine
serum albumin (BSA) or non-fat dry milk in Tris-Buffered Saline
(TBS) or I-Block, with a minute percentage (e.g., 0.1%) of
detergent, such as, e.g., Tween 20 or Triton X-100); exposing the
polypeptides to at least one reagent that detects a RAF1 fusion
protein (e.g., an antibody that recognizes the RAF1 fusion but does
not recognize the wild type RAF1 or the wild type fusion partner);
removing unbound or non-specifically bound reagent by washing; and
detecting the binding of the reagent with the target protein. In
some embodiments, the method comprises two-step detection: exposing
the polypeptides to a primary antibody that specifically binds to a
RAF1 fusion protein; removing unbound or non-specifically hound
primary antibody by washing; exposing the polypeptides to a
secondary antibody that recognizes the primary antibody; removing
unbound or non-specifically bound secondary antibody by washing;
and detecting the binding of the secondary antibody. In some
embodiments, the reagent that detects a RAF1 fusion protein (e.g.,
the fusion specific antibody, or the secondary antibody) is
directly labeled for detection. In other embodiments, the reagent
is linked to an enzyme, and the method further comprises adding a
substrate of the enzyme to the membrane; and developing the
membrane by detecting a detectable signal produced by the reaction
between the enzyme and the substrate. For example, the reagent may
be linked with horseradish peroxidase to cleave a chemiluminescent
agent as a substrate, producing luminescence in proportion to the
amount of the target protein for detection.
[0077] In some embodiments, the method comprises performing ELISA
with polypeptides from a biological sample to detect the presence
of a RAF1 fusion protein (such as, e.g., AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed
herein). In some embodiments, the ELISA is chosen from, e.g.,
direct ELISA, indirect ELISA, sandwich ELISA, and competitive
ELISA.
[0078] In one embodiment, the direct ELISA comprises the steps of:
attaching polypeptides from a biological sample to a surface;
blocking the surface to prevent nonspecific binding by incubating
the surface in a dilute solution of protein; exposing the
polypeptides to an antibody that specifically binds to a RAF1
fusion protein (e.g., an antibody that recognizes the RAF1 fusion
(such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,
CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) but does not
recognize the wild type RAF1 or the wild type fusion partner);
removing unbound or non-specifically bound antibody by washing; and
detecting the binding of the antibody with the target protein. In
some embodiments, the antibody is directly labeled for detection.
In other embodiments, the antibody is linked to an enzyme, and the
method further comprises adding a substrate of the enzyme; and
detecting a detectable signal produced by the reaction between the
enzyme and the substrate.
[0079] In another embodiment, the indirect ELISA comprises the
steps of: attaching polypeptides from a biological sample to a
surface; blocking the surface to prevent nonspecific binding by
incubating the surface in a dilute solution of protein; exposing
the polypeptides to a primary antibody that specifically binds to a
RAF1 fusion protein (such as, e.g., AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed
herein); removing unbound or non-specifically bound primary
antibody by washing; exposing the polypeptides to a secondary
antibody that recognizes the primary antibody; removing unbound or
non-specifically bound secondary antibody by washing; and detecting
the binding of the secondary antibody. In some embodiments, the
secondary antibody is directly labeled for detection. In other
embodiments, the secondary antibody is linked to an enzyme, and the
method further comprises adding a substrate of the enzyme; and
detecting a detectable signal produced by the reaction between the
enzyme and the substrate.
[0080] In some embodiments, the method comprises performing
immunohistochemistry with polypeptides from a biological sample to
detect the presence of a RAF1 fusion protein (such as, e.g.,
AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or
TRAK1:RAF1, as disclosed herein). In some embodiments, the
immunohistochemistry comprises the steps of: fixing a cell or a
tissue section (e.g., by paraformaldehyde or formalin treatment);
permeabilizing the cell or tissue section to allow target
accessibility; blocking the cell or tissue section to prevent
nonspecific binding; exposing the cell or tissue section to at
least one reagent that detects a RAF1 fusion protein (e.g., an
antibody that recognizes the RAF1 fusion but does not recognize the
wild type RAF1 or the wild type fusion partner); removing unbound
or non-specifically bound reagent by washing; and detecting the
binding of the reagent with the target protein. In some
embodiments, the reagent is directly labeled for detection. In
other embodiments, the reagent is linked to an enzyme, and the
method further comprises adding a substrate of the enzyme; and
detecting a detectable signal produced by the reaction between the
enzyme and the substrate. In some embodiments, the
immunohistochemistry may comprise the two-step detection as in the
indirect ELISA.
(2) Size-Based Detection Methods
[0081] In some embodiments, the method of determining the presence
of a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)
comprises analyzing a protein sample by HPLC. The method may
comprise: passing a pressurized liquid solution containing the
sample through a column tilled with a sorbent, wherein the nucleic
acid or protein components in the sample interact differently with
the sorbent, causing different flow rates for the different
components; separating the components as they flow out the column
at different flow rates. In some embodiments, the HPLC is chosen
from, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange
HPLC, and bioaffinity HPLC.
[0082] In some embodiments, the method of determining the presence
of a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,
PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)
comprises analyzing a protein sample by mass spectrometry. The
method may comprise: ionizing the components in the sample (e.g.,
by chemical or electron ionization); accelerating and subjecting
the ionized components to an electric or magnetic field; separating
the ionized components based on their mass-to-charge ratios; and
detecting the separated components by a detector capable of
detecting charged particles (e.g., by an electron multiplier).
[0083] Detection of a RAF1 gene fusion or a RAF1 fusion protein in
a patient can lead to assignment of the patient to the newly
identified patient population that bears the RAF1 fusion. Because
this patient population can suffer from or be susceptible to a
disorder associated with aberrant RAF1 expression or activity or
overexpression of RAF1, detection of the RAF1 fusion can also lead
to diagnosis of such disorder. Thus, a further aspect of the
invention provides a method of stratifying a patient population
(e.g., assigning a patient, to a group or class) and/or diagnosing
a patient, comprising: obtaining a biological sample from the
patient, contacting the sample with at least one reagent that
detects a RAF1 gene fusion or a RAF1 fusion protein to determine
whether a RAF1 fusion is present in the biological sample. The
detection of a RAF1 fusion indicates that the patient belongs to
the newly identified patient population that bears the RAF1 fusion,
and/or the presence of a disorder associated with aberrant RAF1
expression or activity or overexpression of RAF1, such as e.g., a
cancer. The detection of a RAF1 fusion also identities a new
subtype of cancer, which is characterized by the presence of the
RAF1 fusion. In some embodiments, the cancer is melanoma. In sonic
embodiments, the cancer is prostate adenocarcinoma. In certain
embodiments, the RAF1 fusion is AGGF1:RAF1. In other embodiments,
the RAF1 fusion is LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1,
or TRAK1:RAF1. In sonic embodiments, the AGGF1:RAF1 fusion has all
or a part of the nucleotide and/or amino acid sequence (such as,
e.g., the fusion junction) set forth in SEQ ID NO:1 and SEQ ID
NO:2, respectively. In some embodiments, the LMNA:RAF1 fusion has
all or part of the nucleotide and/or amino acid sequence (such as,
e.g., the fusion junction) set forth in SEQ NO:3 and SEQ ID NO:4,
respectively. In some embodiments, the MPRIP:RAF1 fusion has all or
part of the nucleotide and/or amino acid sequence (such as, e.g.,
the fusion junction) set forth in SEQ ID NO:5 and SEQ ID NO:6,
respectively. In some embodiments, the PAPD7:RAF1 fusion has all or
part of the nucleotide and/or amino acid sequence (such as, e.g.,
the fusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:8,
respectively. In some embodiments, the CLCN6:RAF1 fusion has all or
part of the nucleotide and/or amino acid sequence (such as, e.g.,
the fusion junction) set forth in SEQ ID NO:9 and SEQ ID NO:10,
respectively. In some embodiments, the TRAK1:RAF1 fusion has all or
part of the nucleotide and/or amino acid sequence (such as, e.g.,
the fusion junction) set forth in SEQ ID NO:11 and SEQ ID NO:12,
respectively.
[0084] In some embodiments, the RAF1 gene fusion or RAF1 fusion
protein is detected prior to initiating, during, and/or after, a
treatment of a patient with, e.g., a RAF1 inhibitor or a RAF1
fusion inhibitor. In one embodiment, the RAF1 gene fusion or RAF1
fusion protein is detected at the time the patient is diagnosed
with a cancer. In other embodiment, the RAF1 fusion is detected at
a pre-determined interval, e.g., a first point in time and at least
at a subsequent point in time. In certain embodiments, in response
to detection of a RAF1 fusion, such as, e.g., AGGF1:RAF1,
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, the
method further includes one or more of:
[0085] (1) stratifying a patient population (e.g., assigning a
patient, to a group or class);
[0086] (2) identifying or selecting the patient as likely or
unlikely to respond to a treatment, e.g., a RAF1 inhibitor
treatment (e.g., a kinase inhibitor treatment), or a RAF1 fusion
inhibitor treatment as described herein;
[0087] (3) selecting a treatment regimen, e.g., administering or
not administering a preselected therapeutic agent, such as, e.g., a
RAF1 inhibitor, or a RAF1 fusion inhibitor;
[0088] (4) prognosticating the time course of the disease in the
patient e.g., evaluating the likelihood of increased or decreased
patient survival); or
[0089] (5) monitoring the effectiveness of treatment (e.g., by
detecting a reduction in the level of RAF1 gene fusion or fusion
protein in a patient sample).
[0090] In certain embodiments, upon detection of a RAF1 gene fusion
or RAF1 fusion protein in a patient's biological sample, the
patient is identified as likely to respond to a treatment that
comprises a RAF1 inhibitor, or a RAF1 fusion inhibitor. In some
embodiments, the RAF1 fusion detected is an AGGF1:RAF1 fusion. In
alternate embodiments, the RAF1 fusion detected is an MPRIP:RAF1
fusion. In some embodiments, the RAF1 fusion detected is a
PAPD7:RAF1 fusion. In some embodiments, the RAF1 fusion detected is
a LMNA:RAF1 fusion. In some embodiments, the RAF1 fusion detected
is a CLCN6:RAF1 fusion. In sonic embodiments, the RAF1 fusion
detected is a TRAK1:RAF1 fusion.
[0091] A further aspect of the invention provides a method of
selecting a treatment option by detecting a RAF1 fusion. The method
comprises obtaining a biological sample from a patient and exposing
the sample to at least one reagent that detects a RAF1 gene fusion
or fusion protein to determine whether a RAF1 fusion is present in
the biological sample. The detection of the RAF1 fusion indicates
the likelihood of the patient responding to treatment with a RAF1
inhibitor, or a RAF1 fusion inhibitor. The method may be augmented
or personalized by evaluating the effect of a variety of kinase,
RAF1 or RAF1 fusion inhibitors on the biological sample shown to
contain a RAF1 fusion to determine the most appropriate inhibitor
to administer. In certain embodiments, the RAF1 fusion is
AGGF1:RAF1. In other embodiments, the RAF1 fusion is LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1. In some
embodiments, the AGGF1:RAF1 fusion has all or a part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively.
In some embodiments, the LMNA:RAF1 fusion has all or part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.
In some embodiments, the MPRIP:RAF1 fusion has all or part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively.
In some embodiments, the PAPD7:RAF1 fusion has all or part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:7 and SEQ NO:8, respectively. In
some embodiments, the CLCN6:RAF1 fusion has all or part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:10 and SEQ ID NO:11, respectively.
In some embodiments, the TRAK1:RAF1 fusion has all or part of the
nucleotide and/or amino acid sequence (such as, e.g., the fusion
junction) set forth in SEQ ID NO:11 and SEQ ID NO:12,
respectively.
Methods of Treatment
[0092] Alternatively, or in combination with the detection and
diagnostic methods described herein, the invention provides method
for treating the newly identified patient population and the new
RAF1 fusion cancer subtype, which are characterized by the presence
of a RAF1 fusion. The patient population and cancer subtype can be
associated with or predict the onset of a condition mediated by
aberrant RAF1 expression or activity, or overexpression of RAF1,
such as, e.g., a cancer or a tumor harboring a RAF1 fusion. The
methods comprise administering a therapeutic agent, e.g., a RAF1
inhibitor, such as e.g., a kinase inhibitor or an antibody specific
to RAF1; or a RAF1 fusion inhibitor, i.e., an inhibitor that blocks
the activity of the RAF1 fusion but not wild type RAF1 or wild type
fusion partner, such as e.g., an antibody specific to an
AGGF1:RAF1, an LMNA:RAF1, an MPRIP:RAF1, a PAPD7:RAF1, a
CLCN6:RAF1, or a TRAK1:RAF1 fusion protein, e.g., any one of the
antibodies described above, or an RNA inhibitor that recognizes
RAF1 or the fusion junction of a RAF1 gene fusion, including but
not limited to siRNA, dsRNA, shRNA, or any other antisense nucleic
acid inhibitor, alone or in combination with e.g., other
chemotherapeutic agents or procedures, in an amount sufficient to
treat a condition mediated by aberrant RAF1 expression Of activity,
or overexpression of RAF1 by one or more of the following: impeding
growth of a cancer, causing a cancer to shrink by weight or volume,
extending the expected survival time of the patient, inhibiting
tumor growth, reducing tumor mass, reducing size or number of
metastatic lesions, inhibiting the development of new metastatic
lesions, prolonging survival, prolonging progression-free survival,
prolonging time to progression, and/or enhancing quality of
life.
[0093] In certain embodiments, the RAF1 fusion of the invention may
be inhibited by a RAF1 inhibitor or a RAF1 fusion inhibitor. In
some embodiments, the therapeutic agent is a RAF1 inhibitor, such
as, e.g., a compound, biological or chemical, which inhibits,
directly or indirectly, the expression and/or activity of RAF1. For
example, the RAF1 inhibitors may be an antibody (such as, e.g.,
antibodies specific to RAF1) or a small molecule inhibitor (such
as, e.g., a kinase inhibitor). In some embodiments, the inhibitors
may act directly on RAF1 itself, modify the activity of RAF1, or
inhibit the expression of RAF1. In other embodiments, the
inhibitors may indirectly inhibit RAH activity by inhibiting the
activity of proteins or molecules other than RAF1 itself. For
example, the inhibitors may modulate the activity of regulatory
kinases that phosphorylate or dephosphorylate RAF1, interfere with
binding of ligands, or inhibit the activity of interacting or
downstream proteins or molecules.
[0094] In some embodiments, the RAF1 fusion is inhibited by a RAF1
fusion inhibitor, such as, e.g., an antibody that recognizes all or
part of a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described
herein) but does not recognize wild type RAF1 or wild type fusion
partner (e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1). In some
embodiments, the RAF1 fusion protein (such as, e.g., AGGF1:RAF1,
LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as
described herein) is inhibited by an agent that inhibits
transcription or translation of the fusion protein, e.g., an RNA
inhibitor that recognizes the RAF1 coding sequence, the binding
partner (e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1), or the
binding partner: RAF1 fusion junction, including but not limited to
small interfering RNA (siRNA), double stranded RNA (dsRNA),
short-hairpin RNA (shRNA), or any other antisense nucleic acid
inhibitor. In some embodiments, the RAF1 fusion inhibited is
selected from all or a portion of any one of SEQ ID NOs: 1-12.
[0095] As used herein, and unless otherwise specified, a
"therapeutically effective amount" of a compound is an amount
sufficient to provide a therapeutic benefit in the treatment or
management of a condition mediated by aberrant RAF1 expression or
activity, or overexpression of RAF1, such as, delaying or
minimizing one or more symptoms associated with a cancer or a tumor
harboring a RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described
herein). A therapeutically effective amount of a compound means an
amount of therapeutic agent, alone or in combination with other
therapeutic agents, which provides a therapeutic benefit in the
treatment or management of the cancer. The term "therapeutically
effective amount" can encompass an amount that improves overall
therapy, reduces or avoids symptoms or causes of the condition
mediated by aberrant RAF1 expression activity or overexpression of
RAF1, or enhances the therapeutic efficacy of another therapeutic
agent.
[0096] In certain embodiments, the cancer or tumor harboring a RAF1
fusion is melanoma. In some embodiments the cancer or tumor
harboring a RAF1 fusion is a prostate adenocarcinoma.
[0097] In some embodiments, the patient to be treated is suffering
from melanoma, and the method for treating the condition comprises
administering to the patient a therapeutically effective amount of
a RAF1 inhibitor or a RAF1 fusion inhibitor as described above. In
some embodiments, the patient to be treated is suffering from
prostate adenocarcinoma, and the method for treating the condition
comprises administering to the patient a therapeutically effective
amount of a RAF1 inhibitor or a RAF1 fusion inhibitor as described
above.
Screening Methods
[0098] Therapeutic agents, such as, e.g., RAF1 inhibitors, and RAF1
fusion inhibitors (gene fusion and fusion protein), used in the
therapeutic methods of the invention can be evaluated using the
screening assays described herein. Thus, the invention provides a
method of identifying an agent useful for treating a condition
mediated by aberrant RAF1 expression or activity, or overexpression
of RAF1, e.g., cancer or a tumor harboring a RAF1 fusion, such as,
e.g., melanoma or prostate adenocarcinoma, comprising contacting a
cell expressing a RAF1 gene fusion or RAF1 fusion protein with a
candidate agent and using one of the detection methods referenced
above to determine whether the expression level of the fusion is
decreased or a biological function associated with the fusion is
altered. In one embodiment, therapeutic agents can be evaluated in
a cell-free system, e.g., a cell lysate or in a reconstituted
system. In other embodiments, the therapeutic agents are evaluated
in a cell in culture, e.g., a cell expressing a RAF1 fusion (e.g.,
a mammalian cell, a tumor cell or cell line, a recombinant cell).
In yet other embodiments, the therapeutic agents are evaluated in a
cell in vivo (a RAF1 fusion-expressing cell present in a subject,
e.g., an animal subject (e.g., an in vivo animal model).
[0099] Exemplary parameters to evaluate in determining the efficacy
of a therapeutic agent for treating a condition mediated by
aberrant RAF1 expression or activity, or overexpression of RAF1,
such as, e.g., a cancer or a tumor harboring a RAF1 fusion include
one or more of:
[0100] (i) a change in binding activity, e.g., direct binding of
the candidate agent to a RAF1 fusion protein or a binding
competition between a known ligand and the candidate agent to a
RAF1 fusion protein;
[0101] (ii) a change in kinase activity, e.g., phosphorylation
levels of a RAF1 fusion protein (e.g., an increased or decreased
phosphorylation or autophosphorylation) or a change in
phosphorylation of a target of a RAF1 kinase--in certain
embodiments, a change in kinase activity, e.g., phosphorylation, is
detected by any of western blot (e.g., using an anti-RAF1 antibody
or a phosphor-specific antibody, detecting a shift in the molecular
weight of a RAF1 fusion protein), mass spectrometry,
immunoprecipitation, immunohistochemistry, immunomagnetic beads,
among others;
[0102] (iii) a change in an activity of a cell containing a RAF1
fusion (e.g., a tumor cell or a recombinant cell), e.g., a change
in proliferation, morphology, or tumorigenicity of the cell;
[0103] (iv) a change in tumor present in an animal subject, e.g.,
size, appearance, or proliferation of the tumor;
[0104] (v) a change in the level, e.g., expression level, of a RAF1
fusion protein or nucleic acid molecule; or
[0105] (vi) a change in an activity of a signaling pathway
involving RAF1, e.g., phosphorylation or activity of an interacting
or downstream target, or expression level of a target gene.
[0106] In some embodiments, the RAF1 fusion is an AGGF1:RAF1
fusion, an LMNA:RAF1 fusion, an MPRIP:RAF1 fusion, a PAPD7:RAF1
fusion, a CLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion.
[0107] In one embodiment, a change in the activity of a RAF1
fusion, or interaction of a RAF1 fusion with a downstream ligand
detected in a cell free assay in the presence of a candidate agent
indicates that the candidate agent will be effective as a
therapeutic agent for treatment of a condition mediated by aberrant
RAF1 expression or activity, or overexpression of RAF1, such as,
e.g., a cancer or a tumor harboring a RAF1 fusion. In some
embodiments, the cancer or tumor is postate adenocarinoma. In some
embodiments, the cancer or tumor is melanoma.
[0108] In other embodiments, a change in an activity of a cell
expressing a RAF1 fusion, such as, e.g., AGGF1:RAF1, LMNA:RAF1,
MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described
herein, (e.g., a mammalian cell, a tumor cell or cell line, a
recombinant cell) is detected in a cell in culture. In one
embodiment, the cell is a recombinant cell that is modified to
express a RAF1 fusion nucleic acid, e.g., is a recombinant cell
transfected with a RAF1 fusion nucleic acid. The transfected cell
can show a change in response to the expressed RAF1 fusion, e.g.,
increased proliferation, changes in morphology, increased
tumorigenicity, and/or acquired a transformed phenotype. A change
in any of the activities of the cell, e.g., the recombinant cell,
in the presence of the candidate agent can be detected. For
example, a decrease in one or more of: proliferation,
tumorigenicity, or transformed morphology, in the presence of the
candidate agent can be indicative of an inhibitor of a RAF1 fusion.
In other embodiments, a change in binding activity or
phosphorylation of RAF1 or its interacting or downstream proteins
or molecules as described herein is detected.
[0109] In yet other embodiment, a change in a tumor present in an
animal subject (e.g., an in vivo animal model) is detected. In one
embodiment, a tumor containing animal or a xenograft comprising
cells expressing a RAF1 fusion (e.g., tumorigenic cells expressing
a RAF1 fusion) is employed. The therapeutic agents can be
administered to the animal subject and a change in the tumor is
evaluated. In one embodiment, the change in the tumor includes one
Of more of a tumor growth, tumor size, tumor burden, or survival is
evaluated. A decrease in one or more of tumor growth, tumor size,
tumor burden, or an increased survival is indicative that the
candidate agent is an inhibitor or modulator.
[0110] In another aspect of the invention provides a method or
assay for screening for agents that modulate (e.g., inhibit) the
expression or activity of a RAF1 fusion as described herein. The
method includes contacting e.g., a RAF1 fusion, or a cell
expressing a RAF1 fusion, with a candidate agent; and detecting a
change in a parameter associated with a RAF1 fusion, e.g., a change
in the expression or an activity of the RAF1 fusion. The method
can, optionally, include comparing the treated parameter to a
reference value, e.g., a control sample (e.g., comparing a
parameter obtained from a sample with the candidate agent to a
parameter obtained from a sample without the candidate agent). In
one embodiment, if a decrease in expression or activity of the RAF1
fusion is detected, the candidate agent is identified as an
inhibitor. In another embodiment, if an increase in expression or
activity of the RAF1 fusion is detected, the candidate agent is
identified as an activator. In certain embodiments, the RAF1 fusion
is a RAF1 gene fusion or RAF1 fusion protein, such as, e.g., an
AGGF1:RAF1 fusion. an MPRIP:RAF1 fusion, a PAPD7:RAF1 fusion, an
LMNA:RAF1 fusion, a CLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion.
[0111] In one embodiment, the contacting step is detected in a
cell-free system, e.g., a cell lysate or in a reconstituted system.
In other embodiments, the contacting step is detected in a cell in
culture, e.g., a cell expressing a RAF1 fusion (e.g., a mammalian
cell, a tumor cell or cell line, a recombinant cell). In yet other
embodiments, the contacting step is detected in a cell in vivo
(e.g., a RAF1 expressing cell present in a subject, e.g., an animal
subject (e.g., an in vivo animal model)).
[0112] Exemplary parameters evaluated in identifying an agent that
modulates the activity of a RAF1 fusion (e.g., an AGGF1:RAF1
fusion, an MPRIP:RAF1 fusion, a PAPD7:RAF1 fusion, an LMNA:RAF1
fusion, a CLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion) include one or
more of:
[0113] (i) a change in binding activity, e.g., direct binding of
the candidate agent to a RAF1 fusion protein; a binding competition
between a known ligand and the candidate agent to a RAF1 fusion
protein;
[0114] (ii) a change in kinase activity, e.g., phosphorylation
levels of a RAF i fusion protein (e.g., an increased or decreased
phosphorylation or autophosphorylation) or a change in
phosphorylation of a target of a RAF1 kinase--in certain
embodiments, a change in kinase activity, e.g., phosphorylation, is
detected by any of Western blot (e.g., using an anti-RAF1 antibody
or a phosphor-specific antibody, detecting a shift in the molecular
weight of a RAF1 fusion protein), mass spectrometry,
immunoprecipitation, immunohistochemistry, immunomagnetic beads,
among others;
[0115] (iii) a change in an activity of a cell containing a RAF1
fusion (e.g., a tumor cell or a recombinant cell), e.g., a change
in proliferation, morphology, or tumorigenicity of the cell;
[0116] (iv) a change in tumor present in an annual subject, e.g.,
size, appearance, or proliferation of the tumor;
[0117] (v) a change in the level, e.g., expression level, of a RAF1
fusion protein or nucleic acid molecule; or
[0118] (vi) a change in an activity of a signaling pathway
involving RAF1, e.g., phosphorylation or activity of an interacting
or downstream target, or expression level of a target gene.
Methods for Validating RAF1 Fusions
[0119] RAF1 gene fusions (e.g., AGGF1:RAF1 gene fusions, MPRIP:RAF1
gene fusions, PAPD7:RAF1 gene fusions, LMNA:RAF1 gene fusions,
CLCN6:RAF1 gene fusions, or TRAK1:RAF1 gene fusions) may be
evaluated to ensure that the breakpoints are in-frame and can
produce a protein product containing the full kinase domain, i.e.,
that the breakpoint occurs such that complete triplet codons are
intact, and that the RNA sequence will produce a viable protein.
The RAF1 gene fusion can be transfected into cells to confirm that
the protein is functionally active with respect to kinase activity
and oncogenic activity. cDNA encoding the RAF1 fusion protein can
be produced by standard solid-phase DNA synthesis. Alternatively
the RAF1 fusion cDNA can be produced by RT-PCR using tumor mRNA
extracted from samples containing the gene fusion. The DNA
amplified can be subcloned into an appropriate vector and
characterized by DNA sequence analysis or in vitro/in vivo
expression analyses.
[0120] Expression vectors containing the RAF1 gene fusion (such as,
e.g., a RAF1 gene fusion, e.g., an AGGF1:RAF1 gene fusion, an
MPRIP:RAF1 gene fusion, a PAPD7:RAF1 gene fusion, an LMNA:RAF1 gene
fusion, a CLCN6:RAF1 gene fusion, or a TRAK1:RAF1 gene fusion) can
be introduced into host cells to thereby produce a RAF1 fusion
protein (such as, e.g., a RAF1 fusion protein, e.g., an AGGF1:RAF1
fusion protein, an MPRIP:RAF1 fusion protein, a PAPD7:RAF1 fusion
protein, an LMNA:RAF1 fusion protein, a CLCN6:RAF1 fusion protein,
or a TRAK1:RAF1 fusion protein). The RAF1 fusion protein expression
vector can be a yeast expression vector, a vector for expression in
insect cells, e.g., a baculovirus expression vector, or a vector
suitable for expression in mammalian cells. Vector DNA can be
introduced into host 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
(e.g., DNA) into a host cell.
[0121] Cells harboring the expression vector carrying the
recombinant RAF1 gene fusion can then be tested for production of
the unique fusion protein via standard Western blotting using
either an antibody probe that detects the gene product itself or
that recognizes a tag peptide (e.g., FLAG tag) that can be added to
the gene product via the expression vector (using standard,
commercially available reagents). Western blotting can be used to
confirm the ectopic expression of the encoded RAF1 fusion protein
by comparing the samples from cells transfected with the vector
containing the RAF1 gene fusion cDNA to cells transfected with the
empty expression vector. The functional activity can be assessed by
measuring the level of phosphorylation on the kinase or substrate.
Comparison of the level of phosphorylation activity between the
wild type (normal) form of RAF1 and the RAF1 fusion protein can
indicate if the RAF1 fusion protein has elevated activity that
could drive oncogenic activity. Whether the RAF1 gene fusion is
oncogenic can be assessed by measuring capacity of the expressed
RAF1 fusion protein to transform cells, that is, to enable cells to
grow and proliferate under conditions which are not permissive for
growth of normal cells. One commonly used method of measuring the
transforming activity of a kinase is by assessing if expression of
the gene product can allow BaF3 cells to grow in the absence of the
growth factor IL3, which is required for the survival and growth of
BaF3 cells. Another assay for measuring transforming activity is a
soft agar growth assay. This is another standard method which tests
the capacity of an introduce gene product to confer the ability to
grow in a soft agar matrix, or anchorage-independent conditions.
These methods and others can be used to test the oncogenic activity
of a RAF1 gene fusion (such as, e.g., an AGGF1:RAF1 gene fusion, an
MPRIP:RAF1 gene fusion, an LMNA:RAF1 gene fusion, a PAPD7 gene
fusion, a CLCN6:RAF1 gene fusion, or a. TRAK1:RAF1 gene fusion) and
provide a level of validation of a RAF1 fusion protein (such as,
e.g., an AGGF1:RAF1 fusion protein, an MPRIP:RAF1 fusion protein, a
PAPD7:RAF1 fusion protein, an LMNA:RAF1 fusion protein, a
CLCN6:RAF1 fusion protein, or a TRAK1:RAF1 fusion protein) as a
potential target for treating patients that harbor these
fusions.
[0122] A change in an activity of a cell can be detected in a cell
in culture, e.g., a cell expressing a fusion (e.g., a mammalian
cell, a tumor cell or cell line, a recombinant cell). The
transfected cell can show a change in response to the expressed
fusion, e.g., increased proliferation, changes in morphology,
increased tumorigenicity, and/or an acquired transformed
phenotype.
[0123] To further validate the biological implication of the gene
fusion, a change in any of the activities of the cell, e.g., the
recombinant cell, in the presence of a known inhibitor of one of
the fusion partners, e.g., a RAF1 inhibitor, can be detected. For
example, a decrease in one or more of: proliferation,
tumorigenicity, and transformed morphology, in the presence of the
BETA inhibitor can be indicative of an inhibitor of a fusion. In
other embodiments, a change in binding activity or phosphorylation
of RAF1 or its interacting or downstream proteins or molecules is
detected.
[0124] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. To the extent publications and
patents or patent applications incorporated by reference contradict
the disclosure contained in the specification, the specification
will supersede any contradictory material. Unless otherwise
required by context, singular terms shall include the plural and
plural terms shall include the singular. The use of "or" means
"and/or" unless stated otherwise. The use of the term "including,"
as well as other forms, such as "includes" and "included," is not
limiting. All ranges given in the application encompass the
endpoints unless stated otherwise.
[0125] 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
1211983DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 1atggcctcgg aggcgccgtc
cccgccgcgg tcgccgccgc cgcccacctc ccccgagcct 60gagctggccc agctaaggcg
gaaggtggag aagttggaac gtgaactgcg gagctgcaag 120cggcaggtgc
gggagatcga gaagctgctg catcacacag aacggctgta ccagaacgca
180gaaagcaaca accaggagct ccgcacgcag gtggaagaac tcagtaaaat
actccaacgt 240gggagaaatg aagataataa aaagtctgat gtagaagtac
aaacagagaa ccatgctcct 300tggtcaatct cagattattt ttatcagacg
tactacaatg acgttagtct tccaaataaa 360gtgactgaac tgtcagatca
acaagatcaa gctatcgaaa cttctatttt gaattctaaa 420gaccatttac
aagtagaaaa tgatgcttac cctggtaccg atagaacaga aaatgttaaa
480tatagacaag tggaccattt tgcctcaaat tcacaggagc cagcatctgc
attagcaaca 540gaagatacct ccttagaagg ctcatcatta gctgaaagtt
tgagagctgc agcagaagcg 600gctgtatcac agactggatt tagttatgat
gaaaatactg gactgtattt tgaccacagc 660actggtttct attatgattc
tgaaaatcaa ctctattatg atccttccac tggaatttat 720tactattgtg
atgtggaaag tggtcgttat cagtttcatt ctcgagtaga tttgcaacct
780tatccgactt ctagcacaaa acaaagtaaa gataaaaaat tgaagaagaa
aagaaaagat 840ccagattctt ctgcaacaaa tgaggaaaag gatgcaattc
gaagtcacag cgaatcagcc 900tcaccttcag ccctgtccag tagccccaac
aatctgagcc caacaggctg gtcacagccg 960aaaacccccg tgccagcaca
aagagagcgg gcaccagtat ctgggaccca ggagaaaaac 1020aaaattaggc
ctcgtggaca gagagattca agctattatt gggaaataga agccagtgaa
1080gtgatgctgt ccactcggat tgggtcaggc tcttttggaa ctgtttataa
gggtaaatgg 1140cacggagatg ttgcagtaaa gatcctaaag gttgtcgacc
caaccccaga gcaattccag 1200gccttcagga atgaggtggc tgttctgcgc
aaaacacggc atgtgaacat tctgcttttc 1260atggggtaca tgacaaagga
caacctggca attgtgaccc agtggtgcga gggcagcagc 1320ctctacaaac
acctgcatgt ccaggagacc aagtttcaga tgttccagct aattgacatt
1380gcccggcaga cggctcaggg aatggactat ttgcatgcaa agaacatcat
ccatagagac 1440atgaaatcca acaatatatt tctccatgaa ggcttaacag
tgaaaattgg agattttggt 1500ttggcaacag taaagtcacg ctggagtggt
tctcagcagg ttgaacaacc tactggctct 1560gtcctctgga tggccccaga
ggtgatccga atgcaggata acaacccatt cagtttccag 1620tcggatgtct
actcctatgg catcgtattg tatgaactga tgacggggga gcttccttat
1680tctcacatca acaaccgaga tcagatcatc ttcatggtgg gccgaggata
tgcctcccca 1740gatcttagta agctatataa gaactgcccc aaagcaatga
agaggctggt agctgactgt 1800gtgaagaaag taaaggaaga gaggcctctt
tttccccaga tcctgtcttc cattgagctg 1860ctccaacact ctctaccgaa
gatcaaccgg agcgcttccg agccatcctt gcatcgggca 1920gcccacactg
aggatatcaa tgcttgcacg ctgaccacgt ccccgaggct gcctgtcttc 1980tag
19832660PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 2Met Ala Ser Glu Ala Pro
Ser Pro Pro Arg Ser Pro Pro Pro Pro Thr1 5 10 15Ser Pro Glu Pro Glu
Leu Ala Gln Leu Arg Arg Lys Val Glu Lys Leu 20 25 30Glu Arg Glu Leu
Arg Ser Cys Lys Arg Gln Val Arg Glu Ile Glu Lys 35 40 45Leu Leu His
His Thr Glu Arg Leu Tyr Gln Asn Ala Glu Ser Asn Asn 50 55 60Gln Glu
Leu Arg Thr Gln Val Glu Glu Leu Ser Lys Ile Leu Gln Arg65 70 75
80Gly Arg Asn Glu Asp Asn Lys Lys Ser Asp Val Glu Val Gln Thr Glu
85 90 95Asn His Ala Pro Trp Ser Ile Ser Asp Tyr Phe Tyr Gln Thr Tyr
Tyr 100 105 110Asn Asp Val Ser Leu Pro Asn Lys Val Thr Glu Leu Ser
Asp Gln Gln 115 120 125Asp Gln Ala Ile Glu Thr Ser Ile Leu Asn Ser
Lys Asp His Leu Gln 130 135 140Val Glu Asn Asp Ala Tyr Pro Gly Thr
Asp Arg Thr Glu Asn Val Lys145 150 155 160Tyr Arg Gln Val Asp His
Phe Ala Ser Asn Ser Gln Glu Pro Ala Ser 165 170 175Ala Leu Ala Thr
Glu Asp Thr Ser Leu Glu Gly Ser Ser Leu Ala Glu 180 185 190Ser Leu
Arg Ala Ala Ala Glu Ala Ala Val Ser Gln Thr Gly Phe Ser 195 200
205Tyr Asp Glu Asn Thr Gly Leu Tyr Phe Asp His Ser Thr Gly Phe Tyr
210 215 220Tyr Asp Ser Glu Asn Gln Leu Tyr Tyr Asp Pro Ser Thr Gly
Ile Tyr225 230 235 240Tyr Tyr Cys Asp Val Glu Ser Gly Arg Tyr Gln
Phe His Ser Arg Val 245 250 255Asp Leu Gln Pro Tyr Pro Thr Ser Ser
Thr Lys Gln Ser Lys Asp Lys 260 265 270Lys Leu Lys Lys Lys Arg Lys
Asp Pro Asp Ser Ser Ala Thr Asn Glu 275 280 285Glu Lys Asp Ala Ile
Arg Ser His Ser Glu Ser Ala Ser Pro Ser Ala 290 295 300Leu Ser Ser
Ser Pro Asn Asn Leu Ser Pro Thr Gly Trp Ser Gln Pro305 310 315
320Lys Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro Val Ser Gly Thr
325 330 335Gln Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln Arg Asp Ser
Ser Tyr 340 345 350Tyr Trp Glu Ile Glu Ala Ser Glu Val Met Leu Ser
Thr Arg Ile Gly 355 360 365Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly
Lys Trp His Gly Asp Val 370 375 380Ala Val Lys Ile Leu Lys Val Val
Asp Pro Thr Pro Glu Gln Phe Gln385 390 395 400Ala Phe Arg Asn Glu
Val Ala Val Leu Arg Lys Thr Arg His Val Asn 405 410 415Ile Leu Leu
Phe Met Gly Tyr Met Thr Lys Asp Asn Leu Ala Ile Val 420 425 430Thr
Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His Leu His Val Gln 435 440
445Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile Ala Arg Gln Thr
450 455 460Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile Ile His
Arg Asp465 470 475 480Met Lys Ser Asn Asn Ile Phe Leu His Glu Gly
Leu Thr Val Lys Ile 485 490 495Gly Asp Phe Gly Leu Ala Thr Val Lys
Ser Arg Trp Ser Gly Ser Gln 500 505 510Gln Val Glu Gln Pro Thr Gly
Ser Val Leu Trp Met Ala Pro Glu Val 515 520 525Ile Arg Met Gln Asp
Asn Asn Pro Phe Ser Phe Gln Ser Asp Val Tyr 530 535 540Ser Tyr Gly
Ile Val Leu Tyr Glu Leu Met Thr Gly Glu Leu Pro Tyr545 550 555
560Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly
565 570 575Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn Cys Pro
Lys Ala 580 585 590Met Lys Arg Leu Val Ala Asp Cys Val Lys Lys Val
Lys Glu Glu Arg 595 600 605Pro Leu Phe Pro Gln Ile Leu Ser Ser Ile
Glu Leu Leu Gln His Ser 610 615 620Leu Pro Lys Ile Asn Arg Ser Ala
Ser Glu Pro Ser Leu His Arg Ala625 630 635 640Ala His Thr Glu Asp
Ile Asn Ala Cys Thr Leu Thr Thr Ser Pro Arg 645 650 655Leu Pro Val
Phe 66032811DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 3atggagaccc
cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60ctgtcgccca
cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat
120cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc
agggctgcgc 180cttcgcatca ccgagtctga agaggtggtc agccgcgagg
tgtccggcat caaggccgcc 240tacgaggccg agctcgggga tgcccgcaag
acccttgact cagtagccaa ggagcgcgcc 300cgcctgcagc tggagctgag
caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360accaagaagg
agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg
420ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac
gctggagggc 480gagctgcatg atctgcgggg ccaggtggcc aagcttgagg
cagccctagg tgaggccaag 540aagcaacttc aggatgagat gctgcggcgg
gtggatgctg agaacaggct gcagaccatg 600aaggaggaac tggacttcca
gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660cgtcatgaga
cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg
720ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga
gcagtataag 780aaggagctgg agaagactta ttctgccaag ctggacaatg
ccaggcagtc tgctgagagg 840aacagcaacc tggtgggggc tgcccacgag
gagctgcagc agtcgcgcat ccgcatcgac 900agcctctctg cccagctcag
ccagctccag aagcagctgg cagccaagga ggcgaagctt 960cgagacctgg
aggactcact ggcccgtgag cgggacacca gccggcggct gctggcggaa
1020aaggagcggg agatggccga gatgcgggca aggatgcagc agcagctgga
cgagtaccag 1080gagcttctgg acatcaagct ggccctggac atggagatcc
acgcctaccg caagctcttg 1140gagggcgagg aggagaggct acgcctgtcc
cccagcccta cctcgcagcg cagccgtggc 1200cgtgcttcct ctcactcatc
ccagacacag ggtgggggca gcgtcaccaa aaagcgcaaa 1260ctggagtcca
ctgagagccg cagcagcttc tcacagcacg cacgcactag cgggcgcgtg
1320gccgtggagg aggtggatga ggagggcaag tttgtccggc tgcgcaacaa
gtccaatgag 1380gaccagtcca tgggcaattg gcagatcaag cgccagaatg
gagatgatcc cttgctgact 1440taccggttcc caccaaagtt caccctgaag
gctgggcagg tggtgacgat ctgggctgca 1500ggagctgggg ccacccacag
cccccctacc gacctggtgt ggaaggcaca gaacacctgg 1560ggctgcggga
acagcctgcg tacggctctc atcaactcca ctggggaaga agtggccatg
1620cgcaagctgg tgcgctcagt gactgtggtt gaggacgacg aggatgagga
tggagatgac 1680ctgctccatc accaccacga tgcaattcga agtcacagcg
aatcagcctc accttcagcc 1740ctgtccagta gccccaacaa tctgagccca
acaggctggt cacagccgaa aacccccgtg 1800ccagcacaaa gagagcgggc
accagtatct gggacccagg agaaaaacaa aattaggcct 1860cgtggacaga
gagattcaag ctattattgg gaaatagaag ccagtgaagt gatgctgtcc
1920actcggattg ggtcaggctc ttttggaact gtttataagg gtaaatggca
cggagatgtt 1980gcagtaaaga tcctaaaggt tgtcgaccca accccagagc
aattccaggc cttcaggaat 2040gaggtggctg ttctgcgcaa aacacggcat
gtgaacattc tgcttttcat ggggtacatg 2100acaaaggaca acctggcaat
tgtgacccag tggtgcgagg gcagcagcct ctacaaacac 2160ctgcatgtcc
aggagaccaa gtttcagatg ttccagctaa ttgacattgc ccggcagacg
2220gctcagggaa tggactattt gcatgcaaag aacatcatcc atagagacat
gaaatccaac 2280aatatatttc tccatgaagg cttaacagtg aaaattggag
attttggttt ggcaacagta 2340aagtcacgct ggagtggttc tcagcaggtt
gaacaaccta ctggctctgt cctctggatg 2400gccccagagg tgatccgaat
gcaggataac aacccattca gtttccagtc ggatgtctac 2460tcctatggca
tcgtattgta tgaactgatg acgggggagc ttccttattc tcacatcaac
2520aaccgagatc agatcatctt catggtgggc cgaggatatg cctccccaga
tcttagtaag 2580ctatataaga actgccccaa agcaatgaag aggctggtag
ctgactgtgt gaagaaagta 2640aaggaagaga ggcctctttt tccccagatc
ctgtcttcca ttgagctgct ccaacactct 2700ctaccgaaga tcaaccggag
cgcttccgag ccatccttgc atcgggcagc ccacactgag 2760gatatcaatg
cttgcacgct gaccacgtcc ccgaggctgc ctgtcttcta g 28114936PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 4Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly
Ala Gln Ala1 5 10 15Ser Ser Thr Pro Leu Ser Pro Thr Arg Ile Thr Arg
Leu Gln Glu Lys 20 25 30Glu Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala
Val Tyr Ile Asp Arg 35 40 45Val Arg Ser Leu Glu Thr Glu Asn Ala Gly
Leu Arg Leu Arg Ile Thr 50 55 60Glu Ser Glu Glu Val Val Ser Arg Glu
Val Ser Gly Ile Lys Ala Ala65 70 75 80Tyr Glu Ala Glu Leu Gly Asp
Ala Arg Lys Thr Leu Asp Ser Val Ala 85 90 95Lys Glu Arg Ala Arg Leu
Gln Leu Glu Leu Ser Lys Val Arg Glu Glu 100 105 110Phe Lys Glu Leu
Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu Ile 115 120 125Ala Ala
Gln Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135
140Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu
Gly145 150 155 160Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu
Glu Ala Ala Leu 165 170 175Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu
Met Leu Arg Arg Val Asp 180 185 190Ala Glu Asn Arg Leu Gln Thr Met
Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205Asn Ile Tyr Ser Glu Glu
Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210 215 220Arg Leu Val Glu
Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg225 230 235 240Leu
Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val 245 250
255Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp
260 265 270Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly
Ala Ala 275 280 285His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp
Ser Leu Ser Ala 290 295 300Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala
Ala Lys Glu Ala Lys Leu305 310 315 320Arg Asp Leu Glu Asp Ser Leu
Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330 335Leu Leu Ala Glu Lys
Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met 340 345 350Gln Gln Gln
Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys Leu Ala 355 360 365Leu
Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu 370 375
380Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg
Gly385 390 395 400Arg Ala Ser Ser His Ser Ser Gln Thr Gln Gly Gly
Gly Ser Val Thr 405 410 415Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser
Arg Ser Ser Phe Ser Gln 420 425 430His Ala Arg Thr Ser Gly Arg Val
Ala Val Glu Glu Val Asp Glu Glu 435 440 445Gly Lys Phe Val Arg Leu
Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455 460Gly Asn Trp Gln
Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr465 470 475 480Tyr
Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln Val Val Thr 485 490
495Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser Pro Pro Thr Asp Leu
500 505 510Val Trp Lys Ala Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu
Arg Thr 515 520 525Ala Leu Ile Asn Ser Thr Gly Glu Glu Val Ala Met
Arg Lys Leu Val 530 535 540Arg Ser Val Thr Val Val Glu Asp Asp Glu
Asp Glu Asp Gly Asp Asp545 550 555 560Leu Leu His His His His Asp
Ala Ile Arg Ser His Ser Glu Ser Ala 565 570 575Ser Pro Ser Ala Leu
Ser Ser Ser Pro Asn Asn Leu Ser Pro Thr Gly 580 585 590Trp Ser Gln
Pro Lys Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro 595 600 605Val
Ser Gly Thr Gln Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln Arg 610 615
620Asp Ser Ser Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val Met Leu
Ser625 630 635 640Thr Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr
Lys Gly Lys Trp 645 650 655His Gly Asp Val Ala Val Lys Ile Leu Lys
Val Val Asp Pro Thr Pro 660 665 670Glu Gln Phe Gln Ala Phe Arg Asn
Glu Val Ala Val Leu Arg Lys Thr 675 680 685Arg His Val Asn Ile Leu
Leu Phe Met Gly Tyr Met Thr Lys Asp Asn 690 695 700Leu Ala Ile Val
Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His705 710 715 720Leu
His Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile 725 730
735Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile
740 745 750Ile His Arg Asp Met Lys Ser Asn Asn Ile Phe Leu His Glu
Gly Leu 755 760 765Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val
Lys Ser Arg Trp 770 775 780Ser Gly Ser Gln Gln Val Glu Gln Pro Thr
Gly Ser Val Leu Trp Met785 790 795 800Ala Pro Glu Val Ile Arg Met
Gln Asp Asn Asn Pro Phe Ser Phe Gln 805 810 815Ser Asp Val Tyr Ser
Tyr Gly Ile Val Leu Tyr Glu Leu Met Thr Gly 820 825 830Glu Leu Pro
Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met 835 840 845Val
Gly Arg Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn 850 855
860Cys Pro Lys Ala Met Lys Arg Leu Val Ala Asp Cys Val Lys Lys
Val865 870 875 880Lys Glu Glu Arg Pro Leu Phe Pro Gln Ile Leu Ser
Ser Ile Glu Leu 885 890 895Leu Gln His Ser Leu Pro Lys Ile Asn Arg
Ser Ala Ser Glu Pro Ser 900 905 910Leu His Arg Ala Ala His Thr Glu
Asp Ile Asn Ala Cys Thr Leu Thr 915 920 925Thr Ser Pro Arg Leu Pro
Val Phe 930 93554159DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 5aatgtcggca
gccaaggaga acccgtgcag
gaaattccag gccaacatct tcaacaagag 60caagtgtcag aactgcttca agccccgcga
gtcgcatctg ctcaacgacg aggacctgac 120gcaggcaaaa cccatttatg
gcggttggct gctcctggct ccagatggga ccgactttga 180caacccagtg
caccggtctc ggaaatggca gcgacggttc ttcatccttt acgagcacgg
240cctcttgcgc tacgccctgg atgagatgcc cacgaccctt cctcagggca
ccatcaacat 300gaaccagtgc acagatgtgg tggatgggga gggccgcacg
ggccagaagt tctccctgtg 360tattctgacg cctgagaagg agcatttcat
ccgggcggag accaaggaga tcgtcagtgg 420gtggctggag atgctcatgg
tctatccccg gaccaacaag cagaatcaga agaagaaacg 480gaaagtggag
ccccccacac cacaggagcc tgggcctgcc aaggtggctg ttaccagcag
540cagcagcagc agcagcagca gcagcagcat ccccagtgct gagaaagtcc
ccaccaccaa 600gtccacactc tggcaggaag aaatgaggac caaggaccag
ccagatggca gcagcctgag 660tccagctcag agtcccagcc agagccagcc
tcctgctgcc agctccctgc gggaacctgg 720gctagagagc aaagaagagg
agagcgccat gagtagcgac cgcatggact gtggccgcaa 780agtccgggtg
gagagcggct acttctctct ggagaagacc aaacaggact tgaaggctga
840agaacagcag ctgcccccgc cgctctcccc tcccagcccc agcaccccca
accacaggag 900gtcccaggtg attgaaaagt ttgaggcctt ggacattgag
aaggcagagc acatggagac 960caatgcagtg gggccctcac catccagcga
cacacgccag ggccgcagcg agaagagggc 1020gttccctagg aagcgggact
tcaccaatga agccccccca gctcctctcc cagacgcctc 1080ggcttccccc
ctgtctccac accgaagagc caagtcactg gacaggaggt ccacggagcc
1140ctccgtgacg cccgacctgc tgaatttcaa gaaaggctgg ctgactaagc
agtatgagga 1200cggccagtgg aagaaacact ggtttgtcct cgccgatcaa
agcctgagat actacaggga 1260ttcagtggct gaggaggcag ccgacttgga
tggagaaatt gacttgtccg catgttacga 1320tgtcacagag tatccagttc
agagaaacta tggcttccag atacatacaa aggagggcga 1380gtttaccctg
tcggccatga catctgggat tcggcggaac tggatccaga ccatcatgaa
1440gcacgtgcac ccgaccactg ccccggatgt gaccagctcg ttgccagagg
aaaaaaacaa 1500gagcagctgc tcttttgaga cctgcccgag gcctactgag
aagcaagagg cagagctggg 1560ggagccggac cctgagcaga agaggagccg
cgcacgggag cggaggcgag agggccgctc 1620caagaccttt gactgggctg
agttccgtcc catccagcag gccctggctc aggagcgggt 1680gggcggcgtg
gggcctgctg acacccacga gcccctgcgc cctgaggcgg agcctgggga
1740gctggagcgg gagcgtgcac ggaggcggga ggagcgccgc aagcgcttcg
ggatgctcga 1800cgccacagac gggccaggca ctgaggatgc agccctgcgc
atggaggtgg accggagccc 1860agggctgcct atgagcgacc tcaaaacgca
taacgtccac gtggagattg agcagcggtg 1920gcatcaggtg gagaccacac
ctctccggga agagaagcag gtgcccatcg cccccgtcca 1980cctgtcttct
gaagatgggg gtgaccggct ctccacacac gagctgacct ctctgctcga
2040gaaggagctg gagcagagcc agaaggaggc ctcagacctt ctggagcaga
accggctcct 2100gcaggaccag ctgagggtgg ccctgggccg ggagcagagc
gcccgtgagg gctacgtgct 2160gcaggccacg tgcgagcgag ggtttgcagc
aatggaagaa acgcaccaga agaagattga 2220agatctccag aggcagcacc
agcgggagct agagaaactt cgagaagaga aagaccgcct 2280cctagccgag
gagacagcgg ccaccatctc agccatcgaa gccatgaaga acgcccaccg
2340ggaggaaatg gagcgggagc tggagaagag ccagcggtcc cagatcagca
gcgtcaactc 2400ggatgttgag gccctgcggc gccagtacct ggaggagctg
cagtcggtgc agcgggaact 2460ggaggtcctc tcggagcagt actcgcagaa
gtgcctggag aatgcccatc tggcccaggc 2520gctggaggcc gagcggcagg
ccctgcggca gtgccagcgt gagaaccagg agctcaatgc 2580ccacaaccag
gagctgaaca accgcctggc tgcagagatc acacggttgc ggacgctgct
2640gactggggac ggcggtgggg aggccactgg gtcacccctt gcacagggca
aggatgccta 2700tgaactagag gtcttattgc gggtaaagga atcggaaata
cagtacctga aacaggagat 2760tagctccctc aaggatgagc tgcagacggc
actgcgggac aagaagtacg caagtgacaa 2820gtacaaagac atctacacag
agctcagcat cgcgaaggct aaggctgact gtgacatcag 2880caggttgaag
gagcagctca aggctgcaac ggaagcactg ggggagaagt cccctgacag
2940tgccacggtg tccggatatg atataatgaa atctaaaagc aaccctgact
tcttgaagaa 3000agacagatcc tgtgtcaccc ggcaactcag aaacatcagg
tccaaggatg caattcgaag 3060tcacagcgaa tcagcctcac cttcagccct
gtccagtagc cccaacaatc tgagcccaac 3120aggctggtca cagccgaaaa
cccccgtgcc agcacaaaga gagcgggcac cagtatctgg 3180gacccaggag
aaaaacaaaa ttaggcctcg tggacagaga gattcaagct attattggga
3240aatagaagcc agtgaagtga tgctgtccac tcggattggg tcaggctctt
ttggaactgt 3300ttataagggt aaatggcacg gagatgttgc agtaaagatc
ctaaaggttg tcgacccaac 3360cccagagcaa ttccaggcct tcaggaatga
ggtggctgtt ctgcgcaaaa cacggcatgt 3420gaacattctg cttttcatgg
ggtacatgac aaaggacaac ctggcaattg tgacccagtg 3480gtgcgagggc
agcagcctct acaaacacct gcatgtccag gagaccaagt ttcagatgtt
3540ccagctaatt gacattgccc ggcagacggc tcagggaatg gactatttgc
atgcaaagaa 3600catcatccat agagacatga aatccaacaa tatatttctc
catgaaggct taacagtgaa 3660aattggagat tttggtttgg caacagtaaa
gtcacgctgg agtggttctc agcaggttga 3720acaacctact ggctctgtcc
tctggatggc cccagaggtg atccgaatgc aggataacaa 3780cccattcagt
ttccagtcgg atgtctactc ctatggcatc gtattgtatg aactgatgac
3840gggggagctt ccttattctc acatcaacaa ccgagatcag atcatcttca
tggtgggccg 3900aggatatgcc tccccagatc ttagtaagct atataagaac
tgccccaaag caatgaagag 3960gctggtagct gactgtgtga agaaagtaaa
ggaagagagg cctctttttc cccagatcct 4020gtcttccatt gagctgctcc
aacactctct accgaagatc aaccggagcg cttccgagcc 4080atccttgcat
cgggcagccc acactgagga tatcaatgct tgcacgctga ccacgtcccc
4140gaggctgcct gtcttctag 415961385PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 6Met Ser Ala Ala Lys Glu Asn Pro Cys Arg Lys Phe Gln
Ala Asn Ile1 5 10 15Phe Asn Lys Ser Lys Cys Gln Asn Cys Phe Lys Pro
Arg Glu Ser His 20 25 30Leu Leu Asn Asp Glu Asp Leu Thr Gln Ala Lys
Pro Ile Tyr Gly Gly 35 40 45Trp Leu Leu Leu Ala Pro Asp Gly Thr Asp
Phe Asp Asn Pro Val His 50 55 60Arg Ser Arg Lys Trp Gln Arg Arg Phe
Phe Ile Leu Tyr Glu His Gly65 70 75 80Leu Leu Arg Tyr Ala Leu Asp
Glu Met Pro Thr Thr Leu Pro Gln Gly 85 90 95Thr Ile Asn Met Asn Gln
Cys Thr Asp Val Val Asp Gly Glu Gly Arg 100 105 110Thr Gly Gln Lys
Phe Ser Leu Cys Ile Leu Thr Pro Glu Lys Glu His 115 120 125Phe Ile
Arg Ala Glu Thr Lys Glu Ile Val Ser Gly Trp Leu Glu Met 130 135
140Leu Met Val Tyr Pro Arg Thr Asn Lys Gln Asn Gln Lys Lys Lys
Arg145 150 155 160Lys Val Glu Pro Pro Thr Pro Gln Glu Pro Gly Pro
Ala Lys Val Ala 165 170 175Val Thr Ser Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ile Pro Ser 180 185 190Ala Glu Lys Val Pro Thr Thr Lys
Ser Thr Leu Trp Gln Glu Glu Met 195 200 205Arg Thr Lys Asp Gln Pro
Asp Gly Ser Ser Leu Ser Pro Ala Gln Ser 210 215 220Pro Ser Gln Ser
Gln Pro Pro Ala Ala Ser Ser Leu Arg Glu Pro Gly225 230 235 240Leu
Glu Ser Lys Glu Glu Glu Ser Ala Met Ser Ser Asp Arg Met Asp 245 250
255Cys Gly Arg Lys Val Arg Val Glu Ser Gly Tyr Phe Ser Leu Glu Lys
260 265 270Thr Lys Gln Asp Leu Lys Ala Glu Glu Gln Gln Leu Pro Pro
Pro Leu 275 280 285Ser Pro Pro Ser Pro Ser Thr Pro Asn His Arg Arg
Ser Gln Val Ile 290 295 300Glu Lys Phe Glu Ala Leu Asp Ile Glu Lys
Ala Glu His Met Glu Thr305 310 315 320Asn Ala Val Gly Pro Ser Pro
Ser Ser Asp Thr Arg Gln Gly Arg Ser 325 330 335Glu Lys Arg Ala Phe
Pro Arg Lys Arg Asp Phe Thr Asn Glu Ala Pro 340 345 350Pro Ala Pro
Leu Pro Asp Ala Ser Ala Ser Pro Leu Ser Pro His Arg 355 360 365Arg
Ala Lys Ser Leu Asp Arg Arg Ser Thr Glu Pro Ser Val Thr Pro 370 375
380Asp Leu Leu Asn Phe Lys Lys Gly Trp Leu Thr Lys Gln Tyr Glu
Asp385 390 395 400Gly Gln Trp Lys Lys His Trp Phe Val Leu Ala Asp
Gln Ser Leu Arg 405 410 415Tyr Tyr Arg Asp Ser Val Ala Glu Glu Ala
Ala Asp Leu Asp Gly Glu 420 425 430Ile Asp Leu Ser Ala Cys Tyr Asp
Val Thr Glu Tyr Pro Val Gln Arg 435 440 445Asn Tyr Gly Phe Gln Ile
His Thr Lys Glu Gly Glu Phe Thr Leu Ser 450 455 460Ala Met Thr Ser
Gly Ile Arg Arg Asn Trp Ile Gln Thr Ile Met Lys465 470 475 480His
Val His Pro Thr Thr Ala Pro Asp Val Thr Ser Ser Leu Pro Glu 485 490
495Glu Lys Asn Lys Ser Ser Cys Ser Phe Glu Thr Cys Pro Arg Pro Thr
500 505 510Glu Lys Gln Glu Ala Glu Leu Gly Glu Pro Asp Pro Glu Gln
Lys Arg 515 520 525Ser Arg Ala Arg Glu Arg Arg Arg Glu Gly Arg Ser
Lys Thr Phe Asp 530 535 540Trp Ala Glu Phe Arg Pro Ile Gln Gln Ala
Leu Ala Gln Glu Arg Val545 550 555 560Gly Gly Val Gly Pro Ala Asp
Thr His Glu Pro Leu Arg Pro Glu Ala 565 570 575Glu Pro Gly Glu Leu
Glu Arg Glu Arg Ala Arg Arg Arg Glu Glu Arg 580 585 590Arg Lys Arg
Phe Gly Met Leu Asp Ala Thr Asp Gly Pro Gly Thr Glu 595 600 605Asp
Ala Ala Leu Arg Met Glu Val Asp Arg Ser Pro Gly Leu Pro Met 610 615
620Ser Asp Leu Lys Thr His Asn Val His Val Glu Ile Glu Gln Arg
Trp625 630 635 640His Gln Val Glu Thr Thr Pro Leu Arg Glu Glu Lys
Gln Val Pro Ile 645 650 655Ala Pro Val His Leu Ser Ser Glu Asp Gly
Gly Asp Arg Leu Ser Thr 660 665 670His Glu Leu Thr Ser Leu Leu Glu
Lys Glu Leu Glu Gln Ser Gln Lys 675 680 685Glu Ala Ser Asp Leu Leu
Glu Gln Asn Arg Leu Leu Gln Asp Gln Leu 690 695 700Arg Val Ala Leu
Gly Arg Glu Gln Ser Ala Arg Glu Gly Tyr Val Leu705 710 715 720Gln
Ala Thr Cys Glu Arg Gly Phe Ala Ala Met Glu Glu Thr His Gln 725 730
735Lys Lys Ile Glu Asp Leu Gln Arg Gln His Gln Arg Glu Leu Glu Lys
740 745 750Leu Arg Glu Glu Lys Asp Arg Leu Leu Ala Glu Glu Thr Ala
Ala Thr 755 760 765Ile Ser Ala Ile Glu Ala Met Lys Asn Ala His Arg
Glu Glu Met Glu 770 775 780Arg Glu Leu Glu Lys Ser Gln Arg Ser Gln
Ile Ser Ser Val Asn Ser785 790 795 800Asp Val Glu Ala Leu Arg Arg
Gln Tyr Leu Glu Glu Leu Gln Ser Val 805 810 815Gln Arg Glu Leu Glu
Val Leu Ser Glu Gln Tyr Ser Gln Lys Cys Leu 820 825 830Glu Asn Ala
His Leu Ala Gln Ala Leu Glu Ala Glu Arg Gln Ala Leu 835 840 845Arg
Gln Cys Gln Arg Glu Asn Gln Glu Leu Asn Ala His Asn Gln Glu 850 855
860Leu Asn Asn Arg Leu Ala Ala Glu Ile Thr Arg Leu Arg Thr Leu
Leu865 870 875 880Thr Gly Asp Gly Gly Gly Glu Ala Thr Gly Ser Pro
Leu Ala Gln Gly 885 890 895Lys Asp Ala Tyr Glu Leu Glu Val Leu Leu
Arg Val Lys Glu Ser Glu 900 905 910Ile Gln Tyr Leu Lys Gln Glu Ile
Ser Ser Leu Lys Asp Glu Leu Gln 915 920 925Thr Ala Leu Arg Asp Lys
Lys Tyr Ala Ser Asp Lys Tyr Lys Asp Ile 930 935 940Tyr Thr Glu Leu
Ser Ile Ala Lys Ala Lys Ala Asp Cys Asp Ile Ser945 950 955 960Arg
Leu Lys Glu Gln Leu Lys Ala Ala Thr Glu Ala Leu Gly Glu Lys 965 970
975Ser Pro Asp Ser Ala Thr Val Ser Gly Tyr Asp Ile Met Lys Ser Lys
980 985 990Ser Asn Pro Asp Phe Leu Lys Lys Asp Arg Ser Cys Val Thr
Arg Gln 995 1000 1005Leu Arg Asn Ile Arg Ser Lys Asp Ala Ile Arg
Ser His Ser Glu 1010 1015 1020Ser Ala Ser Pro Ser Ala Leu Ser Ser
Ser Pro Asn Asn Leu Ser 1025 1030 1035Pro Thr Gly Trp Ser Gln Pro
Lys Thr Pro Val Pro Ala Gln Arg 1040 1045 1050Glu Arg Ala Pro Val
Ser Gly Thr Gln Glu Lys Asn Lys Ile Arg 1055 1060 1065Pro Arg Gly
Gln Arg Asp Ser Ser Tyr Tyr Trp Glu Ile Glu Ala 1070 1075 1080Ser
Glu Val Met Leu Ser Thr Arg Ile Gly Ser Gly Ser Phe Gly 1085 1090
1095Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val Lys Ile
1100 1105 1110Leu Lys Val Val Asp Pro Thr Pro Glu Gln Phe Gln Ala
Phe Arg 1115 1120 1125Asn Glu Val Ala Val Leu Arg Lys Thr Arg His
Val Asn Ile Leu 1130 1135 1140Leu Phe Met Gly Tyr Met Thr Lys Asp
Asn Leu Ala Ile Val Thr 1145 1150 1155Gln Trp Cys Glu Gly Ser Ser
Leu Tyr Lys His Leu His Val Gln 1160 1165 1170Glu Thr Lys Phe Gln
Met Phe Gln Leu Ile Asp Ile Ala Arg Gln 1175 1180 1185Thr Ala Gln
Gly Met Asp Tyr Leu His Ala Lys Asn Ile Ile His 1190 1195 1200Arg
Asp Met Lys Ser Asn Asn Ile Phe Leu His Glu Gly Leu Thr 1205 1210
1215Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp
1220 1225 1230Ser Gly Ser Gln Gln Val Glu Gln Pro Thr Gly Ser Val
Leu Trp 1235 1240 1245Met Ala Pro Glu Val Ile Arg Met Gln Asp Asn
Asn Pro Phe Ser 1250 1255 1260Phe Gln Ser Asp Val Tyr Ser Tyr Gly
Ile Val Leu Tyr Glu Leu 1265 1270 1275Met Thr Gly Glu Leu Pro Tyr
Ser His Ile Asn Asn Arg Asp Gln 1280 1285 1290Ile Ile Phe Met Val
Gly Arg Gly Tyr Ala Ser Pro Asp Leu Ser 1295 1300 1305Lys Leu Tyr
Lys Asn Cys Pro Lys Ala Met Lys Arg Leu Val Ala 1310 1315 1320Asp
Cys Val Lys Lys Val Lys Glu Glu Arg Pro Leu Phe Pro Gln 1325 1330
1335Ile Leu Ser Ser Ile Glu Leu Leu Gln His Ser Leu Pro Lys Ile
1340 1345 1350Asn Arg Ser Ala Ser Glu Pro Ser Leu His Arg Ala Ala
His Thr 1355 1360 1365Glu Asp Ile Asn Ala Cys Thr Leu Thr Thr Ser
Pro Arg Leu Pro 1370 1375 1380Val Phe 138572226DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 7atgtcccctt gtcctgaaga agcagctatg agaagagagg
tggtgaaacg gatcgaaact 60gtggtgaaag acctttggcc gacggctgat gtacagatat
ttggcagctt tagtacaggt 120ctttatcttc caactagcga catagacctg
gtggtcttcg ggaaatggga gcgtcctcct 180ttacagctgc tggagcaagc
cctgcggaag cacaacgtgg ctgagccgtg ttccatcaaa 240gtccttgaca
aggctacggt accaataata aagctcacag atcaggagac tgaagtgaaa
300gttgacatca gctttaacat ggagacgggc gtccgggcag cggagttcat
caagaattac 360atgaagaaat attcattgct gccttacttg attttagtat
tgaaacagtt ccttctgcag 420agggacctga atgaagtttt tacaggtgga
attagctcat acagcctaat tttaatggcc 480attagctttc tacagttgca
tccaagaatt gatgcccgga gagctgatga aaaccttgga 540atgcttcttg
tagaattttt tgaactctat gggagaaatt ttaattactt gaaaaccggt
600attagaatca aagaaggagg tgcctatatc gccaaagagg agatcatgaa
agccatgacc 660agcgggtaca gaccgtcgat gctgtgcatt gaggaccccc
tgctgccagg gaatgacgtt 720ggccggagct cctatggcgc catgcaggtg
aagcaggtct tcgattatgc ctacatagtg 780ctcagccatg ctgtgtcacc
gctggccagg tcctatccaa acagagacgc cgaaagtact 840ttaggaagaa
tcatcaaagt aactcaggag gtgattgact accggaggtg gatcaaagag
900aagtggggca gcaaagccca cccgtcgcca ggcatggaca gcaggatcaa
gatcaaagag 960cgaatagcca catgcaatgg ggagcagacg cagaaccgag
agcccgagtc tccctatggc 1020cagcgcttga ctttgtcgct gtccagcccc
cagctcctgt cttcaggctc ctcggcctct 1080tctgtgtctt cactttctgg
gagtgacgtt gattcagaca caccgccctg cacaacgccc 1140agtgtttacc
agttcagtct gcaagcgcca gctcctctca tggccggctt acccaccgcc
1200ttgccaatgc ccagtggcaa acctcagccc accacttcca gaacactgat
catgacaacc 1260aacaatcaga ggcctcgtgg acagagagat tcaagctatt
attgggaaat agaagccagt 1320gaagtgatgc tgtccactcg gattgggtca
ggctcttttg gaactgttta taagggtaaa 1380tggcacggag atgttgcagt
aaagatccta aaggttgtcg acccaacccc agagcaattc 1440caggccttca
ggaatgaggt ggctgttctg cgcaaaacac ggcatgtgaa cattctgctt
1500ttcatggggt acatgacaaa ggacaacctg gcaattgtga cccagtggtg
cgagggcagc 1560agcctctaca aacacctgca tgtccaggag accaagtttc
agatgttcca gctaattgac 1620attgcccggc agacggctca gggaatggac
tatttgcatg caaagaacat catccataga 1680gacatgaaat ccaacaatat
atttctccat gaaggcttaa cagtgaaaat tggagatttt 1740ggtttggcaa
cagtaaagtc acgctggagt ggttctcagc aggttgaaca acctactggc
1800tctgtcctct ggatggcccc agaggtgatc cgaatgcagg ataacaaccc
attcagtttc 1860cagtcggatg tctactccta tggcatcgta ttgtatgaac
tgatgacggg ggagcttcct 1920tattctcaca tcaacaaccg agatcagatc
atcttcatgg tgggccgagg atatgcctcc 1980ccagatctta gtaagctata
taagaactgc cccaaagcaa tgaagaggct ggtagctgac 2040tgtgtgaaga
aagtaaagga agagaggcct ctttttcccc agatcctgtc ttccattgag
2100ctgctccaac actctctacc gaagatcaac cggagcgctt ccgagccatc
cttgcatcgg 2160gcagcccaca ctgaggatat caatgcttgc acgctgacca
cgtccccgag
gctgcctgtc 2220ttctag 22268741PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 8Met Ser Pro Cys Pro Glu Glu Ala Ala Met Arg Arg Glu
Val Val Lys1 5 10 15Arg Ile Glu Thr Val Val Lys Asp Leu Trp Pro Thr
Ala Asp Val Gln 20 25 30Ile Phe Gly Ser Phe Ser Thr Gly Leu Tyr Leu
Pro Thr Ser Asp Ile 35 40 45Asp Leu Val Val Phe Gly Lys Trp Glu Arg
Pro Pro Leu Gln Leu Leu 50 55 60Glu Gln Ala Leu Arg Lys His Asn Val
Ala Glu Pro Cys Ser Ile Lys65 70 75 80Val Leu Asp Lys Ala Thr Val
Pro Ile Ile Lys Leu Thr Asp Gln Glu 85 90 95Thr Glu Val Lys Val Asp
Ile Ser Phe Asn Met Glu Thr Gly Val Arg 100 105 110Ala Ala Glu Phe
Ile Lys Asn Tyr Met Lys Lys Tyr Ser Leu Leu Pro 115 120 125Tyr Leu
Ile Leu Val Leu Lys Gln Phe Leu Leu Gln Arg Asp Leu Asn 130 135
140Glu Val Phe Thr Gly Gly Ile Ser Ser Tyr Ser Leu Ile Leu Met
Ala145 150 155 160Ile Ser Phe Leu Gln Leu His Pro Arg Ile Asp Ala
Arg Arg Ala Asp 165 170 175Glu Asn Leu Gly Met Leu Leu Val Glu Phe
Phe Glu Leu Tyr Gly Arg 180 185 190Asn Phe Asn Tyr Leu Lys Thr Gly
Ile Arg Ile Lys Glu Gly Gly Ala 195 200 205Tyr Ile Ala Lys Glu Glu
Ile Met Lys Ala Met Thr Ser Gly Tyr Arg 210 215 220Pro Ser Met Leu
Cys Ile Glu Asp Pro Leu Leu Pro Gly Asn Asp Val225 230 235 240Gly
Arg Ser Ser Tyr Gly Ala Met Gln Val Lys Gln Val Phe Asp Tyr 245 250
255Ala Tyr Ile Val Leu Ser His Ala Val Ser Pro Leu Ala Arg Ser Tyr
260 265 270Pro Asn Arg Asp Ala Glu Ser Thr Leu Gly Arg Ile Ile Lys
Val Thr 275 280 285Gln Glu Val Ile Asp Tyr Arg Arg Trp Ile Lys Glu
Lys Trp Gly Ser 290 295 300Lys Ala His Pro Ser Pro Gly Met Asp Ser
Arg Ile Lys Ile Lys Glu305 310 315 320Arg Ile Ala Thr Cys Asn Gly
Glu Gln Thr Gln Asn Arg Glu Pro Glu 325 330 335Ser Pro Tyr Gly Gln
Arg Leu Thr Leu Ser Leu Ser Ser Pro Gln Leu 340 345 350Leu Ser Ser
Gly Ser Ser Ala Ser Ser Val Ser Ser Leu Ser Gly Ser 355 360 365Asp
Val Asp Ser Asp Thr Pro Pro Cys Thr Thr Pro Ser Val Tyr Gln 370 375
380Phe Ser Leu Gln Ala Pro Ala Pro Leu Met Ala Gly Leu Pro Thr
Ala385 390 395 400Leu Pro Met Pro Ser Gly Lys Pro Gln Pro Thr Thr
Ser Arg Thr Leu 405 410 415Ile Met Thr Thr Asn Asn Gln Arg Pro Arg
Gly Gln Arg Asp Ser Ser 420 425 430Tyr Tyr Trp Glu Ile Glu Ala Ser
Glu Val Met Leu Ser Thr Arg Ile 435 440 445Gly Ser Gly Ser Phe Gly
Thr Val Tyr Lys Gly Lys Trp His Gly Asp 450 455 460Val Ala Val Lys
Ile Leu Lys Val Val Asp Pro Thr Pro Glu Gln Phe465 470 475 480Gln
Ala Phe Arg Asn Glu Val Ala Val Leu Arg Lys Thr Arg His Val 485 490
495Asn Ile Leu Leu Phe Met Gly Tyr Met Thr Lys Asp Asn Leu Ala Ile
500 505 510Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His Leu
His Val 515 520 525Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp
Ile Ala Arg Gln 530 535 540Thr Ala Gln Gly Met Asp Tyr Leu His Ala
Lys Asn Ile Ile His Arg545 550 555 560Asp Met Lys Ser Asn Asn Ile
Phe Leu His Glu Gly Leu Thr Val Lys 565 570 575Ile Gly Asp Phe Gly
Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser 580 585 590Gln Gln Val
Glu Gln Pro Thr Gly Ser Val Leu Trp Met Ala Pro Glu 595 600 605Val
Ile Arg Met Gln Asp Asn Asn Pro Phe Ser Phe Gln Ser Asp Val 610 615
620Tyr Ser Tyr Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Glu Leu
Pro625 630 635 640Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe
Met Val Gly Arg 645 650 655Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu
Tyr Lys Asn Cys Pro Lys 660 665 670Ala Met Lys Arg Leu Val Ala Asp
Cys Val Lys Lys Val Lys Glu Glu 675 680 685Arg Pro Leu Phe Pro Gln
Ile Leu Ser Ser Ile Glu Leu Leu Gln His 690 695 700Ser Leu Pro Lys
Ile Asn Arg Ser Ala Ser Glu Pro Ser Leu His Arg705 710 715 720Ala
Ala His Thr Glu Asp Ile Asn Ala Cys Thr Leu Thr Thr Ser Pro 725 730
735Arg Leu Pro Val Phe 74091260DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 9atggcggggt gcagggggtc tctgtgctgc tgctgcaggt
ggtgctgctg ctgcggtgag 60cgtgagaccc gcacccccga ggagctgacc atccttggag
aaacacagga ggaggaggat 120gagattcttc caaggaaaga ctatgaggat
gcaattcgaa gtcacagcga atcagcctca 180ccttcagccc tgtccagtag
ccccaacaat ctgagcccaa caggctggtc acagccgaaa 240acccccgtgc
cagcacaaag agagcgggca ccagtatctg ggacccagga gaaaaacaaa
300attaggcctc gtggacagag agattcaagc tattattggg aaatagaagc
cagtgaagtg 360atgctgtcca ctcggattgg gtcaggctct tttggaactg
tttataaggg taaatggcac 420ggagatgttg cagtaaagat cctaaaggtt
gtcgacccaa ccccagagca attccaggcc 480ttcaggaatg aggtggctgt
tctgcgcaaa acacggcatg tgaacattct gcttttcatg 540gggtacatga
caaaggacaa cctggcaatt gtgacccagt ggtgcgaggg cagcagcctc
600tacaaacacc tgcatgtcca ggagaccaag tttcagatgt tccagctaat
tgacattgcc 660cggcagacgg ctcagggaat ggactatttg catgcaaaga
acatcatcca tagagacatg 720aaatccaaca atatatttct ccatgaaggc
ttaacagtga aaattggaga ttttggtttg 780gcaacagtaa agtcacgctg
gagtggttct cagcaggttg aacaacctac tggctctgtc 840ctctggatgg
ccccagaggt gatccgaatg caggataaca acccattcag tttccagtcg
900gatgtctact cctatggcat cgtattgtat gaactgatga cgggggagct
tccttattct 960cacatcaaca accgagatca gatcatcttc atggtgggcc
gaggatatgc ctccccagat 1020cttagtaagc tatataagaa ctgccccaaa
gcaatgaaga ggctggtagc tgactgtgtg 1080aagaaagtaa aggaagagag
gcctcttttt ccccagatcc tgtcttccat tgagctgctc 1140caacactctc
taccgaagat caaccggagc gcttccgagc catccttgca tcgggcagcc
1200cacactgagg atatcaatgc ttgcacgctg accacgtccc cgaggctgcc
tgtcttctag 126010419PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 10Met Ala Gly Cys Arg
Gly Ser Leu Cys Cys Cys Cys Arg Trp Cys Cys1 5 10 15Cys Cys Gly Glu
Arg Glu Thr Arg Thr Pro Glu Glu Leu Thr Ile Leu 20 25 30Gly Glu Thr
Gln Glu Glu Glu Asp Glu Ile Leu Pro Arg Lys Asp Tyr 35 40 45Glu Asp
Ala Ile Arg Ser His Ser Glu Ser Ala Ser Pro Ser Ala Leu 50 55 60Ser
Ser Ser Pro Asn Asn Leu Ser Pro Thr Gly Trp Ser Gln Pro Lys65 70 75
80Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro Val Ser Gly Thr Gln
85 90 95Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln Arg Asp Ser Ser Tyr
Tyr 100 105 110Trp Glu Ile Glu Ala Ser Glu Val Met Leu Ser Thr Arg
Ile Gly Ser 115 120 125Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp
His Gly Asp Val Ala 130 135 140Val Lys Ile Leu Lys Val Val Asp Pro
Thr Pro Glu Gln Phe Gln Ala145 150 155 160Phe Arg Asn Glu Val Ala
Val Leu Arg Lys Thr Arg His Val Asn Ile 165 170 175Leu Leu Phe Met
Gly Tyr Met Thr Lys Asp Asn Leu Ala Ile Val Thr 180 185 190Gln Trp
Cys Glu Gly Ser Ser Leu Tyr Lys His Leu His Val Gln Glu 195 200
205Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile Ala Arg Gln Thr Ala
210 215 220Gln Gly Met Asp Tyr Leu His Ala Lys Asn Ile Ile His Arg
Asp Met225 230 235 240Lys Ser Asn Asn Ile Phe Leu His Glu Gly Leu
Thr Val Lys Ile Gly 245 250 255Asp Phe Gly Leu Ala Thr Val Lys Ser
Arg Trp Ser Gly Ser Gln Gln 260 265 270Val Glu Gln Pro Thr Gly Ser
Val Leu Trp Met Ala Pro Glu Val Ile 275 280 285Arg Met Gln Asp Asn
Asn Pro Phe Ser Phe Gln Ser Asp Val Tyr Ser 290 295 300Tyr Gly Ile
Val Leu Tyr Glu Leu Met Thr Gly Glu Leu Pro Tyr Ser305 310 315
320His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly Tyr
325 330 335Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn Cys Pro Lys
Ala Met 340 345 350Lys Arg Leu Val Ala Asp Cys Val Lys Lys Val Lys
Glu Glu Arg Pro 355 360 365Leu Phe Pro Gln Ile Leu Ser Ser Ile Glu
Leu Leu Gln His Ser Leu 370 375 380Pro Lys Ile Asn Arg Ser Ala Ser
Glu Pro Ser Leu His Arg Ala Ala385 390 395 400His Thr Glu Asp Ile
Asn Ala Cys Thr Leu Thr Thr Ser Pro Arg Leu 405 410 415Pro Val
Phe112088DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 11atggcattgg
tttttcaatt cgggcagccc gtcagggctc agcctctgcc aggactctgc 60cacggcaagc
tcattcggac aaacgcctgt gatgtgtgca acagcaccga tcttccggaa
120gtcgagatca ttagcctgct ggaggagcag ctgccccatt ataagttaag
agccgacacc 180atctacggtt atgaccacga cgactggctc catacacctc
tcatttctcc agatgccaac 240attgacctca caaccgagca aattgaagag
acgttaaaat acttcctttt atgtgctgaa 300agagttggcc agatgactaa
gacatataat gacatagatg ctgtcactcg gcttcttgag 360gagaaagagc
gggatttaga attggccgct cgcatcggcc agtcgttgtt gaagaagaac
420aagaccctaa ccgagaggaa cgagctgctg gaggagcagg tggaacacat
cagggaggag 480gtgtctcagc tccggcatga gctgtccatg aaggatgagc
tgcttcagtt ctacaccagc 540gctgcggagg agagtgagcc cgagtccgtt
tgctcaaccc cgttgaagag gaatgagtcg 600tcctcctcag tccagaatta
ctttcatttg gattctcttc aaaagaagct gaaagacctt 660gaagaggaga
atgttgtact tcgatccgag gccagccagc tgaagacaga gaccatcacc
720tatgaggaga aggagcagca gctggtcaat gactgcgtga aggagctgag
ggatgccaat 780gtccagattg ctagtatctc agaggaactg gccaagaaga
cggaagatgc tgcccgccag 840caagaggaga tcacacacct gctatcgcaa
atagttgatt tgcagaaaaa ggcaaaagct 900tgcgcagtgg aaaatgaaga
acttgtccag catctggggg ctgctaagga tgcccagcgg 960cagctcacag
ccgaggatgc aattcgaagt cacagcgaat cagcctcacc ttcagccctg
1020tccagtagcc ccaacaatct gagcccaaca ggctggtcac agccgaaaac
ccccgtgcca 1080gcacaaagag agcgggcacc agtatctggg acccaggaga
aaaacaaaat taggcctcgt 1140ggacagagag attcaagcta ttattgggaa
atagaagcca gtgaagtgat gctgtccact 1200cggattgggt caggctcttt
tggaactgtt tataagggta aatggcacgg agatgttgca 1260gtaaagatcc
taaaggttgt cgacccaacc ccagagcaat tccaggcctt caggaatgag
1320gtggctgttc tgcgcaaaac acggcatgtg aacattctgc ttttcatggg
gtacatgaca 1380aaggacaacc tggcaattgt gacccagtgg tgcgagggca
gcagcctcta caaacacctg 1440catgtccagg agaccaagtt tcagatgttc
cagctaattg acattgcccg gcagacggct 1500cagggaatgg actatttgca
tgcaaagaac atcatccata gagacatgaa atccaacaat 1560atatttctcc
atgaaggctt aacagtgaaa attggagatt ttggtttggc aacagtaaag
1620tcacgctgga gtggttctca gcaggttgaa caacctactg gctctgtcct
ctggatggcc 1680ccagaggtga tccgaatgca ggataacaac ccattcagtt
tccagtcgga tgtctactcc 1740tatggcatcg tattgtatga actgatgacg
ggggagcttc cttattctca catcaacaac 1800cgagatcaga tcatcttcat
ggtgggccga ggatatgcct ccccagatct tagtaagcta 1860tataagaact
gccccaaagc aatgaagagg ctggtagctg actgtgtgaa gaaagtaaag
1920gaagagaggc ctctttttcc ccagatcctg tcttccattg agctgctcca
acactctcta 1980ccgaagatca accggagcgc ttccgagcca tccttgcatc
gggcagccca cactgaggat 2040atcaatgctt gcacgctgac cacgtccccg
aggctgcctg tcttctag 208812695PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 12Met Ala Leu Val Phe Gln Phe Gly Gln Pro Val Arg Ala
Gln Pro Leu1 5 10 15Pro Gly Leu Cys His Gly Lys Leu Ile Arg Thr Asn
Ala Cys Asp Val 20 25 30Cys Asn Ser Thr Asp Leu Pro Glu Val Glu Ile
Ile Ser Leu Leu Glu 35 40 45Glu Gln Leu Pro His Tyr Lys Leu Arg Ala
Asp Thr Ile Tyr Gly Tyr 50 55 60Asp His Asp Asp Trp Leu His Thr Pro
Leu Ile Ser Pro Asp Ala Asn65 70 75 80Ile Asp Leu Thr Thr Glu Gln
Ile Glu Glu Thr Leu Lys Tyr Phe Leu 85 90 95Leu Cys Ala Glu Arg Val
Gly Gln Met Thr Lys Thr Tyr Asn Asp Ile 100 105 110Asp Ala Val Thr
Arg Leu Leu Glu Glu Lys Glu Arg Asp Leu Glu Leu 115 120 125Ala Ala
Arg Ile Gly Gln Ser Leu Leu Lys Lys Asn Lys Thr Leu Thr 130 135
140Glu Arg Asn Glu Leu Leu Glu Glu Gln Val Glu His Ile Arg Glu
Glu145 150 155 160Val Ser Gln Leu Arg His Glu Leu Ser Met Lys Asp
Glu Leu Leu Gln 165 170 175Phe Tyr Thr Ser Ala Ala Glu Glu Ser Glu
Pro Glu Ser Val Cys Ser 180 185 190Thr Pro Leu Lys Arg Asn Glu Ser
Ser Ser Ser Val Gln Asn Tyr Phe 195 200 205His Leu Asp Ser Leu Gln
Lys Lys Leu Lys Asp Leu Glu Glu Glu Asn 210 215 220Val Val Leu Arg
Ser Glu Ala Ser Gln Leu Lys Thr Glu Thr Ile Thr225 230 235 240Tyr
Glu Glu Lys Glu Gln Gln Leu Val Asn Asp Cys Val Lys Glu Leu 245 250
255Arg Asp Ala Asn Val Gln Ile Ala Ser Ile Ser Glu Glu Leu Ala Lys
260 265 270Lys Thr Glu Asp Ala Ala Arg Gln Gln Glu Glu Ile Thr His
Leu Leu 275 280 285Ser Gln Ile Val Asp Leu Gln Lys Lys Ala Lys Ala
Cys Ala Val Glu 290 295 300Asn Glu Glu Leu Val Gln His Leu Gly Ala
Ala Lys Asp Ala Gln Arg305 310 315 320Gln Leu Thr Ala Glu Asp Ala
Ile Arg Ser His Ser Glu Ser Ala Ser 325 330 335Pro Ser Ala Leu Ser
Ser Ser Pro Asn Asn Leu Ser Pro Thr Gly Trp 340 345 350Ser Gln Pro
Lys Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro Val 355 360 365Ser
Gly Thr Gln Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln Arg Asp 370 375
380Ser Ser Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val Met Leu Ser
Thr385 390 395 400Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys
Gly Lys Trp His 405 410 415Gly Asp Val Ala Val Lys Ile Leu Lys Val
Val Asp Pro Thr Pro Glu 420 425 430Gln Phe Gln Ala Phe Arg Asn Glu
Val Ala Val Leu Arg Lys Thr Arg 435 440 445His Val Asn Ile Leu Leu
Phe Met Gly Tyr Met Thr Lys Asp Asn Leu 450 455 460Ala Ile Val Thr
Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His Leu465 470 475 480His
Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile Ala 4