U.S. patent application number 15/839560 was filed with the patent office on 2018-10-04 for novel kif5b-ret fusion molecules and uses thereof.
The applicant listed for this patent is FOUNDATION MEDICINE, INC.. Invention is credited to Sean R. Downing, Matthew J. Hawryluk, Doron Lipson, Alexander N. Parker, Philip James Stephens.
Application Number | 20180280391 15/839560 |
Document ID | / |
Family ID | 47746850 |
Filed Date | 2018-10-04 |
United States Patent
Application |
20180280391 |
Kind Code |
A1 |
Downing; Sean R. ; et
al. |
October 4, 2018 |
NOVEL KIF5B-RET FUSION MOLECULES AND USES THEREOF
Abstract
Novel RET fusion molecules and uses are disclosed.
Inventors: |
Downing; Sean R.; (Methuen,
MA) ; Hawryluk; Matthew J.; (Cambridge, MA) ;
Lipson; Doron; (Cambridge, MA) ; Parker; Alexander
N.; (Boston, MA) ; Stephens; Philip James;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FOUNDATION MEDICINE, INC. |
Cambridge |
MA |
US |
|
|
Family ID: |
47746850 |
Appl. No.: |
15/839560 |
Filed: |
December 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15045716 |
Feb 17, 2016 |
9884060 |
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15839560 |
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14187280 |
Feb 23, 2014 |
9297011 |
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15045716 |
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PCT/US2012/051978 |
Aug 23, 2012 |
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14187280 |
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61594739 |
Feb 3, 2012 |
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61542112 |
Sep 30, 2011 |
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61537024 |
Sep 20, 2011 |
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61526613 |
Aug 23, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C07K 2319/00 20130101; C12Q 2600/156 20130101; C12N 9/14 20130101;
A61K 31/47 20130101; C12N 15/1135 20130101; C12Q 2600/136 20130101;
C12Q 2600/158 20130101; A61K 31/404 20130101; A61K 31/4412
20130101; A61K 31/513 20130101; A61K 31/444 20130101; C07K 14/82
20130101; A61K 31/517 20130101 |
International
Class: |
A61K 31/517 20060101
A61K031/517; C12N 15/113 20060101 C12N015/113; A61K 31/513 20060101
A61K031/513; C07K 14/82 20060101 C07K014/82; A61K 31/4412 20060101
A61K031/4412; A61K 31/404 20060101 A61K031/404; A61K 31/47 20060101
A61K031/47; C12Q 1/6886 20060101 C12Q001/6886; C12N 9/14 20060101
C12N009/14; A61K 31/444 20060101 A61K031/444 |
Claims
1. A method of reducing an activity of a Kinesin Family Member 5B
(KIF5B)-RET fusion polypeptide or a nucleic acid molecule encoding
the KIF5B-RET fusion polypeptide, comprising: optionally, acquiring
knowledge of the presence of the KIF5B-RET fusion polypeptide or a
nucleic acid molecule encoding the KIF5B-RET fusion polypeptide;
and contacting the KIF5B-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide, or a
KIF5B-RET-expressing cell, with an agent that inhibits an activity
or expression of KIF5B-RET.
2.-5. (canceled)
6. A method of treating a subject having cancer, comprising:
acquiring knowledge of the presence of a Kinesin Family Member 5B
(KIF5B)-RET fusion polypeptide or a nucleic acid molecule encoding
the KIF5B-RET fusion polypeptide in said subject; and administering
to the subject an effective amount of an anti-cancer agent, thereby
treating cancer in the subject.
7. The method of claim 6, wherein said anti-cancer agent is a
kinase inhibitor.
8. The method of claim 7, wherein the kinase inhibitor: (i) is
administered responsive to the determination of presence of the
KIF5B-RET fusion polypeptide or a nucleic acid molecule encoding
the KIF5B-RET fusion polypeptide in a tumor sample from said
subject; (ii) inhibits the expression of the nucleic acid encoding
the KIF5B-RET fusion polypeptide; (iii) is selected from an
antisense molecule, a ribozyme, an RNAi molecule, or a triple helix
molecule, each of which hybridizes to a nucleic acid molecule
encoding the KIF5B-RET fusion polypeptide, or a transcription
regulatory region that blocks or reduces mRNA expression of the
nucleic acid molecule encoding the KIF5B-RET fusion polypeptide; or
(iv) is administered in combination with a second therapeutic agent
or a different therapeutic modality.
9. The method of claim 6, wherein: (i) said administration is
administered responsive to acquiring knowledge or information of
the presence of the KIF5B-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide in said subject;
(ii) said administration is administered responsive to acquiring
knowledge or information of the presence of the KIF5B-RET fusion
polypeptide or a nucleic acid molecule encoding the KIF5B-RET
fusion polypeptide in said subject from another party; (iii) said
administration is administered responsive to an identification of
the presence of the KIF5B-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide in the subject,
wherein said identification arises from collaboration with another
party; (iv) the method comprises receiving a communication of the
presence of the KIF5B-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide in the subject;
or (v) the method comprises determining the presence of the
KIF5B-RET fusion polypeptide or a nucleic acid molecule encoding
the KIF5B-RET fusion polypeptide by sequencing.
10.-13. (canceled)
14. The method of claim 6, wherein: (i) said cancer is chosen from
pancreatic cancer, melanoma, colorectal cancer, esophageal-gastric
cancer, thyroid cancer, or adenocarcinoma; or (ii) said cancer is
lung cancer, and wherein said lung cancer is chosen from: small
cell lung cancer (SCLC), non-small cell lung cancer (NSCLC),
squamous cell carcinoma (SCC), adenocarcinoma of the lung,
bronchogenic carcinoma, or a combination thereof.
15.-18. (canceled)
19. The method of claim 6, wherein the anti-cancer agent is: (i) a
multi-kinase inhibitor; (ii) a RET-specific inhibitor; (iii)
sorafenib (NEXAVAR.RTM.); (iv) sunitinib (SUTENT.RTM., SU11248);
(v) vandetanib (CAPRELSA.RTM., ZD6474); or (vi) NVP-AST487.
20.-35. (canceled)
36. A method of determining the presence of a Kinesin Family Member
5B (KIF5B)-RET fusion polypeptide or a nucleic acid molecule
encoding the KIF5B-RET fusion polypeptide comprising: directly
acquiring knowledge that a KIF5B-RET fusion polypeptide or a
nucleic acid molecule encoding the KIF5B-RET fusion polypeptide is
present in a sample from a subject.
37. The method of claim 36, wherein said sample: (i) comprises
fluid, a cell, a circulating tumor cell, tissue, a tumor tissue, or
a tumor biopsy; or (ii) is a nucleic acid sample or a protein
sample.
38.-41. (canceled)
42. The method of claim 36, wherein the subject has a cancer,
wherein the cancer: (i) is chosen from a colorectal cancer, an
esophageal-gastric cancer, a thyroid cancer, an adenocarcinoma or a
melanoma; or (ii) is a lung cancer, and wherein the lung cancer is
chosen from: a NSCLC, a SCLC, a SCC, or a combination thereof.
43.-44. (canceled)
45. The method of claim 36, wherein: (i) a nucleic acid molecule
encoding the KIF5B-RET fusion polypeptide is detected, wherein the
detection comprises sequencing by a method chosen from one or more
of: a nucleic acid hybridization assay, an amplification-based
assay, a PCR-RFLP assay, real-time PCR, sequencing, screening
analysis, FISH, spectral karyotyping, MFISH, comparative genomic
hybridization, in situ hybridization, SSP, HPLC or
mass-spectrometric genotyping; (ii) the KIF5B-RET fusion
polypeptide is detected; or (iii) the level or activity of the
KIF5B-RET fusion polypeptide or a nucleic acid molecule encoding
the KIF5B-RET fusion polypeptide is evaluated.
46. (canceled)
47. The method of claim 36, comprising acquiring a sequence for a
position in a nucleic acid molecule by sequencing at least one
nucleotide of the nucleic acid molecule, wherein the sequence
acquired is compared to a reference sequence, or a wild type
reference sequence, thereby determining that a nucleic acid
molecule encoding a KIF5B-RET fusion polypeptide is present.
48.-49. (canceled)
50. The method of claim 36, comprising: contacting a protein sample
with a reagent which specifically binds to a KIF5B-RET fusion
polypeptide; and detecting the formation of a complex of the
KIF5B-RET fusion polypeptide and the reagent, wherein: the reagent
is labeled with a detectable group to facilitate detection of the
bound and unbound reagent; or the reagent is an antibody
molecule.
51.-53. (canceled)
54. The method of claim 36, wherein the KIF5B-RET fusion
polypeptide or a nucleic acid molecule encoding the KIF5B-RET
fusion polypeptide is detected: (i) prior to initiating, during, or
after, a treatment in the subject; (ii) at the time of diagnosing
the subject with a cancer; or (iii) at a pre-determined interval,
wherein the pre-determined interval comprises a first point in time
and at least at a subsequent point in time.
55.-56. (canceled)
57. The method of claim 36, comprising, responsive to a
determination of the presence of the KIF5B-RET fusion polypeptide
or a nucleic acid molecule encoding the KIF5B-RET fusion
polypeptide, one or more of: (1) stratifying a population
comprising the subject, wherein stratifying comprises assigning the
subject to a class, wherein the class comprises a candidate
classified to receive a treatment that comprises a kinase
inhibitor; (2) identifying or selecting the subject as likely or
unlikely to respond to a treatment; (3) selecting a treatment
option for the subject; or (4) prognosticating the time course of
the disease in the subject.
58. The method of claim 57, wherein the treatment option comprises
administering a kinase inhibitor, and optionally wherein the kinase
inhibitor is chosen from: sorafenib (NEXAVAR.RTM.); sunitinib
(SUTENT.RTM., SU11248); vandetanib (ZACTIMA.RTM., ZD6474); or
NVP-AST487.
59.-62. (canceled)
63. The method of claim 36, wherein responsive to the direct
acquisition of knowledge of the presence of the KIF5B-RET fusion
polypeptide or a nucleic acid molecule encoding the KIF5B-RET
fusion polypeptide: (i) the subject is classified as a candidate to
receive a treatment that comprises a kinase inhibitor; (ii) the
subject is assigned to a class, wherein the class comprises a
candidate classified to receive a treatment that comprises a kinase
inhibitor, if a KIF5B-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide is identified in
a sample from the subject; or (iii) the subject is identified as
likely to respond to a treatment that comprises a kinase
inhibitor.
64.-66. (canceled)
67. A method of evaluating a subject, comprising: identifying,
selecting, or obtaining information or knowledge that the patient
has participated in a clinical trial or has been treated for
cancer; acquiring genotype information that identifies a Kinesin
Family Member 5B (KIF5B)-RET fusion polypeptide or a nucleic acid
molecule encoding the KIF5B-RET fusion polypeptide as being in the
subject, wherein the presence of the KIF5B-RET fusion polypeptide
or a nucleic acid molecule encoding the KIF5B-RET fusion
polypeptide identifies the subject as having an increased risk for,
or having, a cancer associated with the KIF5B-RET fusion
polypeptide or a nucleic acid molecule encoding the KIF5B-RET
fusion polypeptide; and treating the subject with a kinase
inhibitor.
68. The method of claim 67, further comprising providing a report
to a party.
69. The method of claim 68, wherein: (i) the party is the patient,
a caregiver, a physician, an oncologist, a hospital, clinic,
third-party payor, insurance company or government office; (ii)
said report is in electronic, web-based or paper form; or (iii) the
report identifies the presence or absence of an alteration, and
optionally includes an identifier for the subject.
70.-71. (canceled)
72. The method of claim 68, wherein said report comprises:
information on the role of the KIF5B-RET fusion polypeptide or a
nucleic acid molecule encoding the KIF5B-RET fusion polypeptide, or
wildtype KIF5B or RET sequence, in disease; information on
prognosis, resistance, or potential or suggested therapeutic
options; information on the likely effectiveness of a therapeutic
option, the acceptability of a therapeutic option, or the
advisability of applying a therapeutic option to the subject; or
information, or a recommendation on, the administration of a drug
to the subject.
73. (canceled)
74. An isolated or purified nucleic acid molecule that encodes a
Kinesin Family Member 5B (KIF5B)-RET fusion polypeptide or a
nucleic acid molecule encoding the KIF5B-RET fusion polypeptide or
a breakpoint comprising fragment thereof.
75. (canceled)
76. An isolated or purified vector comprising the nucleic acid
molecule of claim 74.
77. A host cell comprising a vector of claim 76.
78. A nucleic acid molecule that specifically reduces or inhibits
the expression of the nucleic acid molecule of claim 74.
79. (canceled)
80. A nucleic acid molecule suitable as a probe, primer, bait or
library member that can specifically identify, capture, detect, or
isolate the nucleic acid molecule of claim 74.
81.-87. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/045,716, filed Feb. 17, 2016, now allowed, which is a
divisional of U.S. application Ser. No. 14/187,280, filed Feb. 23,
2014, now U.S. Pat. No. 9,297,011, which is a continuation of
International Application No. PCT/US2012/051978, filed Aug. 23,
2012, which claims the benefit of U.S. Provisional Application No.
61/526,613, filed Aug. 23, 2011; U.S. Provisional Application No.
61/537,024, filed Sep. 20, 2011; U.S. Provisional Application No.
61/542,112, filed Sep. 30, 2011; and U.S. Provisional Application
No. 61/594,739, filed Feb. 3, 2012. The contents of all these prior
applications are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 17, 2016, is named F2036-702540 Sequence Listing.txt and is
59,049 bytes in size.
BACKGROUND
[0003] Cancer represents the phenotypic end-point of multiple
genetic lesions that endow cells with a full range of biological
properties required for tumorigenesis. Indeed, a hallmark genomic
feature of many cancers, including, for example, B cell cancer,
lung cancer, breast cancer, ovarian cancer, pancreatic cancer, and
colon cancer, is the presence of numerous complex chromosome
structural aberrations, including translocations, intra-chromosomal
inversions, point mutations, deletions, gene copy number changes,
gene expression level changes, and germline mutations, among
others.
[0004] The need still exists for identifying novel genetic lesions
associated with cancer. Such genetic lesions can be an effective
approach to develop compositions, methods and assays for evaluating
and treating cancer patients.
SUMMARY
[0005] The invention is based, at least in part, on the discovery
of novel inversion events that include a fragment of a KIF5B gene
("Kinesin Family Member 5B-RET proto-oncogene") and a fragment of a
RET proto-oncogene in a cancer, e.g., a lung cancer, referred to
herein as "KIF5B-RET." The term "KIF5B-RET" or "KIF5B-RET fusion"
is used generically herein, and includes any fusion molecule (e.g.,
gene, gene product (e.g., cDNA, mRNA, or polypeptide), and variant
thereof) that includes a fragment of KIF5B and a fragment of RET,
including, e.g., a 5'KIF5B-3'RET.
[0006] In one embodiment, a KIF5B-RET fusion includes an in-frame
fusion of an exon of KIF5B (e.g., one more exons encoding a kinesin
motor domain or a fragment thereof) and an exon of RET (e.g., one
or more exons encoding a RET tyrosine kinase domain or a fragment
thereof). For example, the KIF5B-RET fusion can include an in-frame
fusion of at least exon 15 of KIF5B or a fragment thereof (e.g.,
exons 1-15 of KIF5B or a fragment thereof) with at least exon 12 of
RET or a fragment thereof (e.g., exons 12-20 of RET or a fragment
thereof). In certain embodiments, the KIF5B-RET fusion is in a
5'-KIF5B to 3'-RET configuration referred to herein as
"5'KIF5B-3'RET." A KIF5B-RET fusion polypeptide encoded by a
5'KIF5B-3'RET nucleic acid is sometimes referred to herein as a
5'KIF5B-3'RET polypeptide. In an embodiment, the 5'KIF5B-3'RET
fusion comprises sufficient KIF5B and sufficient RET sequence such
that the 5'KIF5B-3'RET fusion has kinase activity, e.g., has
elevated activity, e.g., kinase activity, as compared with wild
type RET, e.g., in a cell of a cancer referred to herein. In one
embodiment, the 5'KIF5B-3'RET fusion comprises at least 1, 2, 3, 4,
5, 6, 7, 9, 10, or 11 exons from KIF5B and at least 1, 2, 3, 4, 5,
6, 7, 9, or 10, RET exons. In one embodiment, the 5'KIF5B-3'RET
fusion polypeptide includes a kinesin motor domain, a coiled coil
domain, or a functional fragment thereof, and a RET tyrosine kinase
domain or a functional fragment thereof.
[0007] The RET proto-oncogene is associated with cancerous
phenotypes, including papillary thyroid carcinomas (PTC), multiple
endocrine neoplasias (MEN), phaeochromocytoma, among others. For
example, chromosomal rearrangements that generate a fusion gene
resulting in the juxtaposition of the C-terminal region of the RET
protein with an N-terminal portion of another protein (known as
RET/PTC) are known to be associated with PTC (Nikiforov, Y E (2002)
Endocr. Pathol. 13 (1):3-16). The RET/PTC has been shown to cause
constitute activation of the RET kinase domain, which is a likely
contributor to tumorigenicity. The KIF5B-RET fusions disclosed
herein (e.g., the 5'-KIF5B to 3'-RET fusions that include a RET
tyrosine kinase domain) are associated with cancers, e.g., lung
cancer. Elevated expression of the 3' end of RET beginning in exon
12 is detected in lung tumor samples, suggesting that the KIF5B-RET
fusion transcript can result in RET kinase domain
overexpression.
[0008] In other embodiments, the KIF5B-RET fusion includes an
in-frame fusion of at least exon 11 of RET or a fragment thereof
(e.g., exons 1-11 of RET or a fragment thereof) with at least exon
16 or a fragment thereof (e.g., exons 16-25 of KIF5B or a fragment
thereof). In certain embodiments, the KIF5B-RET fusion is in a
5'-RET to 3'-KIF5B configuration referred to herein as
"5'RET-3'KIF5B"). The 5'RET-3'KIF5B configuration of the fusion
molecule is not believed to be expressed.
[0009] Accordingly, the invention provides, methods of:
identifying, assessing or detecting a KIF5B-RET fusion; methods of
identifying, assessing, evaluating, and/or treating a subject
having a cancer, e.g., a cancer having a KIF5B-RET fusion; isolated
KIF5B-RET nucleic acid molecules, nucleic acid constructs, host
cells containing the nucleic acid molecules; purified KIF5B-RET
polypeptides and binding agents; detection reagents (e.g., probes,
primers, antibodies, kits, capable, e.g., of specific detection of
a KIF5B-RET nucleic acid or protein); screening assays for
identifying molecules that interact with, e.g., inhibit,
5'KIF5B-3'RET fusions, e.g., novel kinase inhibitors; as well as
assays and kits for evaluating, identifying, assessing and/or
treating a subject having a cancer, e.g., a cancer having a
KIF5B-RET fusion. The compositions and methods identified herein
can be used, for example, to identify new KIF5B-RET inhibitors; to
evaluate, identify or select subject, e.g., a patient, having a
cancer; and to treat or prevent a cancer.
KIF5B-RET Nucleic Acid Molecules
[0010] In one aspect, the invention features a nucleic acid
molecule (e.g., an isolated or purified) nucleic acid molecule that
includes a fragment of a KIF5B gene and a fragment of a RET
proto-oncogene. In one embodiment, the nucleic acid molecule
includes a fusion, e.g., an in-frame fusion, of an exon of KIF5B
(e.g., one more exons encoding a kinesin motor domain or a fragment
thereof), and an exon of RET (e.g., one or more exons encoding a
RET tyrosine kinase domain or a fragment thereof).
[0011] In an embodiment the 5'KIF5B-3'RET nucleic acid molecule
comprises sufficient KIF5B and sufficient RET sequence such that
the encoded 5'KIF5B-3'RET fusion has kinase activity, e.g., has
elevated activity, e.g., kinase activity, as compared with wild
type RET, e.g., in a cell of a cancer referred to herein. In an
embodiment the encoded 5'KIF5B-3'RET fusion comprises at least 1,
2, 3, 4, 5, 6, 7, 9, 10, or 11 exons from KIF5B and at least 1, 2,
3, 4, 5, 6, 7, 9, or 10, RET exons. In one embodiment, the encoded
5'KIF5B-3'RET fusion polypeptide includes a kinesin motor domain, a
coiled coil domain, or a functional fragment thereof, and a RET
tyrosine kinase domain or a functional fragment thereof.
[0012] In one embodiment, the nucleic acid molecule includes a
nucleotide sequence that has an in-frame fusion of exon 15 of KIF5B
with exon 12 of RET (e.g., a sequence within an 11 MB pericentric
inversion on chromosome 10). In other embodiments, the nucleic acid
molecules includes a nucleotide sequence in the region of
32,316,376-32,316,416 of chromosome 10 coupled to (e.g., juxtaposed
to) nucleotides in the region of nucleotides 43,611,042-43,611,118
of chromosome 10. In another embodiment, the nucleic acid molecule
includes a nucleotide sequence that includes a breakpoint, e.g., a
breakpoint identified in FIGS. 1, 2 and 3A-3D. For example, the
nucleic acid molecule includes a nucleotide sequence that includes
the fusion junction between the KIF5B transcript and the RET
transcript, e.g., a nucleotide sequence that includes a portion of
SEQ ID NO:1 (e.g., a nucleotide sequence within exons 1-15 of a
KIF5B gene and 12-20 of a RET gene) (e.g., a portion of SEQ ID NO:1
comprising nucleotides 1720-1731, 1717-1734, or 1714-1737 of SEQ ID
NO:1 (see FIG. 3B)).
[0013] In other embodiments, the nucleic acid molecule includes a
KIF5B-RET fusion having a configuration shown in FIGS. 2B and
3A-3D. For example, the KIF5B-RET fusion can include an in-frame
fusion of at least exon 15 of KIF5B or a fragment thereof (e.g.,
exons 1-15 of KIF5B or a fragment thereof) with at least exon 12 of
RET or a fragment thereof (e.g., exons 12-20 of RET or a fragment
thereof). In certain embodiments, the KIF5B-RET fusion is in a
5'-KIF5B to 3'-RET configuration referred to herein as
"5'KIF5B-3'RET"). In one embodiment, the nucleic acid molecule
includes the nucleotide sequence of nucleotides 1-1725 of SEQ ID
NO:1 (corresponding to exons 1-15 of the KIF5B gene), or a fragment
thereof, or a sequence substantially identical thereto. In another
embodiment, the nucleic acid molecule includes the nucleotide
sequence of nucleotides 1726-2934 of SEQ ID NO:1 (e.g.,
corresponding to exons 12-20 of the RET gene), or a fragment
thereof, or a sequence substantially identical thereto. In yet
other embodiments, the nucleic acid molecule includes the
nucleotide sequence shown in FIGS. 3A-3D (e.g., SEQ ID NO:1) or a
fragment thereof, or a sequence substantially identical thereto. In
one embodiment, the nucleic acid molecule is complementary to at
least a portion of a nucleotide sequence disclosed herein, e.g., is
capable of hybridizing under a stringency condition described
herein to SEQ ID NO:1 or a fragment thereof. In yet other
embodiment, the nucleic acid molecule hybridizes to a nucleotide
sequence that is complementary to at least a portion of a
nucleotide sequence disclosed herein, e.g., is capable of
hybridizing under a stringency condition described herein to a
nucleotide sequence complementary to SEQ ID NO:1 or a fragment
thereof. The nucleotide sequence of a cDNA encoding an exemplary
5'KIF5B-3'RET fusion is shown in SEQ ID NO:1, and the amino acid
sequence is shown in SEQ ID NO:2.
[0014] In other embodiments, the nucleic acid molecule includes a
nucleotide sequence encoding a KIF5B-RET fusion polypeptide that
includes a fragment of a KIF5B gene and a fragment of a RET
proto-oncogene. In one embodiment, the nucleotide sequence encodes
a KIF5B-RET fusion polypeptide that includes a kinesin motor domain
or a functional fragment thereof, and a RET tyrosine kinase domain
or a functional fragment thereof. In another embodiment, the
nucleotide sequence encodes a fragment of the KIF5B polypeptide
including the amino acid sequence of amino acids 1-575 of SEQ ID
NO:2 or a fragment thereof, or a sequence substantially identical
thereto. For example, the nucleic acid molecule can include a
nucleotide sequence encoding a kinesin motor domain of KIF5B-RET
fusion polypeptide that includes amino acids 6-325 of SEQ ID NO:2
or a fragment thereof. In other embodiments, the nucleic acid
molecule includes a fragment of the RET gene encoding the amino
acid sequence of amino acids 576-977 of SEQ ID NO:2 or a fragment
thereof, or a sequence substantially identical thereto. For
example, the nucleic acid molecule can include a nucleotide
sequence encoding a RET kinase domain of a KIF5B-RET fusion
polypeptide that includes amino acids 587-868 of SEQ ID NO:2 or a
fragment thereof. In yet other embodiments, the nucleic acid
molecule includes a nucleotide sequence encoding the amino acid
sequence shown in FIGS. 3A-3D (e.g., SEQ ID NO:2) or a fragment
thereof, or a sequence substantially identical thereto.
[0015] In another embodiment, the nucleic acid molecule includes a
KIF5B-RET fusion having the configuration shown in FIGS. 2A and
4A-4D. In one embodiment, the nucleic acid molecule includes a
nucleotide sequence that include a fusion junction between the RET
transcript and the KIF5B transcript, e.g., a nucleotide sequence
within SEQ ID NO:3 (e.g., a sequence comprising nucleotides
2131-2142, 2128-2145, or 2125-2148 of SEQ ID NO:3 (see FIG. 4B)).
In another embodiment, the nucleic acid molecule includes a fusion,
e.g., an in-frame fusion, of at least exon 11 of RET or a fragment
thereof (e.g., exons 1-11 of RET or a fragment thereof), and at
least exon 16 or a fragment thereof (e.g., exons 16-25 of KIF5B or
a fragment thereof). In certain embodiments, the KIF5B-RET fusion
is in a 5'-RET to 3'-KIF5B configuration referred to herein as
"5'RET-3'KIF5B"). In one embodiment, the nucleic acid molecule
includes the nucleotides sequence of 1-2136 of SEQ ID NO:3
(corresponding to exons 1-11 of a RET gene) or a fragment thereof,
or a sequence substantially identical thereto. In another
embodiment, the nucleic acid molecule includes the nucleotides
sequence of 2137-3360 of SEQ ID NO:3 (corresponding to exons 16-25
of the a KIF5B gene) or a fragment thereof, or a sequence
substantially identical thereto. In yet other embodiments, the
nucleic acid molecule includes the nucleotide sequence shown in
FIGS. 4A-4D (e.g., SEQ ID NO:3) or a fragment thereof, or a
sequence substantially identical thereto. In one embodiment, the
nucleic acid molecule is complementary to at least a portion of a
nucleotide sequence disclosed herein, e.g., is capable of
hybridizing under a stringency condition described herein to SEQ ID
NO:3 or a fragment thereof. In yet other embodiment, the nucleic
acid molecule hybridizes to a nucleotide sequence that is
complementary to at least a portion of a nucleotide sequence
disclosed herein, e.g., is capable of hybridizing under a
stringency condition to a nucleotide sequence complementary to SEQ
ID NO:3 or a fragment thereof. The nucleotide sequence of a cDNA
encoding an exemplary 5'RET-3'KIF5B fusion is shown in SEQ ID NO:3,
and the predicted amino acid sequence is shown in SEQ ID NO:4.
RT-PCR studies have shown that the RET exon 11 and KIF5B exon 16
did not yield a transcription product.
[0016] In a related aspect, the invention features nucleic acid
constructs that include the KIF5B-RET nucleic acid molecules
described herein. In certain embodiments, the nucleic acid
molecules are operatively linked to a native or a heterologous
regulatory sequence. Also included are vectors and host cells that
include the KIF5B-RET nucleic acid molecules described herein,
e.g., vectors and host cells suitable for producing the nucleic
acid molecules and polypeptides described herein.
[0017] In a related aspect, methods of producing the nucleic acid
molecules and polypeptides described herein are also described.
[0018] In another aspect, the invention features nucleic acid
molecules that reduces or inhibits the expression of a nucleic acid
molecule that encodes a KIF5B-RET fusion described herein. Examples
of such nucleic acid molecules include, for example, antisense
molecules, ribozymes, RNAi, triple helix molecules that hybridize
to a nucleic acid encoding KIF5B-RET, or a transcription regulatory
region of KIF5B-RET, and blocks or reduces mRNA expression of
KIF5B-RET.
Nucleic Acid Detection and Capturing Reagents
[0019] The invention also features a nucleic acid molecule, e.g.,
nucleic acid fragment, suitable as probe, primer, bait or library
member that includes, flanks, hybridizes to, which are useful for
identifying, or are otherwise based on, the KIF5B-RET fusions
described herein. In certain embodiments, the probe, primer or bait
molecule is an oligonucleotide that allows capture, detection or
isolation of a KIF5B-RET fusion nucleic acid molecule described
herein. The oligonucleotide can comprise a nucleotide sequence
substantially complementary to a fragment of the KIF5B-RET fusion
nucleic acid molecules described herein. The sequence identity
between the nucleic acid fragment, e.g., the oligonucleotide, and
the target KIF5B-RET sequence need not be exact, so long as the
sequences are sufficiently complementary to allow the capture,
detection 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. 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.
[0020] In one embodiment, the nucleic acid fragment can be used to
identify or capture, e.g., by hybridization, a KIF5B-RET fusion.
For example, the nucleic acid fragment can be a probe, a primer, or
a bait, for use in identifying or capturing, e.g., by
hybridization, a KIF5B-RET fusion described herein. In one
embodiment, the nucleic acid fragment can be useful for identifying
or capturing a KIF5B-RET breakpoint, e.g., as identified in FIG. 1.
In one embodiment, the nucleic acid fragment hybridizes to a
nucleotide sequence within a chromosomal rearrangement that creates
an in-frame fusion of exon 15 of KIF5B with exon 12 of RET (e.g., a
sequence within an 11 MB pericentric inversion on chromosome 10).
In one embodiment, the nucleic acid fragment hybridizes to a
nucleotide sequence in the region of 32,316,376-32,316,416 of
chromosome 10 coupled to (e.g., juxtaposed to) nucleotides in the
region of nucleotides 43,611,042-43,611,118 of chromosome 10. In
one embodiment, the nucleic acid fragment hybridizes to a
nucleotide sequence that includes a breakpoint, e.g., a breakpoint
as identified in FIGS. 1, 2 and 3A-3D. For example, the nucleic
acid fragment can hybridize to a nucleotide sequence that includes
the fusion junction between the KIF5B transcript and the RET
transcript, e.g., a nucleotide sequence that includes a portion of
SEQ ID NO:1 (e.g., a nucleotide sequence within exons 1-15 of a
KIF5B gene and 12-20 of a RET gene) (e.g., a portion of SEQ ID NO:1
comprising nucleotides 1720-1731, 1717-1734, or 1714-1737 of SEQ ID
NO:1 (see FIG. 3B)).
[0021] The probes or primers described herein can be used, for
example, for FISH detection or PCR amplification. In one exemplary
embodiment where detection is based on PCR, amplification of the
KIF5B-RET fusion junction can be performed using a primer or a
primer pair, e.g., for amplifying a sequence flanking the KIF5B-RET
fusion junctions described herein, e.g., the mutations or the
junction of a chromosomal rearrangement described herein. In one
embodiment, a pair of isolated oligonucleotide primers can amplify
a region containing or adjacent to a position in the KIF5B-RET
fusion. For example, forward primers can be designed to hybridize
to a nucleotide sequence within KIF5B genomic or mRNA sequence
(e.g., a nucleotide sequence within exons 1-15 of a KIF5B gene, or
nucleotides 1-1725 of SEQ ID NO:1), and the reverse primers can be
designed to hybridize to a nucleotide sequence within RET (e.g., a
nucleotide sequence within exons 12-20 of RET, or nucleotides
1725-2934 of SEQ ID NO:1).
[0022] In another embodiment, the nucleic acid fragments can be
used to identify, e.g., by hybridization, a 5'RET-3'KIF5B fusion.
In one embodiment, the nucleic acid fragment hybridizes to a
nucleotide sequence that include a fusion junction between the RET
transcript and the KIF5B transcript, e.g., a nucleotide sequence
within SEQ ID NO:3 (e.g., a sequence comprising nucleotides
2131-2142, 2128-2145, or 2125-2148 of SEQ ID NO:3 (see FIG.
4B)).
[0023] In other embodiments, the nucleic acid fragment includes a
bait that comprises a nucleotide sequence that hybridizes to a
KIF5B-RET fusion nucleic acid molecule described herein, and
thereby allows the capture or isolation said nucleic acid molecule.
In one embodiment, a bait is suitable for solution phase
hybridization. In other embodiments, a bait includes a binding
entity, e.g., an affinity tag, that allows capture and separation,
e.g., by binding to a binding entity, of a hybrid formed by a bait
and a nucleic acid hybridized to the bait.
[0024] In other embodiments, the nucleic acid fragment includes a
library member comprising a KIF5B-RET nucleic acid molecule
described herein. In one embodiment, the library member includes a
rearrangement that results in a KIF5B-RET fusion described
herein.
[0025] The nucleic acid fragment 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, or
can include an affinity tag; a tag, or identifier (e.g., an
adaptor, barcode or other sequence identifier).
KIF5B-RET Fusion Polypeptides
[0026] In another aspect, the invention features a KIF5B-RET fusion
polypeptide (e.g., a purified KIF5B-RET fusion polypeptide), a
biologically active or antigenic fragment thereof, as well as
reagents (e.g., antibody molecules that bind to a KIF5B-RET fusion
polypeptide), methods for modulating a KIF5B-RET polypeptide
activity and detection of a KIF5B-RET polypeptide.
[0027] In one embodiment, the KIF5B-RET fusion polypeptide has at
least one biological activity, e.g., a RET kinase activity, and/or
a dimerizing or multimerizing activity. In one embodiment, at least
one biological activity of the KIF5B-RET fusion polypeptide is
reduced or inhibited by an anti-cancer drug, e.g., a kinase
inhibitor (e.g., a multikinase inhibitor or a RET-specific
inhibitor). In one embodiment, at least one biological activity of
the KIF5B-RET fusion polypeptide is reduced or inhibited by a
kinase inhibitor chosen from lenvatinib (E7080), sorafenib
(NEXAVAR.RTM.), sunitinib (SUTENT.RTM., SU11248), vandetanib
(CAPRELSA.RTM., ZACTIMA.RTM., ZD6474), NVP-AST487, regorafenib
(BAY-73-4506), motesanib (AMG 706), cabozantinib (XL-184), apatinib
(YN-968D1), or DCC-2157.
[0028] In other embodiments, the KIF5B-RET fusion polypeptide
includes a fragment of a KIF5B polypeptide and a fragment of a RET
polypeptide. In one embodiment, the KIF5B-RET fusion polypeptide
includes amino acids 578-575 of SEQ ID NO:2 or a fragment thereof
(e.g., amino acids 1-575 of SEQ ID NO:2 or a fragment thereof), and
amino acids 576-624 of SEQ ID NO:2 or a fragment thereof (e.g.,
amino acids 576-977 of SEQ ID NO:2 or a fragment thereof). In yet
other embodiments, the KIF5B-RET fusion polypeptide includes an
amino acid sequence substantially identical to an in-frame fusion
of amino acids 578-575 of SEQ ID NO:2 or a fragment thereof (e.g.,
amino acids 1-575 of SEQ ID NO:2 or a fragment thereof), and amino
acids 576-624 of SEQ ID NO:2 or a fragment thereof (e.g., amino
acids 576-977 of SEQ ID NO:2 or a fragment thereof).
[0029] In other embodiments, the KIF5B-RET fusion polypeptide
includes a KIF5B kinesin motor domain or a fragment thereof, and a
RET kinase domain or a fragment thereof. In another embodiment, the
KIF5B-RET fusion polypeptide includes the amino acid sequence of
amino acids 1-575 of SEQ ID NO:2 or a fragment thereof, or a
sequence substantially identical thereto. For example, the
KIF5B-RET fusion polypeptide can include a kinesin motor domain of
KIF5B that includes amino acids 6-325 of SEQ ID NO:2 or a fragment
thereof. In other embodiments, the KIF5B-RET fusion polypeptide
includes the amino acid sequence of amino acids 576-977 of SEQ ID
NO:2 or a fragment thereof, or a sequence substantially identical
thereto. For example, the KIF5B-RET fusion polypeptide can include
a RET kinase domain that includes amino acids 587-868 of SEQ ID
NO:2 or a fragment thereof. In yet other embodiments, the KIF5B-RET
fusion polypeptide includes the amino acid sequence shown in FIGS.
3A-3D (e.g., SEQ ID NO:2) or a fragment thereof, or a sequence
substantially identical thereto.
[0030] In yet other embodiments, the KIF5B-RET fusion polypeptide
is encoded by a nucleic acid molecule described herein. In one
embodiment, the KIF5B-RET fusion polypeptide is encoded by an
in-frame fusion of exon 15 of KIF5B with exon 12 of RET (e.g., a
sequence within an 11 MB pericentric inversion on chromosome 10).
In other embodiments, the KIF5B-RET fusion polypeptide is encoded
by a nucleotide sequence in the region of 32,316,376-32,316,416 of
chromosome 10 coupled to (e.g., juxtaposed to) nucleotides in the
region of nucleotides 43,611,042-43,611,118 of chromosome 10. In
another embodiment, the KIF5B-RET fusion polypeptide includes an
amino acid sequence encoded by a nucleotide sequence comprising a
fusion junction between the KIF5B transcript and the RET
transcript, e.g., a nucleotide sequence that includes a portion of
SEQ ID NO:1 (e.g., a nucleotide sequence within exons 1-15 of a
KIF5B gene and 12-20 of a RET gene) (e.g., a portion of SEQ ID NO:1
comprising nucleotides 1720-1731, 1717-1734, or 1714-1737 of SEQ ID
NO:1 (see FIG. 3B)).
[0031] In yet other embodiments, the KIF5B-RET fusion polypeptide
is encoded by a 5'-RET to 3'-KIF5B nucleic acid molecule described
herein. In one embodiment, the KIF5B-RET fusion polypeptide is
encoded by a nucleotide sequence that include a fusion junction
between the RET transcript and the KIF5B transcript, e.g., a
nucleotide sequence within SEQ ID NO:3 (e.g., a sequence comprising
nucleotides 2131-2142, 2128-2145, or 2125-2148 of SEQ ID NO:3 (see
FIG. 4B)). In yet other embodiments, the KIF5B-RET fusion
polypeptide is encoded by the nucleotide sequence shown in FIGS.
4A-4D (e.g., SEQ ID NO:3) or a fragment thereof, or a sequence
substantially identical thereto.
[0032] In an embodiment, the 5'KIF5B-3'RET fusion polypeptide
comprises sufficient KIF5B and sufficient RET sequence such that it
has kinase activity, e.g., has elevated activity, e.g., kinase
activity, as compared with wild type RET, e.g., in a cell of a
cancer referred to herein. In an embodiment the 5'KIF5B-3'RET
fusion polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 9, 10,
or 11 exons from KIF5B and at least 1, 2, 3, 4, 5, 6, 7, 9, or 10,
RET exons. In one embodiment, the 5'KIF5B-3'RET fusion polypeptide
includes a kinesin motor domain or a functional fragment thereof,
and a RET tyrosine kinase domain or a functional fragment thereof.
In a related aspect, the invention features KIF5B-RET fusion
polypeptide or fragments operatively linked to heterologous
polypeptides to form fusion proteins.
[0033] In another embodiment, the KIF5B-RET fusion polypeptide or
fragment is a peptide, e.g., an immunogenic peptide or protein,
that contains a fusion junction described herein. Such immunogenic
peptides or proteins can be used to raise antibodies specific to
the fusion protein. In other embodiments, such immunogenic peptides
or proteins can be used for vaccine preparation. The vaccine
preparation can include other components, e.g., an adjuvant.
[0034] In another aspect, the invention features antibody molecules
that bind to a KIF5B-RET fusion polypeptide or fragment described
herein. In embodiments the antibody can distinguish wild type RET
(or KIF5B) from KIF5B-RET.
Methods Reducing a KIF5B-RET Activity
[0035] In another aspect, the invention features a method of
reducing an activity of a KIF5B-RET fusion. The method includes
contacting the KIF5B-RET fusion, or a KIF5B-RET-expressing cell,
with an agent that inhibits an activity or expression of KIF5B-RET
(e.g., a kinase inhibitor). In one embodiment, the contacting step
can be effected in vitro, e.g., in a cell lysate or in a
reconstituted system. Alternatively, the method can be performed on
cells in culture, e.g., in vitro or ex vivo. In other embodiments,
the method can be performed on KIF5B-RET-expressing cells present
in a subject, e.g., as part of an in vivo (e.g., therapeutic or
prophylactic) protocol. In an embodiment the method is practiced on
an animal subject (e.g., an in vivo animal model). In certain
embodiments, the KIF5B-RET fusion is a nucleic acid molecule or a
polypeptide as described herein.
[0036] In a related aspect, a method of inhibiting, reducing, or
treating a hyperproliferative disorder, e.g., a cancer, in a
subject is provided. The method includes administering to the
subject a preselected therapeutic agent, e.g., an anti-cancer agent
(e.g., a kinase inhibitor), as a single agent, or in combination,
in an amount sufficient to reduce, inhibit or treat the activity or
expression of KIF5B-RET (e.g., a KIF5B-RET fusion described
herein), thereby inhibiting, reducing, or treating the
hyperproliferative disorder in the subject. "Treatment" as used
herein includes, but is not limited to, inhibiting tumor growth,
reducing tumor mass, reducing size or number of metastatic lesions,
inhibiting the development of new metastatic lesions, prolonged
survival, prolonged progression-free survival, prolonged time to
progression, and/or enhanced quality of life.
[0037] In one embodiment, the kinase inhibitor is administered
based on a determination that a KIF5B-RET fusion is present in a
subject, e.g., based on its present in a subject's sample. Thus,
treatment can be combined with a KIF5B-RET detection or evaluation
method, e.g., as described herein, or administered in response to a
determination made by a KIF5B-RET detection or evaluation method,
e.g., as described herein. In certain embodiments, the kinase
inhibitor is administered responsive to acquiring knowledge or
information of the presence of the KIF5B-RET fusion in a subject.
In one embodiment, the kinase inhibitor is administered responsive
to acquiring knowledge or information on the subject's genotype,
e.g., acquiring knowledge or information that the patient's
genotype has a KIF5B-RET fusion. In other embodiments, the kinase
inhibitor is administered responsive to receiving a communication
(e.g., a report) of the presence of the KIF5B-RET fusion in a
subject (e.g., a subject's sample). In yet other embodiments, the
kinase inhibitor is administered responsive to information obtained
from a collaboration with another party that identifies the
presence of the KIF5B-RET fusion in a subject (e.g., a subject's
sample). In other embodiments, the kinase inhibitor is administered
responsive to a determination that the KIF5B-RET fusion is present
in a subject. In one embodiment, the determination of the presence
of the KIF5B-RET fusion is carried out using one or more of the
methods, e.g., the sequencing methods, described herein. In other
embodiments, the determination of the presence of the KIF5B-RET
fusion includes receiving information on the subject's KIF5B-RET
fusion genotype, e.g., from another party or source.
[0038] The methods can, optionally, further include the step(s) of
identifying (e.g., evaluating, diagnosing, screening, and/or
selecting) a subject at risk of having, or having, a KIF5B-RET
fusion. In one embodiment, the method further includes one or more
of: acquiring knowledge or information of the presence of the
KIF5B-RET fusion in a subject (e.g., a subject's sample); acquiring
knowledge or information on the subject's genotype, e.g., acquiring
knowledge or information that the patient's genotype has a
KIF5B-RET fusion; receiving a communication (e.g., a report) of the
presence of the KIF5B-RET fusion in a subject (e.g., a subject's
sample); or collaborating with another party that identifies the
presence of the KIF5B-RET fusion in a subject.
[0039] In one embodiment, the subject treated has a KIF5B-RET
fusion; e.g., the subject has a tumor or cancer harboring a
KIF5B-RET fusion. In other embodiments, the subject has been
previously identified as having a KIF5B-RET fusion. In yet other
embodiments, the subject has been previously identified as being
likely or unlikely to respond to treatment with a protein kinase
inhibitor, e.g., a subject that has previously participated in a
clinical trial. In other embodiments, the subject has been
previously identified as being likely or unlikely to respond to
treatment with a protein kinase inhibitor, based on the presence of
the KIF5B-RET fusion. In one embodiment, the subject is a mammal,
e.g., a human. In one embodiment, the subject has, or at risk of
having a cancer at any stage of disease. In other embodiments, the
subject is a patient, e.g., a cancer patient.
[0040] In other embodiments, the subject treated is a cancer
patient who has participated in a clinical trial. For example, the
subject participated in a clinical trial that evaluated a kinase
inhibitor (e.g., a multikinase inhibitor, a RET kinase inhibitor).
In other embodiment, the subject participated in a clinical trial
that evaluates upstream or downstream targets of RET. In one
embodiment, said cancer patient responded to the kinase inhibitor
evaluated.
[0041] In certain embodiments, the cancer is a solid tumor, a soft
tissue tumor, or a metastatic lesion. In one embodiment, the cancer
is chosen from a lung cancer, a pancreatic cancer, melanoma, a
colorectal cancer, an esophageal-gastric cancer, a thyroid cancer,
or an adenocarcinoma. In other embodiment, the lung cancer is
chosen from one or more of the following: non-small cell lung
cancer (NSCLC), small cell lung cancer (SCLC), squamous cell
carcinoma (SCC), adenocarcinoma of the lung, bronchogenic
carcinoma, or a combination thereof. In one embodiment, the lung
cancer is NSCLC or SCC.
[0042] In one embodiment, the anti-cancer agent is a kinase
inhibitor. For example, the kinase inhibitor is a multi-kinase
inhibitor or a RET-specific inhibitor. In one embodiment, the
kinase inhibitor is chosen from lenvatinib (E7080) (Eisai Co.),
sorafenib (NEXAVAR.RTM.), sunitinib (SUTENT.RTM., SU11248),
vandetanib (CAPRELSA.RTM., ZACTIMA.RTM., ZD6474), NVP-AST487,
regorafenib (BAY-73-4506), motesanib (AMG 706), cabozantinib
(XL-184), apatinib (YN-968D1), or DCC-2157.
[0043] In other embodiments, the anti-cancer agent is a KIF5B-RET
antagonist inhibits the expression of nucleic acid encoding
KIF5B-RET. Examples of such KIF5B-RET antagonists include nucleic
acid molecules, for example, antisense molecules, ribozymes, RNAi,
triple helix molecules that hybridize to a nucleic acid encoding
KIF5B-RET, or a transcription regulatory region, and blocks or
reduces mRNA expression of KIF5B-RET.
[0044] In other embodiments, the kinase inhibitor is administered
in combination with a second therapeutic agent or a different
therapeutic modality, e.g., anti-cancer agents, and/or in
combination with surgical and/or radiation procedures. For example,
the second therapeutic agent can be a cytotoxic or a cytostatic
agent. Exemplary cytotoxic agents include antimicrotubule agents,
topoisomerase inhibitors, or taxanes, antimetabolites, mitotic
inhibitors, alkylating agents, intercalating agents, agents capable
of interfering with a signal transduction pathway, agents that
promote apoptosis and radiation. In yet other embodiments, the
methods can be used in combination with immunodulatory agents,
e.g., IL-1, 2, 4, 6, or 12, or interferon alpha or gamma, or immune
cell growth factors such as GM-CSF.
Screening Methods
[0045] In another aspect, the invention features a method, or
assay, for screening for agents that modulate, e.g., inhibit, the
expression or activity of a KIF5B-RET fusion, e.g., a KIF5B-RET
fusion as described herein. The method includes contacting a
KIF5B-RET fusion, or a cell expressing a KIF5B-RET fusion, with a
candidate agent; and detecting a change in a parameter associated
with a KIF5B-RET fusion, e.g., a change in the expression or an
activity of the KIF5B-RET 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 KIF5B-RET fusion is
detected, the candidate agent is identified as an inhibitor. In
another embodiment, if an increase in expression or activity of the
KIF5B-RET fusion is detected, the candidate agent is identified as
an activator. In certain embodiments, the KIF5B-RET fusion is a
nucleic acid molecule or a polypeptide as described herein.
[0046] In one embodiment, the contacting step is effected in a
cell-free system, e.g., a cell lysate or in a reconstituted system.
In other embodiments, the contacting step is effected in a cell in
culture, e.g., a cell expressing a KIF5B-RET fusion (e.g., a
mammalian cell, a tumor cell or cell line, a recombinant cell). In
yet other embodiments, the contacting step is effected in a cell in
vivo (a KIF5B-RET-expressing cell present in a subject, e.g., an
animal subject (e.g., an in vivo animal model).
[0047] Exemplary parameters evaluated include one or more of:
[0048] (i) a change in binding activity, e.g., direct binding of
the candidate agent to a KIF5B-RET fusion polypeptide; a binding
competition between a known ligand and the candidate agent to a
KIF5B-RET fusion polypeptide;
[0049] (ii) a change in kinase activity, e.g., phosphorylation
levels of a KIF5B-RET fusion polypeptide (e.g., an increased or
decreased autophosphorylation); or a change in phosphorylation of a
target of a RET kinase, e.g., focal adhesion kinase (FAK),
persephin or glial derived neurotrophic factor (GDNF), In certain
embodiments, a change in kinase activity, e.g., phosphorylation, is
detected by any of Western blot (e.g., using an anti-KIF5B or
anti-RET antibody; a phosphor-specific antibody, detecting a shift
in the molecular weight of a KIF5B-RET fusion polypeptide), mass
spectrometry, immunoprecipitation, immunohistochemistry,
immunomagnetic beads, among others;
[0050] (iii) a change in an activity of a cell containing a
KIF5B-RET fusion (e.g., a tumor cell or a recombinant cell), e.g.,
a change in proliferation, morphology or tumorigenicity of the
cell;
[0051] (iv) a change in tumor present in an animal subject, e.g.,
size, appearance, proliferation, of the tumor; or
[0052] (v) a change in the level, e.g., expression level, of a
KIF5B-RET fusion polypeptide or nucleic acid molecule.
[0053] In one embodiment, a change in a cell free assay in the
presence of a candidate agent is evaluated. For example, an
activity of a KIF5B-RET fusion, or interaction of a KIF5B-RET
fusion with a downstream ligand can be detected. In one embodiment,
a KIF5B-RET fusion polypeptide is contacted with a ligand, e.g., in
solution, and a candidate agent is monitored for an ability to
modulate, e.g., inhibit, an interaction, e.g., binding, between the
KIF5B-RET fusion polypeptide and the ligand.
[0054] In other embodiments, a change in an activity of a cell is
detected in a cell in culture, e.g., a cell expressing a KIF5B-RET
fusion (e.g., a mammalian cell, a tumor cell or cell line, a
recombinant cell). In one embodiment, the cell is a recombinant
cell that is modified to express a KIF5B-RET fusion nucleic acid,
e.g., is a recombinant cell transfected with a KIF5B-RET fusion
nucleic acid. The transfected cell can show a change in response to
the expressed KIF5B-RET 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, transformed morphology, in the
presence of the candidate agent can be indicative of an inhibitor
of a KIF5B-RET fusion. In other embodiments, a change in binding
activity or phosphorylation as described herein is detected.
[0055] 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, the animal model is a tumor containing animal or a
xenograft comprising cells expressing a KIF5B-RET fusion (e.g.,
tumorigenic cells expressing a KIF5B-RET fusion). The candidate
agent can be administered to the animal subject and a change in the
tumor is detected. In one embodiment, the change in the tumor
includes one or more of a tumor growth, tumor size, tumor burden,
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.
[0056] In other embodiments, a change in expression of a KIF5B-RET
fusion can be monitored by detecting the nucleic acid or protein
levels, e.g., using the methods described herein.
[0057] In certain embodiments, the screening methods described
herein can be repeated and/or combined. In one embodiment, a
candidate agent that is evaluated in a cell-free or cell-based
described herein can be further tested in an animal subject.
[0058] In one embodiment, the candidate agent is a small molecule
compound, e.g., a kinase inhibitor, a nucleic acid (e.g.,
antisense, siRNA, aptamer, ribozymes, microRNA), an antibody
molecule (e.g., a full antibody or antigen binding fragment thereof
that binds to KIF5B5 or RET). The candidate agent can be obtained
from a library (e.g., a commercial library of kinase inhibitors) or
rationally designed (e.g., based on the RET kinase domain).
Methods for Detecting KIF5B-RET Fusions
[0059] In another aspect, the invention features a method of
determining the presence of a KIF5B-RET fusion, e.g., a KIF5B-RET
fusion as described herein. In one embodiment, the KIF5B-RET fusion
is detected in a nucleic acid molecule or a polypeptide. The method
includes detecting whether a KIF5B-RET fusion nucleic acid molecule
or polypeptide is present in a cell (e.g., a circulating 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.
[0060] In one embodiment, the sample is, or has been, classified as
non-malignant using other diagnostic techniques, e.g.,
immunohistochemistry.
[0061] In one embodiment, the sample is acquired from a subject
(e.g., a subject having or at risk of having a cancer, e.g., a
patient), or alternatively, the method further includes acquiring a
sample from the subject. The sample can be chosen from one or more
of: tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole
blood, serum, plasma, buccal scrape, sputum, saliva, cerebrospinal
fluid, urine, stool, circulating tumor cells, circulating nucleic
acids, or bone marrow. In certain embodiments, the sample is a
tissue (e.g., a tumor biopsy), a circulating tumor cell or nucleic
acid.
[0062] In embodiments, the tumor is from a cancer described herein,
e.g., is chosen from a lung cancer, a colorectal cancer, an
esophageal-gastric cancer, a thyroid cancer, an adenocarcinoma or a
melanoma. In one embodiment, the tumor is from a lung cancer, e.g.,
a NSCLC, a SCLC, a SCC, or a combination thereof.
[0063] In one embodiment, the subject is at risk of having, or has
a cancer (e.g., a patient with a cancer described herein). For
example, in one embodiment, the subject is at risk for having, or
has a lung cancer.
[0064] In other embodiments, the KIF5B-RET fusion is detected in a
nucleic acid molecule by a method chosen from one or more of:
nucleic acid hybridization assay, amplification-based assays (e.g.,
polymerase chain reaction (PCR)), PCR-RFLP assay, real-time PCR,
sequencing, screening analysis (including metaphase cytogenetic
analysis by standard karyotype methods, FISH (e.g., break away
FISH), spectral karyotyping or MFISH, comparative genomic
hybridization), in situ hybridization, SSP, HPLC or
mass-spectrometric genotyping.
[0065] 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, cDNA), obtained from the subject, with a
nucleic acid fragment (e.g., a probe or primer as described herein
(e.g., an exon-specific probe or primer) under conditions suitable
for hybridization, and determining the presence or absence of the
KIF5B-RET nucleic acid molecule. The method can, optionally,
include enriching a sample for the gene or gene product.
[0066] In a related aspect, a method for determining the presence
of a KIF5B-RET fusion nucleic acid molecule is provided. The method
includes: acquiring a sequence for a position in a nucleic acid
molecule, e.g., by sequencing at least one nucleotide of the
nucleic acid molecule (e.g., sequencing at least one nucleotide in
the nucleic acid molecule that comprises the KIF5B-RET fusion),
thereby determining that the KIF5B-RET 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 nucleic acid molecule is from a
cell (e.g., a circulating cell), a tissue (e.g., a tumor), or any
sample from a subject (e.g., blood or plasma sample). In other
embodiments, the nucleic acid molecule from a tumor sample (e.g., a
tumor or cancer sample) is sequenced. In one embodiment, the
sequence is determined by a next generation sequencing method. The
method further can further include acquiring, e.g., directly or
indirectly acquiring, a sample, e.g., a tumor or cancer sample,
from a subject (e.g., a patient). In certain embodiments, the
cancer is chosen from a lung cancer, colorectal cancer,
esophageal-gastric cancer or melanoma.
[0067] In another aspect, the invention features a method of
analyzing a tumor or a circulating tumor cell. The method includes
acquiring a nucleic acid sample from the tumor or the circulating
cell; and sequencing, e.g., by a next generation sequencing method,
a nucleic acid molecule, e.g., a nucleic acid molecule that
includes a KIF5B-RET fusion described herein.
[0068] In yet another embodiment, a KIF5B-RET fusion polypeptide is
detected. The method includes: contacting a protein sample with a
reagent which specifically binds to a KIF5B-RET fusion polypeptide;
and detecting the formation of a complex of the KIF5B-RET fusion
polypeptide and the reagent. In one embodiment, the reagent is
labeled with a detectable group to facilitate detection of the
bound and unbound reagent. In one embodiment, the reagent is an
antibody molecule, e.g., is selected from the group consisting of
an antibody, and antibody derivative, and an antibody fragment.
[0069] In yet another embodiment, the level (e.g., expression
level) or activity the KIF5B-RET fusion is evaluated. For example,
the level (e.g., expression level) or activity of the KIF5B-RET
fusion (e.g., mRNA or polypeptide) is detected and (optionally)
compared to a pre-determined value, e.g., a reference value (e.g.,
a control sample).
[0070] In yet another embodiment, the KIF5B-RET fusion is detected
prior to initiating, during, or after, a treatment in a subject,
e.g., a treatment with a kinase inhibitor. In one embodiment, the
KIF5B-RET fusion is detected at the time of diagnosis with a
cancer. In other embodiment, the KIF5B-RET fusion is detected at a
pre-determined interval, e.g., a first point in time and at least
at a subsequent point in time.
[0071] In certain embodiments, responsive to a determination of the
presence of the KIF5B-RET fusion, the method further includes one
or more of:
[0072] (1) stratifying a patient population (e.g., assigning a
subject, e.g., a patient, to a group or class);
[0073] (2) identifying or selecting the subject as likely or
unlikely to respond to a treatment, e.g., a kinase inhibitor
treatment as described herein;
[0074] (3) selecting a treatment option, e.g., administering or not
administering a preselected therapeutic agent, e.g., a kinase
inhibitor as described herein; or
[0075] (4) prognosticating the time course of the disease in the
subject (e.g., evaluating the likelihood of increased or decreased
patient survival).
[0076] In certain embodiments, the kinase inhibitor is a
multi-kinase inhibitor or a RET-specific inhibitor. In one
embodiment, the kinase inhibitor is chosen from lenvatinib (E7080),
sorafenib (NEXAVAR.RTM.), sunitinib (SUTENT.RTM., SU11248),
vandetanib (CAPRELSA.RTM., ZACTIMA.RTM., ZD6474), NVP-AST487,
regorafenib (BAY-73-4506), motesanib (AMG 706), cabozantinib
(XL-184), apatinib (YN-968D1), or DCC-2157.
[0077] In certain embodiments, responsive to the determination of
the presence of the KIF5B-RET fusion, the subject is classified as
a candidate to receive treatment with a kinase inhibitor, e.g., a
kinase inhibitor as described herein. In one embodiment, responsive
to the determination of the presence of the KIF5B-RET fusion, the
subject, e.g., a patient, can further be assigned to a particular
class if a KIF5B-RET fusion is identified in a sample of the
patient. For example, a patient identified as having a KIF5B-RET
fusion can be classified as a candidate to receive treatment with a
kinase inhibitor, e.g., a kinase inhibitor as described herein. In
one embodiment, the subject, e.g., a patient, is assigned to a
second class if the mutation is not present. For example, a patient
who has a lung tumor that does not contain a KIF5B-RET fusion, may
be determined as not being a candidate to receive a kinase
inhibitor, e.g., a kinase inhibitor as described herein.
[0078] In another embodiment, responsive to the determination of
the presence of the KIF5B-RET fusion, the subject is identified as
likely to respond to a treatment that comprises a kinase inhibitor
e.g., a kinase inhibitor as described herein.
[0079] In yet another embodiment, responsive to the determination
of the presence of the KIF5B-RET fusion, the method includes
administering a kinase inhibitor, e.g., a kinase inhibitor as
described herein, to the subject.
Method of Evaluating a Tumor or a Subject
[0080] In another aspect, the invention features a method of
evaluating a subject (e.g., a patient), e.g., for risk of having or
developing a cancer, e.g., a lung cancer, colorectal cancer or skin
cancer. The method includes: acquiring information or knowledge of
the presence of a KIF5B-RET fusion in a subject (e.g., acquiring
genotype information of the subject that identifies a KIF5B-RET
fusion as being present in the subject); acquiring a sequence for a
nucleic acid molecule identified herein (e.g., a nucleic acid
molecule that includes a KIF5B-RET fusion sequence); or detecting
the presence of a KIF5B-RET fusion nucleic acid or polypeptide in
the subject), wherein the presence of the KIF5B-RET fusion is
positively correlated with increased risk for, or having, a cancer
associated with the KIF5B-RET fusion.
[0081] The method can further include the step(s) of identifying
(e.g., evaluating, diagnosing, screening, and/or selecting) the
subject as being positively correlated with increased risk for, or
having, a cancer associated with the KIF5B-RET fusion. In one
embodiment, the subject is identified or selected as likely or
unlikely to respond to a treatment, e.g., a kinase inhibitor
treatment as described herein.
[0082] The method can further include treating the subject with a
kinase inhibitor, e.g., a kinase inhibitor as described herein.
[0083] The method can further include acquiring, e.g., directly or
indirectly, a sample from a patient and evaluating the sample for
the present of a KIF5B-RET fusion as described herein.
[0084] In certain embodiments, the subject is a patient or patient
population that has participated in a clinical trial. In one
embodiment, the subject has participated in a clinical trial for
evaluating a kinase inhibitor (e.g., a multikinase inhibitor or a
RET inhibitor). In one embodiment, the clinical trial is
discontinued or terminated. In other embodiments, the subject has
participated in a clinical trial that evaluates a RET kinase, a
KIF5B inhibitor (e.g., a kinesin inhibitor), an upstream or
downstream component of RET or KIF5B. In one embodiment, the
subject responded favorably to the clinical trial, e.g., experience
an improvement in at least one symptom of a cancer (e.g., decreased
in tumor size, rate of tumor growth, increased survival). In other
embodiments, the subject did not respond in a detectable way to the
clinical trial.
[0085] In a related aspect, a method of evaluating a patient or a
patient population is provided. The method includes: identifying,
selecting, or obtaining information or knowledge that the patient
or patient population has participated in a clinical trial;
[0086] acquiring information or knowledge of the presence of a
KIF5B-RET fusion in the patient or patient population (e.g.,
acquiring genotype information of the subject that identifies a
KIF5B-RET fusion as being present in the subject); acquiring a
sequence for a nucleic acid molecule identified herein (e.g., a
nucleic acid molecule that includes a KIF5B-RET fusion sequence);
or detecting the presence of a KIF5B-RET fusion nucleic acid or
polypeptide in the subject), wherein the presence of the KIF5B-RET
fusion is identifies the patient or patient population as having an
increased risk for, or having, a cancer associated with the
KIF5B-RET fusion.
[0087] In certain embodiments, the subject is a patient or patient
population that has participated in a clinical trial. In one
embodiment, the subject has participated in a clinical trial for
evaluating a kinase inhibitor (e.g., a multikinase inhibitor or a
RET inhibitor). In one embodiment, the clinical trial is
discontinued or terminated. In other embodiments, the subject has
participated in a clinical trial that evaluates a RET kinase, a
KIF5B inhibitor (e.g., a kinesin inhibitor), an upstream or
downstream component of RET or KIF5B. In one embodiment, the
subject responded favorably to the clinical trial, e.g., experience
an improvement in at least one symptom of a cancer (e.g., decreased
in tumor size, rate of tumor growth, increased survival). In other
embodiments, the subject did not respond in a detectable way to the
clinical trial.
[0088] In embodiments, the method further includes treating the
subject with a kinase inhibitor, e.g., a kinase inhibitor as
described herein.
Reporting
[0089] Methods described herein can include providing a report,
such as, in electronic, web-based, or paper form, to the patient or
to another person or entity, e.g., a caregiver, e.g., a physician,
e.g., an oncologist, a hospital, clinic, third-party payor,
insurance company or government office. The report can include
output from the method, e.g., the identification of nucleotide
values, the indication of presence or absence of a KIF5B-RET fusion
as described herein, or wildtype sequence. In one embodiment, a
report is generated, such as in paper or electronic form, which
identifies the presence or absence of an alteration described
herein, and optionally includes an identifier for the patient from
which the sequence was obtained.
[0090] The report can also include information on the role of a
sequence, e.g., a KIF5B-RET fusion as described herein, or wildtype
sequence, in disease. Such information can include information on
prognosis, resistance, or potential or suggested therapeutic
options. The report can include information on the likely
effectiveness of a therapeutic option, the acceptability of a
therapeutic option, or the advisability of applying the therapeutic
option to a patient, e.g., a patient having a sequence, alteration
or mutation identified in the test, and in embodiments, identified
in the report. For example, the report can include information, or
a recommendation on, the administration of a drug, e.g., the
administration at a preselected dosage or in a preselected
treatment regimen, e.g., in combination with other drugs, to the
patient. In an embodiment, not all mutations identified in the
method are identified in the report. For example, the report can be
limited to mutations in genes having a preselected level of
correlation with the occurrence, prognosis, stage, or
susceptibility of the cancer to treatment, e.g., with a preselected
therapeutic option. The report can be delivered, e.g., to an entity
described herein, within 7, 14, or 21 days from receipt of the
sample by the entity practicing the method.
[0091] In another aspect, the invention features a method for
generating a report, e.g., a personalized cancer treatment report,
by obtaining a sample, e.g., a tumor sample, from a subject,
detecting a KIF5B-RET fusion as described herein in the sample, and
selecting a treatment based on the mutation identified. In one
embodiment, a report is generated that annotates the selected
treatment, or that lists, e.g., in order of preference, two or more
treatment options based on the mutation identified. In another
embodiment, the subject, e.g., a patient, is further administered
the selected method of treatment.
[0092] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
the example are illustrative only and not intended to be
limiting.
[0093] The details of one or more embodiments featured in the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages featured
in the invention will be apparent from the description and
drawings, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0094] FIG. 1 is a snapshot of the sequencing reads illustrating
that there are RET read pairs that map on one end to 5'KIF5B and on
the other end to 3'RET, and other RET read pairs that map on one
end to 5'RET and on the other end to 3'KIFB.
[0095] FIGS. 2A and 2B are schematic diagrams illustrating the
inversion event that brings together exon 15 of KIF5B and exon 12
of RET.
[0096] FIGS. 3A to 3D are the predicted cDNA sequence (SEQ ID NO:1)
and protein sequence of the KIF5B-RET fusion (SEQ ID NO:2). The
KIF5B portion of the cDNA sequence is equivalent to nucleotides
471-2195 of RefSeq NM_004521.2, and the KIF5B portion of the
protein sequence is equivalent to amino acids 1-575 of RefSeq
NP_004512. The KIF5B portions of the cDNA and protein sequences are
underlined. The RET portion of the cDNA sequence is equivalent to
nucleotides 2327-3535 of RefSeq NM_020975, and the RET portion of
the protein sequence is equivalent to amino acids 713-1114 of
RefSeq NP_066124.1. The intact tyrosine kinase domain of RET is
represented by a gray box (see FIG. 3C).
[0097] FIGS. 4A to 4D are the predicted cDNA sequence (SEQ ID NO:3)
and protein sequence of the RET-KIF5B fusion (SEQ ID NO:4). The
cDNA sequence of the RET portion of the fusion transcript is
equivalent to nucleotides 190-2326 of RefSeq NM_020975, and the
protein sequence is equivalent to amino acids 1-712 of RefSeq
NP_066124.1. The cDNA sequence of the KIF5B portion of the fusion
transcript is equivalent to nucleotides 2196-3362 of RefSeq
NM_004521.2, and the protein portion is equivalent to amino acids
576-963 of RefSeq NP_004512. The KIF5B portions of the cDNA and
protein sequences are underlined.
[0098] FIG. 5 is a summary of the DNA alterations identified in
colorectal cancer cells.
[0099] FIG. 6A provides a schematic representation of the
11,294,741 bp inversion in NSCLC that generates an in-frame
KIF5B-RET gene fusion (not to scale). The inverted region of
chromosome 10 starts at 32,316,377 bps (within KIF5B intron 15) and
ends at 43,611,118 bps (within RET intron 11).
[0100] FIG. 6B is a schematic of the protein domain structure of
the RET-KIF5B and KIF5B-RET gene fusions. The cadherin domain of
RET is included in the predicted RET-KIF5B protein. The kinesin and
coiled coil domains of KIF5B and the tyrosine kinase domain of RET
are included in the KIF5B-RET fusion protein. RT-PCR of primer
designed to RET exon 11 and KIF5B exon 16 yielded no product.
RT-PCR of primer designed to KIF5B exon 15 and RET exon 12 yielded
a strong product (data not shown).
[0101] FIG. 6C is a photograph depicting the distribution of RET
expression in the case of NSCLC with confirmed RET fusion on DNA
sequencing by NGS. Note focal moderate cytoplasmic immunoreactivity
for RET protein expression (avidin-biotin perxodase.times.200).
High magnification detail of cytoplasmic immunostaining for RET is
shown in the insert at the lower right.
[0102] FIG. 6D is a summary of the exons present or absent in the
KIF5B-RET fusion.
[0103] FIG. 7A provides another schematic representation of the
KIF5B-RET fusion. This variant was identified in 8 CRC cases, where
KIF5B exon 15 is fused in-frame to RET exon 12. The predicted full
length fusion protein is 977 amino acids in length, with amino
acids 1-575 derived from KIF5B and amino acids 576-977 derived from
RET (shown above). Capillary sequence confirmation of the exon
junction boundaries derived from cDNA is shown below.
[0104] FIG. 7B is another representation of the predicted KIF5B-RET
variant amino acid sequence.
[0105] FIG. 8 is a graph showing the effects of different drugs on
Ba/F3 cells transfected with KIF5B-RET fusion constructs.
[0106] FIG. 9 is a Western blot showing the effects of sunitinib
and gefitinib on RET phosphorylation in Ba/R3 cells expressing
KIF5B-RET.
[0107] The Tables are described herein.
[0108] Table 1 is a summary of the distribution of 125 CRC
mutations across 21 mutated cancer genes
[0109] Table 2 provides a summary of NSCLC patients analyzed by RET
immunohistochemistry.
[0110] Supplementary Table 1a provides a listing of the 145 genes
sequenced across the entire coding sequence.
[0111] Supplementary Table 1b provides a listing of the 14 genes
sequenced across selected introns.
[0112] Supplementary Table 2a provides a detailed summary of the
alterations detected in 40 colorectal cancer cases.
[0113] Supplementary Table 2b provides a detailed summary of the
alterations detected in 24 NSCLC cases.
[0114] Supplementary Table 3 provides a summary of the alterations
that could be linked to a clinical treatment option or a clinical
trial of novel targeted therapies.
[0115] Supplementary Table 4 provides a distribution of 51
mutations across 21 mutated NSCLC genes.
[0116] Supplementary Table 5 provides a summary of NSCLC
alterations that could be linked to a clinical treatment option or
clinical trial or novel targeted therapies.
DETAILED DESCRIPTION
[0117] The invention is based, at least in part, on the discovery
of a novel chromosomal inversion event and its association with
cancer, e.g., lung cancer. In one embodiment, Applicants have
discovered an inversion on chromosome 10 that results in an
in-frame fusion of a fragment of a KIF5B gene and a fragment of a
RET gene.
[0118] The term "KIF5B-RET" or "KIF5B-RET fusion" is used
generically herein, and includes any fusion molecule (e.g., gene,
gene product (e.g., cDNA, mRNA, polypeptide), and variant thereof)
that includes a fragment of KIF5B and a fragment of RET, in any
configuration, including, e.g., a 5'KIF5B-3'RET or a 5'RET-3'KIF5B
fusion molecule.
[0119] In one embodiment, a KIF5B-RET fusion includes an in-frame
fusion of an exon of KIF5B (e.g., one more exons encoding a kinesin
motor domain or a fragment thereof) and an exon of RET (e.g., one
or more exons encoding a RET tyrosine kinase domain or a fragment
thereof). For example, the KIF5B-RET fusion can include an in-frame
fusion of at least exon 15 of KIF5B or a fragment thereof (e.g.,
exons 1-15 of KIF5B or a fragment thereof) with at least exon 12 of
RET or a fragment thereof (e.g., exons 12-20 of RET or a fragment
thereof). In certain embodiments, the KIF5B-RET fusion is in a
5'-KIF5B to 3'-RET configuration referred to herein as
"5'KIF5B-3'RET."
[0120] In other embodiments, the KIF5B-RET fusion includes an
in-frame fusion of at least exon 11 of RET or a fragment thereof
(e.g., exons 1-11 of RET or a fragment thereof) with at least exon
16 or a fragment thereof (e.g., exons 16-25 of KIF5B or a fragment
thereof). In certain embodiments, the KIF5B-RET fusion is in a
5'-RET to 3'-KIF5B configuration referred to herein as
"5'RET-3'KIF5B").
[0121] The RET proto-oncogene is known to be associated with
cancerous phenotypes, including papillary thyroid carcinomas (PTC),
multiple endocrine neoplasias (MEN), phaeochromocytoma, among
others. For example, chromosomal rearrangements that generate a
fusion gene resulting in the juxtaposition of the C-terminal region
of the RET protein with an N-terminal portion of another protein
(known as RET/PTC) are known to be associated with PTC (Nikiforov,
Y E (2002) Endocr. Pathol. 13 (1):3-16). Thus, the KIF5B-RET
fusions disclosed herein (e.g., the 5'-KIF5B to 3'-RET fusions that
include a RET tyrosine kinase domain) are likely to be associated
with cancers, e.g., lung cancer.
[0122] Accordingly, the invention provides, at least in part,
isolated KIF5B-RET nucleic acid molecules, nucleic acid constructs,
host cells containing the nucleic acid molecules; purified
KIF5B-RET polypeptides and binding agents; detection reagents
(e.g., probes, primers, antibodies, kits); screening assays for
identifying novel kinase inhibitors; as well as methods, assays and
kits for evaluating, identifying, assessing and/or treating a
subject having a cancer, e.g., a cancer having a KIF5B-RET fusion
disclosed herein. The compositions and methods identified herein
can be used, for example, to identify new KIF5B-RET inhibitors; to
treat or prevent a cancer; as well as in methods or assays for
evaluating a cancer (e.g., evaluating one or more of: cancer
progression, cancer treatment response or resistance to cancer
treatment; selecting a treatment option, stratifying a patient
population, and/or more effectively monitoring, treating or
preventing a cancer).
[0123] Certain terms are first defined. Additional terms are
defined throughout the specification.
[0124] As used herein, the articles "a" and "an" refer to one or to
more than one (e.g., to at least one) of the grammatical object of
the article.
[0125] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or", unless context clearly
indicates otherwise.
[0126] "About" and "approximately" shall generally mean an
acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Exemplary degrees of error
are within 20 percent (%), typically, within 10%, and more
typically, within 5% of a given value or range of values.
[0127] "Acquire" or "acquiring" as the terms are used herein, refer
to obtaining possession of a physical entity, or a value, e.g., a
numerical value, by "directly acquiring" or "indirectly acquiring"
the physical entity or value. "Directly acquiring" means performing
a process (e.g., performing a synthetic or analytical method) to
obtain the physical entity or value. "Indirectly acquiring" refers
to receiving the physical entity or value from another party or
source (e.g., a third party laboratory that directly acquired the
physical entity or value). Directly acquiring a physical entity
includes performing a process that includes a physical change in a
physical substance, e.g., a starting material. Exemplary changes
include making a physical entity from two or more starting
materials, shearing or fragmenting a substance, separating or
purifying a substance, combining two or more separate entities into
a mixture, performing a chemical reaction that includes breaking or
forming a covalent or non-covalent bond. Directly acquiring a value
includes performing a process that includes a physical change in a
sample or another substance, e.g., performing an analytical process
which includes a physical change in a substance, e.g., a sample,
analyte, or reagent (sometimes referred to herein as "physical
analysis"), performing an analytical method, e.g., a method which
includes one or more of the following: separating or purifying a
substance, e.g., an analyte, or a fragment or other derivative
thereof, from another substance; combining an analyte, or fragment
or other derivative thereof, with another substance, e.g., a
buffer, solvent, or reactant; or changing the structure of an
analyte, or a fragment or other derivative thereof, e.g., by
breaking or forming a covalent or non-covalent bond, between a
first and a second atom of the analyte; or by changing the
structure of a reagent, or a fragment or other derivative thereof,
e.g., by breaking or forming a covalent or non-covalent bond,
between a first and a second atom of the reagent.
[0128] "Acquiring a sequence" as the term is used herein, refers to
obtaining possession of a nucleotide sequence or amino acid
sequence, by "directly acquiring" or "indirectly acquiring" the
sequence. "Directly acquiring a sequence" means performing a
process (e.g., performing a synthetic or analytical method) to
obtain the sequence, such as performing a sequencing method (e.g.,
a Next Generation Sequencing (NGS) method). "Indirectly acquiring a
sequence" refers to receiving information or knowledge of, or
receiving, the sequence from another party or source (e.g., a third
party laboratory that directly acquired the sequence). The sequence
acquired need not be a full sequence, e.g., sequencing of at least
one nucleotide, or obtaining information or knowledge, that
identifies a KIF5B-RET fusion disclosed herein as being present in
a subject constitutes acquiring a sequence.
[0129] Directly acquiring a sequence includes performing a process
that includes a physical change in a physical substance, e.g., a
starting material, such as a tissue sample, e.g., a biopsy, or an
isolated nucleic acid (e.g., DNA or RNA) sample. Exemplary changes
include making a physical entity from two or more starting
materials, shearing or fragmenting a substance, such as a genomic
DNA fragment; separating or purifying a substance (e.g., isolating
a nucleic acid sample from a tissue); combining two or more
separate entities into a mixture, performing a chemical reaction
that includes breaking or forming a covalent or non-covalent bond.
Directly acquiring a value includes performing a process that
includes a physical change in a sample or another substance as
described above.
[0130] "Acquiring a sample" as the term is used herein, refers to
obtaining possession of a sample, e.g., a tissue sample or nucleic
acid sample, by "directly acquiring" or "indirectly acquiring" the
sample. "Directly acquiring a sample" means performing a process
(e.g., performing a physical method such as a surgery or
extraction) to obtain the sample. "Indirectly acquiring a sample"
refers to receiving the sample from another party or source (e.g.,
a third party laboratory that directly acquired the sample).
Directly acquiring a sample includes performing a process that
includes a physical change in a physical substance, e.g., a
starting material, such as a tissue, e.g., a tissue in a human
patient or a tissue that has was previously isolated from a
patient. Exemplary changes include making a physical entity from a
starting material, dissecting or scraping a tissue; separating or
purifying a substance (e.g., a sample tissue or a nucleic acid
sample); combining two or more separate entities into a mixture;
performing a chemical reaction that includes breaking or forming a
covalent or non-covalent bond. Directly acquiring a sample includes
performing a process that includes a physical change in a sample or
another substance, e.g., as described above.
[0131] "Binding entity" means any molecule to which molecular tags
can be directly or indirectly attached that is capable of
specifically binding to an analyte. The binding entity can be an
affinity tag on a nucleic acid sequence. In certain embodiments,
the binding entity allows for separation of the nucleic acid from a
mixture, such as an avidin molecule, or an antibody that binds to
the hapten or an antigen-binding fragment thereof. Exemplary
binding entities include, but are not limited to, a biotin
molecule, a hapten, an antibody, an antibody binding fragment, a
peptide, and a protein.
[0132] "Complementary" refers to sequence complementarity between
regions of two nucleic acid strands or between two regions of the
same nucleic acid strand. It is known that an adenine residue of a
first nucleic acid region is capable of forming specific hydrogen
bonds ("base pairing") with a residue of a second nucleic acid
region which is antiparallel to the first region if the residue is
thymine or uracil. Similarly, it is known that a cytosine residue
of a first nucleic acid strand is capable of base pairing with a
residue of a second nucleic acid strand which is antiparallel to
the first strand if the residue is guanine. A first region of a
nucleic acid is complementary to a second region of the same or a
different nucleic acid if, when the two regions are arranged in an
antiparallel fashion, at least one nucleotide residue of the first
region is capable of base pairing with a residue of the second
region. In certain embodiments, the first region comprises a first
portion and the second region comprises a second portion, whereby,
when the first and second portions are arranged in an antiparallel
fashion, at least about 50%, at least about 75%, at least about
90%, or at least about 95% of the nucleotide residues of the first
portion are capable of base pairing with nucleotide residues in the
second portion. In other embodiments, all nucleotide residues of
the first portion are capable of base pairing with nucleotide
residues in the second portion.
[0133] The term "cancer" or "tumor" is used interchangeably herein.
These terms refer to the presence of cells possessing
characteristics typical of cancer-causing cells, such as
uncontrolled proliferation, immortality, metastatic potential,
rapid growth and proliferation rate, and certain characteristic
morphological features. Cancer cells are often in the form of a
tumor, but such cells can exist alone within an animal, or can be a
non-tumorigenic cancer cell, such as a leukemia cell. These terms
include a solid tumor, a soft tissue tumor, or a metastatic lesion.
As used herein, the term "cancer" includes premalignant, as well as
malignant cancers.
[0134] Cancer is "inhibited" if at least one symptom of the cancer
is alleviated, terminated, slowed, or prevented. As used herein,
cancer is also "inhibited" if recurrence or metastasis of the
cancer is reduced, slowed, delayed, or prevented.
[0135] "Chemotherapeutic agent" means a chemical substance, such as
a cytotoxic or cytostatic agent, that is used to treat a condition,
particularly cancer.
[0136] As used herein, "cancer therapy" and "cancer treatment" are
synonymous terms.
[0137] As used herein, "chemotherapy" and "chemotherapeutic" and
"chemotherapeutic agent" are synonymous terms.
[0138] The terms "homology" or "identity," as used interchangeably
herein, refer to sequence similarity between two polynucleotide
sequences or between two polypeptide sequences, with identity being
a more strict comparison. The phrases "percent identity or
homology" and "% identity or homology" refer to the percentage of
sequence similarity found in a comparison of two or more
polynucleotide sequences or two or more polypeptide sequences.
"Sequence similarity" refers to the percent similarity in base pair
sequence (as determined by any suitable method) between two or more
polynucleotide sequences. Two or more sequences can be anywhere
from 0-100% similar, or any integer value there between. Identity
or similarity can be determined by comparing a position in each
sequence that can be aligned for purposes of comparison. When a
position in the compared sequence is occupied by the same
nucleotide base or amino acid, then the molecules are identical at
that position. A degree of similarity or identity between
polynucleotide sequences is a function of the number of identical
or matching nucleotides at positions shared by the polynucleotide
sequences. A degree of identity of polypeptide sequences is a
function of the number of identical amino acids at positions shared
by the polypeptide sequences. A degree of homology or similarity of
polypeptide sequences is a function of the number of amino acids at
positions shared by the polypeptide sequences. The term
"substantially identical," as used herein, refers to an identity or
homology of at least 75%, at least 80%, at least 85%, at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
[0139] "Likely to" or "increased likelihood," as used herein,
refers to an increased probability that an item, object, thing or
person will occur. Thus, in one example, a subject that is likely
to respond to treatment with a kinase inhibitor, alone or in
combination, has an increased probability of responding to
treatment with the inhibitor alone or in combination, relative to a
reference subject or group of subjects.
[0140] "Unlikely to" refers to a decreased probability that an
event, item, object, thing or person will occur with respect to a
reference. Thus, a subject that is unlikely to respond to treatment
with a kinase inhibitor, alone or in combination, has a decreased
probability of responding to treatment with a kinase inhibitor,
alone or in combination, relative to a reference subject or group
of subjects.
[0141] "Sequencing" a nucleic acid molecule requires determining
the identity of at least 1 nucleotide in the molecule. In
embodiments, the identity of less than all of the nucleotides in a
molecule are determined. In other embodiments, the identity of a
majority or all of the nucleotides in the molecule is
determined.
[0142] "Next-generation sequencing or NGS or NG sequencing" as used
herein, refers to any sequencing method that determines the
nucleotide sequence of either individual nucleic acid molecules
(e.g., in single molecule sequencing) or clonally expanded proxies
for individual nucleic acid molecules in a highly parallel fashion
(e.g., greater than 10.sup.5 molecules are sequenced
simultaneously). In one embodiment, the relative abundance of the
nucleic acid species in the library can be estimated by counting
the relative number of occurrences of their cognate sequences in
the data generated by the sequencing experiment. Next generation
sequencing methods are known in the art, and are described, e.g.,
in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46,
incorporated herein by reference. Next generation sequencing can
detect a variant present in less than 5% of the nucleic acids in a
sample.
[0143] "Sample," "tissue sample," "patient sample," "patient cell
or tissue sample" or "specimen" each refers to a collection of
similar cells obtained from a tissue of a subject or patient. The
source of the tissue sample can be solid tissue as from a fresh,
frozen and/or preserved organ, tissue sample, biopsy, or aspirate;
blood or any blood constituents; bodily fluids such as cerebral
spinal fluid, amniotic fluid, peritoneal fluid or interstitial
fluid; or cells from any time in gestation or development of the
subject. The tissue sample can contain compounds that are not
naturally intermixed with the tissue in nature such as
preservatives, anticoagulants, buffers, fixatives, nutrients,
antibiotics or the like. In one embodiment, the sample is preserved
as a frozen sample or as formaldehyde- or paraformaldehyde-fixed
paraffin-embedded (FFPE) tissue preparation. For example, the
sample can be embedded in a matrix, e.g., an FFPE block or a frozen
sample.
[0144] A "tumor nucleic acid sample" as used herein, refers to
nucleic acid molecules from a tumor or cancer sample. Typically, it
is DNA, e.g., genomic DNA, or cDNA derived from RNA, from a tumor
or cancer sample. In certain embodiments, the tumor nucleic acid
sample is purified or isolated (e.g., it is removed from its
natural state).
[0145] A "control" or "reference" "nucleic acid sample" as used
herein, refers to nucleic acid molecules from a control or
reference sample. Typically, it is DNA, e.g., genomic DNA, or cDNA
derived from RNA, not containing the alteration or variation in the
gene or gene product, e.g., not containing a KIF5B-RET fusion. In
certain embodiments, the reference or control nucleic acid sample
is a wild type or a non-mutated sequence. In certain embodiments,
the reference nucleic acid sample is purified or isolated (e.g., it
is removed from its natural state). In other embodiments, the
reference nucleic acid sample is from a non-tumor sample, e.g., a
blood control, a normal adjacent tumor (NAT), or any other
non-cancerous sample from the same or a different subject.
[0146] Headings, e.g., (a), (b), (i) etc, are presented merely for
ease of reading the specification and claims. The use of headings
in the specification or claims does not require the steps or
elements be performed in alphabetical or numerical order or the
order in which they are presented.
[0147] Various aspects of the invention are described in further
detail below. Additional definitions are set out throughout the
specification.
Isolated Nucleic Acid Molecules
[0148] One aspect of the invention pertains to isolated nucleic
acid molecules that include a KIF5B-RET fusion, including nucleic
acids which encode a KIF5B-RET fusion polypeptide or a portion of
such a polypeptide. The nucleic acid molecules include those
nucleic acid molecules which reside in genomic regions identified
herein. As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,
mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-stranded or
double-stranded; in certain embodiments the nucleic acid molecule
is double-stranded DNA.
[0149] Isolated nucleic acid molecules also include nucleic acid
molecules sufficient for use as hybridization probes or primers to
identify nucleic acid molecules that correspond to a KIF5B-RET
fusion, e.g., those suitable for use as PCR primers for the
amplification or mutation of nucleic acid molecules.
[0150] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. In certain
embodiments, an "isolated" nucleic acid molecule is free of
sequences (such as protein-encoding sequences) which naturally
flank the nucleic acid (i.e., sequences located at the 5' and 3'
ends of the nucleic acid) in the genomic DNA of the organism from
which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5 kB, less than about 4 kB, less than about 3 kB, less
than about 2 kB, less than about 1 kB, less than about 0.5 kB or
less than about 0.1 kB of nucleotide sequences which naturally
flank the nucleic acid molecule in genomic DNA of the cell from
which the nucleic acid is derived. Moreover, an "isolated" nucleic
acid molecule, such as a cDNA molecule, can be substantially free
of other cellular material or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0151] The language "substantially free of other cellular material
or culture medium" includes preparations of nucleic acid molecule
in which the molecule is separated from cellular components of the
cells from which it is isolated or recombinantly produced. Thus,
nucleic acid molecule that is substantially free of cellular
material includes preparations of nucleic acid molecule having less
than about 30%, less than about 20%, less than about 10%, or less
than about 5% (by dry weight) of other cellular material or culture
medium.
[0152] A KIF5B-RET fusion nucleic acid molecule can be isolated
using standard molecular biology techniques and the sequence
information in the database records described herein. Using all or
a portion of such nucleic acid sequences, KIF5B-RET fusion nucleic
acid molecules can be isolated using standard hybridization and
cloning techniques (e.g., as described in Sambrook et al., ed.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0153] A KIF5B-RET fusion nucleic acid molecule can be amplified
using cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid molecules so amplified can be cloned
into an appropriate vector and characterized by DNA sequence
analysis. Furthermore, oligonucleotides corresponding to all or a
portion of a nucleic acid molecule of the invention can be prepared
by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
[0154] In another embodiment, a KIF5B-RET fusion nucleic acid
molecule comprises a nucleic acid molecule which has a nucleotide
sequence complementary to the nucleotide sequence of a KIF5B-RET
fusion nucleic acid molecule or to the nucleotide sequence of a
nucleic acid encoding a KIF5B-RET fusion protein. A nucleic acid
molecule which is complementary to a given nucleotide sequence is
one which is sufficiently complementary to the given nucleotide
sequence that it can hybridize to the given nucleotide sequence
thereby forming a stable duplex.
[0155] Moreover, a KIF5B-RET fusion nucleic acid molecule can
comprise only a portion of a nucleic acid sequence, wherein the
full length nucleic acid sequence or which encodes a KIF5B-RET
fusion polypeptide. Such nucleic acid molecules can be used, for
example, as a probe or primer. The probe/primer typically is used
as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7, at
least about 15, at least about 25, at least about 50, at least
about 75, at least about 100, at least about 125, at least about
150, at least about 175, at least about 200, at least about 250, at
least about 300, at least about 350, at least about 400, at least
about 500, at least about 600, at least about 700, at least about
800, at least about 900, at least about 1 kb, at least about 2 kb,
at least about 3 kb, at least about 4 kb, at least about 5 kb, at
least about 6 kb, at least about 7 kb, at least about 8 kb, at
least about 9 kb, at least about 10 kb, at least about 15 kb, at
least about 20 kb, at least about 25 kb, at least about 30 kb, at
least about 35 kb, at least about 40 kb, at least about 45 kb, at
least about 50 kb, at least about 60 kb, at least about 70 kb, at
least about 80 kb, at least about 90 kb, at least about 100 kb, at
least about 200 kb, at least about 300 kb, at least about 400 kb,
at least about 500 kb, at least about 600 kb, at least about 700
kb, at least about 800 kb, at least about 900 kb, at least about 1
mb, at least about 2 mb, at least about 3 mb, at least about 4 mb,
at least about 5 mb, at least about 6 mb, at least about 7 mb, at
least about 8 mb, at least about 9 mb, at least about 10 mb or more
consecutive nucleotides of a KIF5B-RET fusion nucleic acid.
[0156] The invention further encompasses nucleic acid molecules
that are substantially identical to the gene mutations and/or gene
products described herein, e.g., KIF5B-RET fusion having a
nucleotide sequence of SEQ ID NO:1 or 2, or an amino acid sequence
of SEQ ID NO:3 or 4) such that they are at least 70%, at least 75%,
at least 80%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, at least 99.5% or greater. In other
embodiments, the invention further encompasses nucleic acid
molecules that are substantially homologous to the KIF5B-RET fusion
gene mutations and/or gene products described herein, such that
they differ by only or at least 1, at least 2, at least 3, at least
4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 30, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 200, at
least 300, at least 400, at least 500, at least 600 nucleotides or
any range in between.
[0157] In another embodiment, an isolated KIF5B-RET fusion nucleic
acid molecule is at least 7, at least 15, at least 20, at least 25,
at least 30, at least 35, at least 40, at least 45, at least 50, at
least 55, at least 60, at least 65, at least 70, at least 75, at
least 80, at least 85, at least 90, at least 95, at least 100, at
least 125, at least 150, at least 175, at least 200, at least 250,
at least 300, at least 350, at least 400, at least 450, at least
550, at least 650, at least 700, at least 800, at least 900, at
least 1000, at least 1200, at least 1400, at least 1600, at least
1800, at least 2000, at least 2200, at least 2400, at least 2600,
at least 2800, at least 3000, or more nucleotides in length and
hybridizes under stringent conditions to a KIF5B-RET fusion nucleic
acid molecule or to a nucleic acid molecule encoding a protein
corresponding to a marker of the invention.
[0158] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, or at least 85%
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in sections 6.3.1-6.3.6 of Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989). Another,
non-limiting example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50-65.degree. C.
[0159] The invention also includes molecular beacon nucleic acid
molecules having at least one region which is complementary to a
KIF5B-RET fusion nucleic acid molecule, such that the molecular
beacon is useful for quantitating the presence of the nucleic acid
molecule of the invention in a sample. A "molecular beacon" nucleic
acid is a nucleic acid molecule comprising a pair of complementary
regions and having a fluorophore and a fluorescent quencher
associated therewith. The fluorophore and quencher are associated
with different portions of the nucleic acid in such an orientation
that when the complementary regions are annealed with one another,
fluorescence of the fluorophore is quenched by the quencher. When
the complementary regions of the nucleic acid molecules are not
annealed with one another, fluorescence of the fluorophore is
quenched to a lesser degree. Molecular beacon nucleic acid
molecules are described, for example, in U.S. Pat. No.
5,876,930.
Probes
[0160] The invention also provides isolated KIF5B-RET nucleic acid
molecules useful as probes.
[0161] Probes based on the sequence of a KIF5B-RET fusion nucleic
acid molecule can be used to detect transcripts or genomic
sequences corresponding to one or more markers of the invention.
The probe comprises a label group attached thereto, e.g., a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as part of a test kit for
identifying cells or tissues which express the KIF5B-RET protein,
such as by measuring levels of a nucleic acid molecule encoding the
protein in a sample of cells from a subject, e.g., detecting mRNA
levels or determining whether a gene encoding the protein has been
mutated or deleted.
[0162] Probes featured in the invention include those that will
specifically hybridize to a gene sequence described in the Example,
e.g., a KIF5B-RET fusion. Typically these probes are 12 to 20,
e.g., 17 to 20 nucleotides in length (longer for large insertions)
and have the nucleotide sequence corresponding to the region of the
mutations at their respective nucleotide locations on the gene
sequence. Such molecules can be labeled according to any technique
known in the art, such as with radiolabels, fluorescent labels,
enzymatic labels, sequence tags, biotin, other ligands, etc. As
used herein, a probe that "specifically hybridizes" to a KIF5B-RET
fusion gene sequence will hybridize under high stringency
conditions.
[0163] A probe will typically contain one or more of the specific
mutations described herein. Typically, a nucleic acid probe will
encompass only one mutation. Such molecules may be labeled and can
be used as allele-specific probes to detect the mutation of
interest.
[0164] In one aspect, the invention features a probe or probe set
that specifically hybridizes to a nucleic acid comprising an
inversion resulting in a KIF5B-RET fusion.
[0165] Isolated pairs of allele specific oligonucleotide probes are
also provided, where the first probe of the pair specifically
hybridizes to the mutant allele, and the second probe of the pair
specifically hybridizes to the wildtype allele. For example, in one
exemplary probe pair, one probe will recognize the fusion junction
in the KI5B-RET fusion, and the other probe will recognize a
sequence downstream or upstream of KIF5B or RET, neither of which
includes the fusion junction. These allele-specific probes are
useful in detecting a RET somatic mutation in a tumor sample, e.g.,
a lung tumor sample.
Primers
[0166] The invention also provides isolated nucleic acid molecules
useful as primers.
[0167] The term "primer" as used herein refers to a sequence
comprising two or more deoxyribonucleotides or ribonucleotides,
e.g., more than three, and more than eight, or at least 20
nucleotides of a gene described in the Example, where the sequence
corresponds to a sequence flanking one of the mutations or a wild
type sequence of a gene identified in the Example, e.g., a KIF5B or
RET gene. Primers may be used to initiate DNA synthesis via the PCR
(polymerase chain reaction) or a sequencing method. Primers
featured in the invention include the sequences recited and
complementary sequences which would anneal to the opposite DNA
strand of the sample target. Since both strands of DNA are
complementary and mirror images of each other, the same segment of
DNA will be amplified.
[0168] Primers can be used to sequence a nucleic acid, e.g., an
isolated nucleic acid described herein, such as by an NGS method,
or to amplify a gene described in the Example, such as by PCR. The
primers can specifically hybridize, for example, to the ends of the
exons or to the introns flanking the exons. The amplified segment
can then be further analyzed for the presence of the mutation such
as by a sequencing method. The primers are useful in directing
amplification of a target polynucleotide prior to sequencing. In
another aspect, the invention features a pair of oligonucleotide
primers that amplify a region that contains or is adjacent to a
fusion junction identified in the Example. Such primers are useful
in directing amplification of a target region that includes a
fusion junction identified in the Example, e.g., prior to
sequencing. The primer typically contains 12 to 20, or 17 to 20, or
more nucleotides, although a primer may contain fewer
nucleotides.
[0169] A primer is typically single stranded, e.g., for use in
sequencing or amplification methods, but may be double stranded. If
double stranded, the primer may first be treated to separate its
strands before being used to prepare extension products. A primer
must be sufficiently long to prime the synthesis of extension
products in the presence of the inducing agent for polymerization.
The exact length of primer will depend on many factors, including
applications (e.g., amplification method), temperature, buffer, and
nucleotide composition. A primer typically contains 12-20 or more
nucleotides, although a primer may contain fewer nucleotides.
[0170] Primers are typically designed to be "substantially"
complementary to each strand of a genomic locus to be amplified.
Thus, the primers must be sufficiently complementary to
specifically hybridize with their respective strands under
conditions which allow the agent for polymerization to perform. In
other words, the primers should have sufficient complementarity
with the 5' and 3' sequences flanking the mutation to hybridize
therewith and permit amplification of the genomic locus.
[0171] The term "substantially complementary to" or "substantially
the sequence" refers to sequences that hybridize to the sequences
provided under stringent conditions and/or sequences having
sufficient homology with a sequence comprising a fusion junction
identified in the Example, or the wildtype counterpart sequence,
such that the allele specific oligonucleotides hybridize to the
sequence. In one embodiment, a sequence is substantially
complementary to a fusion junction in an inversion event, e.g., to
a fusion junction in SEQ ID NO:1. "Substantially the same" as it
refers to oligonucleotide sequences also refers to the functional
ability to hybridize or anneal with sufficient specificity to
distinguish between the presence or absence of the mutation. This
is measurable by the temperature of melting being sufficiently
different to permit easy identification of whether the
oligonucleotide is binding to the normal or mutant gene sequence
identified in the Example.
[0172] In one aspect, the invention features a primer or primer set
for amplifying a nucleic acid comprising an inversion resulting in
a KIF5B-RET fusion.
[0173] Isolated pairs of allele specific oligonucleotide primer are
also provided, where the first primer of the pair specifically
hybridizes to the mutant allele, and the second primer of the pair
specifically hybridizes to a sequence upstream or downstream of a
mutation, or a fusion junction resulting from, e.g., an inversion,
duplication, deletion, insertion or translocation. For example, in
one exemplary primer pair, one probe will recognize the a KIF5B-RET
inversion, such as by hybridizing to a sequence at the fusion
junction between the KIF5B and RET transcripts, and the other
primer will recognize a sequence upstream or downstream of the
fusion junction. These allele-specific primers are useful for
amplifying a KIF5B-RET fusion sequence from a tumor sample, e.g.,
an adenocarcinoma, such as an adenocarcinoma of the lung.
[0174] In another exemplary primer pair, one primer can recognize a
RET-KIF5B inversion (e.g., the reciprocal of the KIF5B-RET
inversion), such as by hybridizing to a sequence at the fusion
junction between the RET and KIF5B transcripts, and the other
primer will recognize a sequence upstream or downstream of the
fusion junction. These allele-specific primers are useful for
amplifying a RET-KIF5B fusion sequence from a tumor sample, e.g.,
an adenocarcinoma, such as an adenocarcinoma of the lung.
[0175] Primers can be prepared using any suitable method, such as
conventional phosphotriester and phosphodiester methods or
automated embodiments thereof. In one such automated embodiment,
diethylphosphoramidites are used as starting materials and may be
synthesized as described by Beaucage, et al., Tetrahedron Letters,
22:1859-1862, (1981). One method for synthesizing oligonucleotides
on a modified solid support is described in U.S. Pat. No.
4,458,066.
[0176] An oligonucleotide probe or primer that hybridizes to a
mutant or wildtype allele is said to be the complement of the
allele. As used herein, a probe exhibits "complete complementarity"
when every nucleotide of the probe is complementary to the
corresponding nucleotide of the allele. Two polynucleotides are
said to be "minimally complementary" if they can hybridize to one
another with sufficient stability to permit them to remain annealed
to one another under at least conventional "low-stringency"
conditions. Similarly, the polynucleotides are said to be
"complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are known to those skilled in
the art and can be found, for example in Molecular Cloning: A
Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook,
D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press,
2000.
[0177] Departures from complete complementarity are therefore
permissible, as long as such departures do not completely preclude
the capacity of a probe to hybridize to an allele. Thus, in order
for a polynucleotide to serve as a primer or probe it need only be
sufficiently complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed. Appropriate stringency conditions which
promote DNA hybridization are, for example, 6.0.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times.SSC at 50.degree. C. Such conditions are known to
those skilled in the art and can be found, for example in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
Salt concentration and temperature in the wash step can be adjusted
to alter hybridization stringency. For example, conditions may vary
from low stringency of about 2.0.times.SSC at 40.degree. C. to
moderately stringent conditions of about 2.0.times.SSC at
50.degree. C. to high stringency conditions of about 0.2.times.SSC
at 50.degree. C.
KIF5B-RET Fusion Proteins and Antibodies
[0178] One aspect of the invention pertains to purified KIF5B-RET
fusion polypeptides, and biologically active portions thereof. In
one embodiment, the native KIF5B-RET fusion polypeptide can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, a KIF5B-RET fusion polypeptide is produced
by recombinant DNA techniques. Alternative to recombinant
expression, a KIF5B-RET fusion polypeptide can be synthesized
chemically using standard peptide synthesis techniques.
[0179] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, less than about 20%, less than about 10%, or less than
about 5% (by dry weight) of heterologous protein (also referred to
herein as a "contaminating protein"). When the protein or
biologically active portion thereof is recombinantly produced, it
can be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation. When the protein
is produced by chemical synthesis, it can substantially be free of
chemical precursors or other chemicals, i.e., it is separated from
chemical precursors or other chemicals which are involved in the
synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, less than about 20%, less than
about 10%, less than about 5% (by dry weight) of chemical
precursors or compounds other than the polypeptide of interest.
[0180] Biologically active portions of a KIF5B-RET fusion
polypeptide include polypeptides comprising amino acid sequences
sufficiently identical to or derived from the amino acid sequence
of the KIF5B-RET fusion protein, which include fewer amino acids
than the full length protein, and exhibit at least one activity of
the corresponding full-length protein, e.g., a kinase activity, or
a dimerizing or multimerizing activity. A biologically active
portion of a protein of the invention can be a polypeptide which
is, for example, 10, 25, 50, 100 or more amino acids in length.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of a polypeptide.
[0181] In certain embodiments, the KIF5B-RET fusion polypeptide has
an amino acid sequence of a protein encoded by a nucleic acid
molecule disclosed herein. Other useful proteins are substantially
identical (e.g., at least 60, at least 65, at least 70, at least
75, at least 80, at least 85, at least 86, at least 87, at least
88, at least 89, at least 90, at least 91, at least 92, at least
93, at least 94, at least 95, at least 96, at least 97, at least
98, at least 99, at least 99.5% or greater) to one of these
sequences and retain the functional activity of the protein of the
corresponding full-length protein yet differ in amino acid
sequence.
[0182] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment the two sequences are the
same length.
[0183] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. Another,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules. When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See online at ncbi.nlm.nih.gov. Another non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller, (1988) Comput Appl
Biosci, 4:11-7. Such an algorithm is incorporated into the ALIGN
program (version 2.0) which is part of the GCG sequence alignment
software package. When utilizing the ALIGN program for comparing
amino acid sequences, a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used. Yet another
useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.
When using the FASTA algorithm for comparing nucleotide or amino
acid sequences, a PAM120 weight residue table can, for example, be
used with a k-tuple value of 2.
[0184] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0185] An isolated KIF5B-RET fusion polypeptide, or a fragment
thereof, can be used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. The full-length KIF5B-RET fusion polypeptide can be
used or, alternatively, the invention provides antigenic peptide
fragments for use as immunogens. The antigenic peptide of a protein
of the invention comprises at least 8 (or at least 10, at least 15,
at least 20, or at least 30 or more) amino acid residues of the
amino acid sequence of one of the polypeptides of the invention,
and encompasses an epitope of the protein such that an antibody
raised against the peptide forms a specific immune complex with a
marker of the invention to which the protein corresponds. Exemplary
epitopes encompassed by the antigenic peptide are regions that are
located on the surface of the protein, e.g., hydrophilic regions.
Hydrophobicity sequence analysis, hydrophilicity sequence analysis,
or similar analyses can be used to identify hydrophilic
regions.
[0186] An immunogen typically is used to prepare antibodies by
immunizing a suitable (i.e., immunocompetent) subject such as a
rabbit, goat, mouse, or other mammal or vertebrate. An appropriate
immunogenic preparation can contain, for example,
recombinantly-expressed or chemically-synthesized polypeptide. The
preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or a similar immunostimulatory
agent.
[0187] Accordingly, another aspect of the invention pertains to
antibodies directed against a KIF5B-RET fusion polypeptide. In one
embodiment, the antibody molecule specifically binds to KIF5B-RET
fusion, e.g., specifically binds to an epitope formed by the
KIF5B-RET fusion. In embodiments the antibody can distinguish wild
type RET (or KIF5B) from KIF5B-RET.
[0188] The terms "antibody" and "antibody molecule" as used
interchangeably herein refer to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site which specifically
binds an antigen, such as a polypeptide of the invention. A
molecule which specifically binds to a given polypeptide of the
invention is a molecule which binds the polypeptide, but does not
substantially bind other molecules in a sample, e.g., a biological
sample, which naturally contains the polypeptide. Examples of
immunologically active portions of immunoglobulin molecules include
F(ab) and F(ab').sub.2 fragments which can be generated by treating
the antibody with an enzyme such as pepsin. The invention provides
polyclonal and monoclonal antibodies. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope.
[0189] Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a polypeptide of the invention
as an immunogen. Antibody-producing cells can be obtained from the
subject and used to prepare monoclonal antibodies by standard
techniques, such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497, the human B cell
hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72),
the EBV-hybridoma technique (see Cole et al., pp. 77-96 In
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985)
or trioma techniques. The technology for producing hybridomas is
well known (see generally Current Protocols in Immunology, Coligan
et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells
producing a monoclonal antibody of the invention are detected by
screening the hybridoma culture supernatants for antibodies that
bind the polypeptide of interest, e.g., using a standard ELISA
assay.
[0190] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody can be identified and isolated by
screening a recombinant combinatorial immunoglobulin library (e.g.,
an antibody phage display library) with the polypeptide of
interest. Kits for generating and screening phage display libraries
are commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP
Phage Display Kit, Catalog No. 240612). Additionally, examples of
methods and reagents particularly amenable for use in generating
and screening antibody display library can be found in, for
example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619;
PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791;
PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288;
PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690;
PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85;
Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993)
EMBO J. 12:725-734.
[0191] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions can be made using standard recombinant DNA
techniques. Such chimeric and humanized monoclonal antibodies can
be produced by recombinant DNA techniques known in the art, for
example using methods described in PCT Publication No. WO 87/02671;
European Patent Application 184,187; European Patent Application
171,496; European Patent Application 173,494; PCT Publication No.
WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559; Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0192] Completely human antibodies can be produced using transgenic
mice which are incapable of expressing endogenous immunoglobulin
heavy and light chains genes, but which can express human heavy and
light chain genes. For an overview of this technology for producing
human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol.
13:65-93). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., U.S. Pat. No.
5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition,
companies such as Abgenix, Inc. (Freemont, Calif.), can be engaged
to provide human antibodies directed against a selected antigen
using technology similar to that described above.
[0193] An antibody directed against a KIF5B-RET fusion polypeptide
(e.g., a monoclonal antibody) can be used to isolate the
polypeptide by standard techniques, such as affinity chromatography
or immunoprecipitation. Moreover, such an antibody can be used to
detect the marker (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the level and pattern of expression of the
marker. The antibodies can also be used diagnostically to monitor
protein levels in tissues or body fluids (e.g., in a tumor
cell-containing body fluid) as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include, but are not limited to, various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include, but are not limited to, horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include, but are not limited to, streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include,
but are not limited to, umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin; an example of a luminescent
material includes, but is not limited to, luminol; examples of
bioluminescent materials include, but are not limited to,
luciferase, luciferin, and aequorin, and examples of suitable
radioactive materials include, but are not limited to, .sup.125I,
.sup.131I, .sup.35S or .sup.3H.
Antigens and Vaccines
[0194] Embodiments of the invention include preparations, e.g.,
antigenic preparations, of the entire fusion or a fragment thereof,
e.g., a fragment capable of raising antibodies specific to the
fusion protein, e.g., a fusion junction containing fragment
(collectively referred to herein as a fusion specific polypeptides
or FSP). The preparation can include an adjuvant or other
component.
[0195] An FSP can be used as an antigen or vaccine. E.g., an FSP
can be used as an antigen to immunize an animal, e.g., a rodent,
e.g., a mouse or rat, rabbit, horse, goat, dog, or non-human
primate, to obtain antibodies, e.g., fusion protein specific
antibodies. In an embodiment a fusion specific antibody molecule is
an antibody molecule described herein, e.g., a polyclonal. In other
embodiments a fusion specific antibody molecule is monospecific,
e.g., monoclonal, human, humanized, chimeric or other monospecific
antibody molecule. The fusion protein specific antibody molecules
can be used to treat a subject having cancer, e.g., a cancer
described herein.
[0196] Embodiments of the invention include vaccine preparations
that comprise an FSP capable of stimulating an immune response in a
subject, e.g., by raising, in the subject, antibodies specific to
the fusion protein. The vaccine preparation can include other
components, e.g., an adjuvant. The vaccine preparations can be used
to treat a subject having cancer, e.g., a cancer described
herein.
Expression Vectors, Host Cells and Recombinant Cells
[0197] In another aspect, the invention includes vectors (e.g.,
expression vectors), containing a nucleic acid encoding a KIF5B-RET
fusion polypeptide described herein. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked and can include a
plasmid, cosmid or viral vector. The vector can be capable of
autonomous replication or it can integrate into a host DNA. Viral
vectors include, e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses.
[0198] A vector can include a KIF5B-RET fusion nucleic acid in a
form suitable for expression of the nucleic acid in a host cell.
Preferably the recombinant expression vector includes one or more
regulatory sequences operatively linked to the nucleic acid
sequence to be expressed. The term "regulatory sequence" includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors can be
introduced into host cells to thereby produce KIF5B-RET fusion
polypeptide, including fusion proteins or polypeptides encoded by
nucleic acids as described herein, mutant forms thereof, and the
like).
[0199] The term "recombinant host cell" (or simply "host cell" or
"recombinant cell"), as used herein, is intended to refer to a cell
into which a recombinant expression vector has been introduced. It
should be understood that such terms are intended to refer not only
to the particular subject cell, but to the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used
herein.
[0200] The recombinant expression vectors can be designed for
expression of KIF5B-RET fusion polypeptide in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, (1990) Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0201] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0202] Purified KIF5B-RET fusion polypeptides can be used in
activity assays (e.g., direct assays or competitive assays
described in detail below), or to generate antibodies specific for
KIF5B-RET fusion polypeptides.
[0203] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S.,
(1990) Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0204] The KIF5B-RET fusion polypeptide 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.
[0205] When used in mammalian cells, the expression vector's
control functions can be provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0206] In another embodiment, the promoter is an inducible
promoter, e.g., a promoter regulated by a steroid hormone, by a
polypeptide hormone (e.g., by means of a signal transduction
pathway), or by a heterologous polypeptide (e.g., the
tetracycline-inducible systems, "Tet-On" and "Tet-Off"; see, e.g.,
Clontech Inc., C A, Gossen and Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983). In
another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially
in a particular cell type (e.g., tissue-specific regulatory
elements are used to express the nucleic acid). Non-limiting
examples of suitable tissue-specific promoters include the albumin
promoter (liver-specific; Pinkert et al. (1987) Genes Dev.
1:268-277), lymphoid-specific promoters (Calame and Eaton (1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0207] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus.
[0208] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a
KIF5B-RET fusion nucleic acid molecule within a recombinant
expression vector or a KIF5B-RET fusion nucleic acid molecule
containing sequences which allow it to homologous recombination
into a specific site of the host cell's genome.
[0209] A host cell can be any prokaryotic or eukaryotic cell. For
example, a KIF5B-RET fusion polypeptide can be expressed in
bacterial cells (such as E. coli), insect cells, yeast or mammalian
cells (such as Chinese hamster ovary cells (CHO) or COS cells,
e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell
23:175-182). Other suitable host cells are known to those skilled
in the art.
[0210] 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, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0211] A host cell can be used to produce (e.g., express) a
KIF5B-RET fusion polypeptide. Accordingly, the invention further
provides methods for producing a KIF5B-RET fusion polypeptide using
the host cells. In one embodiment, the method includes culturing
the host cell of the invention (into which a recombinant expression
vector encoding a KIF5B-RET fusion polypeptide has been introduced)
in a suitable medium such that a KIF5B-RET fusion polypeptide is
produced. In another embodiment, the method further includes
isolating a KIF5B-RET fusion polypeptide from the medium or the
host cell.
[0212] In another aspect, the invention features, a cell or
purified preparation of cells which include a KIF5B-RET fusion
transgene, or which otherwise misexpress KIF5B-RET fusion. The cell
preparation can consist of human or non-human cells, e.g., rodent
cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In
embodiments, the cell or cells include a KIF5B-RET fusion
transgene, e.g., a heterologous form of a KIF5B-RET fusion, e.g., a
gene derived from humans (in the case of a non-human cell). The
KIF5B-RET fusion transgene can be misexpressed, e.g., overexpressed
or underexpressed. In other preferred embodiments, the cell or
cells include a gene that mis-expresses an endogenous KIF5B-RET
fusion, e.g., a gene the expression of which is disrupted, e.g., a
knockout. Such cells can serve as a model for studying disorders
that are related to mutated or mis-expressed KIF5B-RET fusion
alleles (e.g., cancers) or for use in drug screening, as described
herein.
Therapeutic Methods
[0213] Alternatively, or in combination with the methods described
herein, the invention features a method of treating a cancer or
tumor harboring a KIF5B-RET fusion described herein. The methods
include administering an anti-cancer agent, e.g., a kinase
inhibitor, alone or in combination, e.g., in combination with other
chemotherapeutic agents or procedures, in an amount sufficient to
reduce or inhibit the tumor cell growth, and/or treat or prevent
the cancer(s), in the subject.
[0214] "Treat," "treatment," and other forms of this word refer to
the administration of a kinase inhibitor, alone or in combination
with a second agent to impede growth of a cancer, to cause a cancer
to shrink by weight or volume, to extend the expected survival time
of the subject and or time to progression of the tumor or the like.
In those subjects, treatment can include, but is not limited to,
inhibiting tumor growth, reducing tumor mass, reducing size or
number of metastatic lesions, inhibiting the development of new
metastatic lesions, prolonged survival, prolonged progression-free
survival, prolonged time to progression, and/or enhanced quality of
life.
[0215] As used herein, unless otherwise specified, the terms
"prevent," "preventing" and "prevention" contemplate an action that
occurs before a subject begins to suffer from the re-growth of the
cancer and/or which inhibits or reduces the severity of the
cancer.
[0216] 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 the cancer, or to delay or minimize one or more
symptoms associated with the cancer. 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 cancer, or enhances the therapeutic efficacy of another
therapeutic agent.
[0217] As used herein, and unless otherwise specified, a
"prophylactically effective amount" of a compound is an amount
sufficient to prevent re-growth of the cancer, or one or more
symptoms associated with the cancer, or prevent its recurrence. A
prophylactically effective amount of a compound means an amount of
the compound, alone or in combination with other therapeutic
agents, which provides a prophylactic benefit in the prevention of
the cancer. The term "prophylactically effective amount" can
encompass an amount that improves overall prophylaxis or enhances
the prophylactic efficacy of another prophylactic agent.
[0218] As used herein, the term "patient" or "subject" refers to an
animal, typically a human (i.e., a male or female of any age group,
e.g., a pediatric patient (e.g, infant, child, adolescent) or adult
patient (e.g., young adult, middle-aged adult or senior adult) or
other mammal, such as a primate (e.g., cynomolgus monkey, rhesus
monkey); commercially relevant mammals such as cattle, pigs,
horses, sheep, goats, cats, and/or dogs; and/or birds, including
commercially relevant birds such as chickens, ducks, geese, and/or
turkeys, that will be or has been the object of treatment,
observation, and/or experiment. When the term is used in
conjunction with administration of a compound or drug, then the
patient has been the object of treatment, observation, and/or
administration of the compound or drug.
[0219] In certain embodiments, the cancer includes, but is not
limited to, a solid tumor, a soft tissue tumor, and a metastatic
lesion (e.g., a cancer as described herein). In one embodiment, the
cancer is chosen from a lung cancer, a pancreatic cancer, melanoma,
a colorectal cancer, an esophageal-gastric cancer, a thyroid
cancer, or an adenocarcinoma. In other embodiment, the lung cancer
is chosen from one or more of the following: non-small cell lung
cancer (NSCLC), small cell lung cancer (SCLC), squamous cell
carcinoma (SCC), adenocarcinoma of the lung, bronchogenic
carcinoma, or a combination thereof. In one embodiment, the lung
cancer is NSCLC or SCC.
[0220] In other embodiments, the cancer is chosen from lung cancer,
thyroid cancer, colorectal cancer, adenocarcinoma, melanoma, B cell
cancer, breast cancer, bronchus cancer, cancer of the oral cavity
or pharynx, cancer of hematological tissues, cervical cancer, colon
cancer, esophageal cancer, esophageal-gastric cancer, gastric
cancer, kidney cancer, liver cancer, multiple myeloma, ovarian
cancer, pancreatic cancer, prostate cancer, salivary gland cancer,
small bowel or appendix cancer, stomach cancer, testicular cancer,
urinary bladder cancer, uterine or endometrial cancer, inflammatory
myofibroblastic tumors, gastrointestinal stromal tumor (GIST), and
the like.
[0221] In one embodiment, the anti-cancer agent is a kinase
inhibitor. For example, the kinase inhibitor is a multi-kinase
inhibitor or a RET-specific inhibitor. Exemplary kinase inhibitors
include, but are not limited to, axitinib (AG013736), bosutinib
(SKI-606), cediranib (RECENTIN.TM., AZD2171), dasatinib
(SPRYCEL.RTM., BMS-354825), erlotinib (TARCEVA.RTM.), gefitinib
(IRESSA.RTM.), imatinib (Gleevec.RTM., CGP57148B, STI-571),
lapatinib (TYKERB.RTM., TYVERB.RTM.), lestaurtinib (CEP-701),
lenvatinib (E7080), neratinib (HKI-272), nilotinib (TASIGNA.RTM.),
semaxanib (semaxinib, SU5416), sunitinib (SUTENT.RTM., SU11248),
toceranib (PALLADIA.RTM.), vandetanib, vatalanib (PTK787, PTK/ZK),
sorafenib (NEXAVAR.RTM.), ENMD-2076, PCI-32765, AC220, dovitinib
lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK.TM.), SGX523,
PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470,
BIBF 1120 (VARGATEF.RTM.), AP24534, JNJ-26483327, MGCD265,
DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930,
MM-121, XL-184, XL-647, and XL228.
[0222] In one embodiment, the kinase inhibitor is chosen from
lenvatinib (E7080), sorafenib (NEXAVAR.RTM.), sunitinib
(SUTENT.RTM., SU11248), vandetanib (CAPRELSA.RTM., ZACTIMA.RTM.,
ZD6474), NVP-AST487, regorafenib (BAY-73-4506), motesanib (AMG
706), cabozantinib (XL-184), apatinib (YN-968D1), DCC-2157, or
AST-487.
[0223] In other embodiments, the anti-cancer agent is a KIF5B-RET
antagonist inhibits the expression of nucleic acid encoding
KIF5B-RET. Examples of such KIF5B-RET antagonists include nucleic
acid molecules, for example, antisense molecules, ribozymes, RNAi,
triple helix molecules that hybridize to a nucleic acid encoding
KIF5B-RET, or a transcription regulatory region, and blocks or
reduces mRNA expression of KIF5B-RET.
[0224] In other embodiments, the kinase inhibitor is administered
in combination with a second therapeutic agent or a different
therapeutic modality, e.g., anti-cancer agents, and/or in
combination with surgical and/or radiation procedures.
[0225] By "in combination with," it is not intended to imply that
the therapy or the therapeutic agents must be administered at the
same time and/or formulated for delivery together, although these
methods of delivery are within the scope of the invention. The
pharmaceutical compositions can be administered concurrently with,
prior to, or subsequent to, one or more other additional therapies
or therapeutic agents. In general, each agent will be administered
at a dose and/or on a time schedule determined for that agent. In
will further be appreciated that the additional therapeutic agent
utilized in this combination can be administered together in a
single composition or administered separately in different
compositions. The particular combination to employ in a regimen
will take into account compatibility of the inventive
pharmaceutical composition with the additional therapeutically
active agent and/or the desired therapeutic effect to be
achieved.
[0226] For example, the second therapeutic agent can be a a
cytotoxic or a cytostatic agent. Exemplary cytotoxic agents include
antimicrotubule agents, topoisomerase inhibitors, or taxanes,
antimetabolites, mitotic inhibitors, alkylating agents,
intercalating agents, agents capable of interfering with a signal
transduction pathway, agents that promote apoptosis and radiation.
In yet other embodiments, the methods can be used in combination
with immunodulatory agents, e.g., IL-1, 2, 4, 6, or 12, or
interferon alpha or gamma, or immune cell growth factors such as
GM-CSF.
[0227] Anti-cancer agents, e.g., kinase inhibitors, used in the
therapeutic methods of the invention can be evaluated using the
screening assays described herein. In one embodiment, the
anti-cancer agents are evaluated in a cell-free system, e.g., a
cell lysate or in a reconstituted system. In other embodiments, the
anti-cancer agents are evaluated in a cell in culture, e.g., a cell
expressing a KIF5B-RET fusion (e.g., a mammalian cell, a tumor cell
or cell line, a recombinant cell). In yet other embodiments, the
anti-cancer agents are evaluated cell in vivo (a
KIF5B-RET-expressing cell present in a subject, e.g., an animal
subject (e.g., an in vivo animal model).
[0228] Exemplary parameters evaluated include one or more of:
[0229] (i) a change in binding activity, e.g., direct binding of
the candidate agent to a KIF5B-RET fusion polypeptide; a binding
competition between a known ligand and the candidate agent to a
KIF5B-RET fusion polypeptide;
[0230] (ii) a change in kinase activity, e.g., phosphorylation
levels of a KIF5B-RET fusion polypeptide (e.g., an increased or
decreased autophosphorylation); or a change in phosphorylation of a
target of a RET kinase, e.g., focal adhesion kinase (FAK),
persephin or glial derived neurotrophic factor (GDNF);
[0231] (iii) a change in an activity of a cell containing a
KIF5B-RET fusion (e.g., a tumor cell or a recombinant cell), e.g.,
a change in proliferation, morphology or tumorigenicity of the
cell;
[0232] (iv) a change in tumor present in an animal subject, e.g.,
size, appearance, proliferation, of the tumor; or
[0233] (v) a change in the level, e.g., expression level, of a
KIF5B-RET fusion polypeptide or nucleic acid molecule.
[0234] In one embodiment, a change in a cell free assay in the
presence of a candidate agent is evaluated. For example, an
activity of a KIF5B-RET fusion, or interaction of a KIF5B-RET
fusion with a downstream ligand can be detected.
[0235] In other embodiments, a change in an activity of a cell is
detected in a cell in culture, e.g., a cell expressing a KIF5B-RET
fusion (e.g., a mammalian cell, a tumor cell or cell line, a
recombinant cell). In one embodiment, the cell is a recombinant
cell that is modified to express a KIF5B-RET fusion nucleic acid,
e.g., is a recombinant cell transfected with a KIF5B-RET fusion
nucleic acid. The transfected cell can show a change in response to
the expressed KIF5B-RET 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, transformed morphology, in the
presence of the candidate agent can be indicative of an inhibitor
of a KIF5B-RET fusion. In other embodiments, a change in binding
activity or phosphorylation as described herein is detected.
[0236] 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, the animal model is a tumor containing animal or a
xenograft comprising cells expressing a KIF5B-RET fusion (e.g.,
tumorigenic cells expressing a KIF5B-RET fusion). The anti-cancer
agents can be administered to the animal subject and a change in
the tumor is detected. In one embodiment, the change in the tumor
includes one or more of a tumor growth, tumor size, tumor burden,
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.
[0237] The screening methods and assays are described in more
detail herein below.
Screening Methods
[0238] In another aspect, the invention features a method, or
assay, for screening for agents that modulate, e.g., inhibit, the
expression or activity of a KIF5B-RET fusion, e.g., a KIF5B-RET
fusion as described herein. The method includes contacting a
KIF5B-RET fusion, or a cell expressing a KIF5B-RET fusion, with a
candidate agent; and detecting a change in a parameter associated
with a KIF5B-RET fusion, e.g., a change in the expression or an
activity of the KIF5B-RET 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 KIF5B-RET fusion is
detected, the candidate agent is identified as an inhibitor. In
another embodiment, if an increase in expression or activity of the
KIF5B-RET fusion is detected, the candidate agent is identified as
an activator. In certain embodiments, the KIF5B-RET fusion is a
nucleic acid molecule or a polypeptide as described herein.
[0239] In one embodiment, the contacting step is effected in a
cell-free system, e.g., a cell lysate or in a reconstituted system.
In other embodiments, the contacting step is effected in a cell in
culture, e.g., a cell expressing a KIF5B-RET fusion (e.g., a
mammalian cell, a tumor cell or cell line, a recombinant cell). In
yet other embodiments, the contacting step is effected in a cell in
vivo (a KIF5B-RET-expressing cell present in a subject, e.g., an
animal subject (e.g., an in vivo animal model).
[0240] Exemplary parameters evaluated include one or more of:
[0241] (i) a change in binding activity, e.g., direct binding of
the candidate agent to a KIF5B-RET fusion polypeptide; a binding
competition between a known ligand and the candidate agent to a
KIF5B-RET fusion polypeptide;
[0242] (ii) a change in kinase activity, e.g., phosphorylation
levels of a KIF5B-RET fusion polypeptide (e.g., an increased or
decreased autophosphorylation); or a change in phosphorylation of a
target of a RET kinase, e.g., focal adhesion kinase (FAK),
persephin or glial derived neurotrophic factor (GDNF), In certain
embodiments, a change in kinase activity, e.g., phosphorylation, is
detected by any of Western blot (e.g., using an anti-KIF5B or
anti-RET antibody; a phosphor-specific antibody, detecting a shift
in the molecular weight of a KIF5B-RET fusion polypeptide), mass
spectrometry, immunoprecipitation, immunohistochemistry,
immunomagnetic beads, among others;
[0243] (iii) a change in an activity of a cell containing a
KIF5B-RET fusion (e.g., a tumor cell or a recombinant cell), e.g.,
a change in proliferation, morphology or tumorigenicity of the
cell;
[0244] (iv) a change in tumor present in an animal subject, e.g.,
size, appearance, proliferation, of the tumor; or
[0245] (v) a change in the level, e.g., expression level, of a
KIF5B-RET fusion polypeptide or nucleic acid molecule.
[0246] In one embodiment, a change in a cell free assay in the
presence of a candidate agent is evaluated. For example, an
activity of a KIF5B-RET fusion, or interaction of a KIF5B-RET
fusion with a downstream ligand can be detected. In one embodiment,
a KIF5B-RET fusion polypeptide is contacted with a ligand, e.g., in
solution, and a candidate agent is monitored for an ability to
modulate, e.g., inhibit, an interaction, e.g., binding, between the
KIF5B-RET fusion polypeptide and the ligand. In one exemplary
assay, purified KIF5B-RET fusion protein is contacted with a
ligand, e.g., in solution, and a candidate agent is monitored for
an ability to inhibit interaction of the fusion protein with the
ligand, or to inhibit phosphorylation of the ligand by the fusion
protein. An effect on an interaction between the fusion protein and
a ligand can be monitored by methods known in the art, such as by
absorbance, and an effect on phosphorylation of the ligand can be
assayed, e.g., by Western blot, immunoprecipitation, or
immunomagnetic beads.
[0247] In other embodiments, a change in an activity of a cell is
detected in a cell in culture, e.g., a cell expressing a KIF5B-RET
fusion (e.g., a mammalian cell, a tumor cell or cell line, a
recombinant cell). In one embodiment, the cell is a recombinant
cell that is modified to express a KIF5B-RET fusion nucleic acid,
e.g., is a recombinant cell transfected with a KIF5B-RET fusion
nucleic acid. The transfected cell can show a change in response to
the expressed KIF5B-RET 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, transformed morphology, in the
presence of the candidate agent can be indicative of an inhibitor
of a KIF5B-RET fusion. In other embodiments, a change in binding
activity or phosphorylation as described herein is detected.
[0248] In an exemplary cell-based assay, a nucleic acid comprising
a KIF5B-RET fusion can be expressed in a cell, such as a cell
(e.g., a mammalian cell) in culture. The cell containing a nucleic
acid expressing the KIF5B-RET fusion can be contacted with a
candidate agent, and the cell is monitored for an effect of the
candidate agent. A candidate agent that causes decreased cell
proliferation or cell death can be determined to be a candidate for
treating a tumor (e.g., a cancer) that carries a KIF5B-RET
fusion.
[0249] In one embodiment, a cell containing a nucleic acid
expressing a KIF5B-RET fusion can be monitored for expression of
the KIF5B-RET fusion protein. Protein expression can be monitored
by methods known in the art, such as by, e.g., mass spectrometry
(e.g., tandem mass spectrometry), a reporter assay (e.g., a
fluorescence-based assay), Western blot, and immunohistochemistry.
By one method, decreased KIF5B-RET expression is detected. A
candidate agent that causes decreased expression of the KIF5B-RET
fusion protein as compared to a cell that does not contain the
KIF5B-RET nucleic acid fusion can be determined to be a candidate
for treating a tumor (e.g., a cancer) that carries a KIF5B-RET
fusion.
[0250] A cell containing a nucleic acid expressing a KIF5B-RET
fusion can be monitored for altered KIF5B-RET kinase activity.
Kinase activity can be assayed by measuring the effect of a
candidate agent on a known RET kinase target protein, such as focal
adhesion kinase (FAK), persephin or GDNF (glial-cell-line-derived
neurotrophic factor).
[0251] 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, the animal model is a tumor containing animal or a
xenograft comprising cells expressing a KIF5B-RET fusion (e.g.,
tumorigenic cells expressing a KIF5B-RET fusion). The candidate
agent can be administered to the animal subject and a change in the
tumor is detected. In one embodiment, the change in the tumor
includes one or more of a tumor growth, tumor size, tumor burden,
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.
[0252] In one exemplary animal model, a xenograft is created by
injecting cells into mouse. A candidate agent is administered to
the mouse, e.g., by injection (such as subcutaneous,
intraperitoneal, or tail vein injection, or by injection directly
into the tumor) or oral delivery, and the tumor is observed to
determine an effect of the candidate anti-cancer agent. The health
of the animal is also monitored, such as to determine if an animal
treated with a candidate agent survives longer. A candidate agent
that causes growth of the tumor to slow or stop, or causes the
tumor to shrink in size, or causes decreased tumor burden, or
increases survival time, can be considered to be a candidate for
treating a tumor (e.g., a cancer) that carries a KIF5B-RET
fusion.
[0253] In another exemplary animal assay, cells expressing a
KIF5B-RET fusion are injected into the tail vein, e.g., of a mouse,
to induce metastasis. A candidate agent is administered to the
mouse, e.g., by injection (such as subcutaneous, intraperitoneal,
or tail vein injection, or by injection directly into the tumor) or
oral delivery, and the tumor is observed to determine an effect of
the candidate anti-cancer agent. A candidate agent that inhibits or
prevents or reduces metastasis, or increases survival time, can be
considered to be a candidate for treating a tumor (e.g., a cancer)
that carries a KIF5B-RET fusion.
[0254] Cell proliferation can be measured by methods known in the
art, such as PCNA (Proliferating cell nuclear antigen) assay,
5-bromodeoxyuridine (BrdUrd) incorporation, Ki-67 assay,
mitochondrial respiration, or propidium iodide staining. Cells can
also be measured for apoptosis, such as by use of a TUNEL (Terminal
Deoxynucleotide Transferase dUTP Nick End Labeling) assay. Cells
can also be assayed for presence of angiogenesis using methods
known in the art, such as by measuring endothelial tube formation
or by measuring the growth of blood vessels from subcutaneous
tissue, such as into a solid gel of basement membrane.
[0255] In other embodiments, a change in expression of a KIF5B-RET
fusion can be monitored by detecting the nucleic acid or protein
levels, e.g., using the methods described herein.
[0256] In certain embodiments, the screening methods described
herein can be repeated and/or combined. In one embodiment, a
candidate agent that is evaluated in a cell-free or cell-based
described herein can be further tested in an animal subject.
[0257] In one embodiment, the candidate agent is identified and
re-tested in the same or a different assay. For example, a test
compound is identified in an in vitro or cell-free system, and
re-tested in an animal model or a cell-based assay. Any order or
combination of assays can be used. For example, a high throughput
assay can be used in combination with an animal model or tissue
culture.
[0258] Candidate agents suitable for use in the screening assays
described herein include, e.g., small molecule compounds, nucleic
acids (e.g., siRNA, aptamers, short hairpin RNAs, antisense
oligonucleotides, ribozymes, antagomirs, microRNA mimics or DNA,
e.g., for gene therapy) or polypeptides, e.g., antibodies (e.g.,
full length antibodies or antigen-binding fragments thereof, Fab
fragments, or scFv fragments). The candidate anti-cancer agents can
be obtained from a library (e.g., a commercial library), or can be
rationally designed, such as to target an active site in a
functional domain of RET (e.g., the kinase domain of RET), or a
functional domain of KIF5B (e.g., the oligomerization domain or the
kinesin domain).
[0259] In other embodiments, the method, or assay, includes
providing a step based on proximity-dependent signal generation,
e.g., a two-hybrid assay that includes a first fusion protein
(e.g., a KIF5B-RET fusion protein), and a second fusion protein
(e.g., a ligand), contacting the two-hybrid assay with a test
compound, under conditions wherein said two hybrid assay detects a
change in the formation and/or stability of the complex, e.g., the
formation of the complex initiates transcription activation of a
reporter gene.
[0260] In one non-limiting example, the three-dimensional structure
of the active site of KIF5B-RET fusion is determined by
crystallizing the complex formed by the KIF5B-RET fusion and a
known inhibitor. Rational drug design is then used to identify new
test agents by making alterations in the structure of a known
inhibitor or by designing small molecule compounds that bind to the
active site of the KIF5B-RET fusion.
[0261] The candidate agents can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; peptoid libraries (libraries
of molecules having the functionalities of peptides, but with a
novel, non-peptide backbone which are resistant to enzymatic
degradation but which nevertheless remain bioactive; see, e.g.,
Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85);
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0262] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0263] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Nall Acad
Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0264] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule may simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label may be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means known in the
art (e.g., using a fluorimeter).
[0265] In another embodiment, determining the ability of the
KIF5B-RET fusion protein to bind to a target molecule can be
accomplished using real-time Biomolecular Interaction Analysis
(BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal.
Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.
Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the mass at the binding
surface (indicative of a binding event) result in alterations of
the refractive index of light near the surface (the optical
phenomenon of surface plasmon resonance (SPR)), resulting in a
detectable signal which can be used as an indication of real-time
reactions between biological molecules.
Nucleic Acid Inhibitors
[0266] In yet another embodiment, the KIF5B-RET fusion inhibitor
inhibits the expression of nucleic acid encoding a KIF5B-RET
fusion. Examples of such KIF5B-RET fusion inhibitors include
nucleic acid molecules, for example, antisense molecules,
ribozymes, siRNA, triple helix molecules that hybridize to a
nucleic acid encoding a KIF5B-RET fusion, or a transcription
regulatory region, and blocks or reduces mRNA expression of the
KIF5B-RET fusion.
[0267] In one embodiment, the nucleic acid antagonist is a siRNA
that targets mRNA encoding KIF5B-RET fusion. Other types of
antagonistic nucleic acids can also be used, e.g., a dsRNA, a
ribozyme, a triple-helix former, or an antisense nucleic acid.
Accordingly, isolated nucleic acid molecules that are nucleic acid
inhibitors, e.g., antisense, RNAi, to a KIF5B-RET fusion-encoding
nucleic acid molecule are provided.
[0268] An "antisense" nucleic acid can include a nucleotide
sequence which is complementary to a "sense" nucleic acid encoding
a protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
The antisense nucleic acid can be complementary to an entire
KIF5B-RET fusion coding strand, or to only a portion thereof. In
another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding KIF5B-RET fusion (e.g., the 5' and 3'
untranslated regions). Anti-sense agents can include, for example,
from about 8 to about 80 nucleobases (i.e. from about 8 to about 80
nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to
about 30 nucleobases. Anti-sense compounds include ribozymes,
external guide sequence (EGS) oligonucleotides (oligozymes), and
other short catalytic RNAs or catalytic oligonucleotides which
hybridize to the target nucleic acid and modulate its expression.
Anti-sense compounds can include a stretch of at least eight
consecutive nucleobases that are complementary to a sequence in the
target gene. An oligonucleotide need not be 100% complementary to
its target nucleic acid sequence to be specifically hybridizable.
An oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target interferes with the normal function
of the target molecule to cause a loss of utility, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment
or, in the case of in vitro assays, under conditions in which the
assays are conducted.
[0269] Hybridization of antisense oligonucleotides with mRNA can
interfere with one or more of the normal functions of mRNA. The
functions of mRNA to be interfered with include all key functions
such as, for example, translocation of the RNA to the site of
protein translation, translation of protein from the RNA, splicing
of the RNA to yield one or more mRNA species, and catalytic
activity which may be engaged in by the RNA. Binding of specific
protein(s) to the RNA may also be interfered with by antisense
oligonucleotide hybridization to the RNA.
[0270] Exemplary antisense compounds include DNA or RNA sequences
that specifically hybridize to the target nucleic acid, e.g., the
mRNA encoding KIF5B-RET fusion. The complementary region can extend
for between about 8 to about 80 nucleobases. The compounds can
include one or more modified nucleobases. Modified nucleobases are
known in the art. Descriptions of modified nucleic acid agents are
also available. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and
U.S. Pat. No. 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci
USA; Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff
and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer
Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36;
and Maher (1992) Bioassays 14:807-15.
[0271] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a KIF5B-RET
fusion to thereby inhibit expression of the protein, e.g., by
inhibiting transcription and/or translation. Alternatively,
antisense nucleic acid molecules can be modified to target selected
cells and then administered systemically. For systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies which bind to cell surface receptors or
antigens. The antisense nucleic acid molecules can also be
delivered to cells using the vectors described herein. To achieve
sufficient intracellular concentrations of the antisense molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0272] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0273] siRNAs are small double stranded RNAs (dsRNAs) that
optionally include overhangs. For example, the duplex region of an
siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20,
21, 22, 23, or 24 nucleotides in length. Typically, the siRNA
sequences are exactly complementary to the target mRNA. dsRNAs and
siRNAs in particular can be used to silence gene expression in
mammalian cells (e.g., human cells). siRNAs also include short
hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3'
overhangs. See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci.
USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA
98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et
al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al.
(2005), Nat. Biotechnol. 23(2):227-31; 20040086884; U.S.
20030166282; 20030143204; 20040038278; and 20030224432.
[0274] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
KIF5B-RET fusion-encoding nucleic acid can include one or more
sequences complementary to the nucleotide sequence of a KIF5B-RET
fusion cDNA disclosed herein (i.e., SEQ ID NO:1 or SEQ ID NO:3),
and a sequence having known catalytic sequence responsible for mRNA
cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach
(1988) Nature 334:585-591). For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide
sequence to be cleaved in a KIF5B-RET fusion-encoding mRNA. See,
e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S.
Pat. No. 5,116,742. Alternatively, KIF5B-RET fusion mRNA can be
used to select a catalytic RNA having a specific ribonuclease
activity from a pool of RNA molecules. See, e.g., Bartel, D. and
Szostak, J. W. (1993) Science 261:1411-1418.
[0275] KIF5B-RET fusion gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the KIF5B-RET fusion to form triple helical structures
that prevent transcription of the KIF5B-RET fusion gene in target
cells. See generally, Helene, C. (1991) Anticancer Drug Des.
6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences
that can be targeted for triple helix formation can be increased by
creating a so-called "switchback" nucleic acid molecule. Switchback
molecules are synthesized in an alternating 5'-3', 3'-5' manner,
such that they base pair with first one strand of a duplex and then
the other, eliminating the necessity for a sizeable stretch of
either purines or pyrimidines to be present on one strand of a
duplex.
[0276] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0277] A KIF5B-RET fusion nucleic acid molecule can be modified at
the base moiety, sugar moiety or phosphate backbone to improve,
e.g., the stability, hybridization, or solubility of the molecule.
For non-limiting examples of synthetic oligonucleotides with
modifications see Toulme (2001) Nature Biotech. 19:17 and Faria et
al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite
oligonucleotides can be effective antisense agents.
[0278] For example, the deoxyribose phosphate backbone of the
nucleic acid molecules can be modified to generate peptide nucleic
acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal
Chemistry 4: 5-23). As used herein, the terms "peptide nucleic
acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic,
in which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of a PNA can allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl.
Acad. Sci. 93: 14670-675.
[0279] PNAs of KIF5B-RET fusion nucleic acid molecules can be used
in therapeutic and diagnostic applications. For example, PNAs can
be used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of KIF5B-RET fusion nucleic acid molecules can also be used in the
analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., S1 nucleases
(Hyrup B. et al. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe supra).
[0280] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; WO88/09810) or the blood-brain barrier (see,
e.g., WO 89/10134). In addition, oligonucleotides can be modified
with hybridization-triggered cleavage agents (see, e.g., Krol et
al. (1988) Bio-Techniques 6:958-976) or intercalating agents (See,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
Evaluation of Subjects
[0281] Subjects, e.g., patients, can be evaluated for the presence
of a KIF5B-RET fusion. A patient can be evaluated, for example, by
determining the genomic sequence of the patient, e.g., by an NGS
method. Alternatively, or in addition, evaluation of a patient can
include directly assaying for the presence of a KIF5B-RET fusion in
the patient, such as by an assay to detect a KIF5B-RET nucleic acid
(e.g., DNA or RNA), such as by, Southern blot, Northern blot, or
RT-PCR, e.g., qRT-PCR. Alternatively, or in addition, a patient can
be evaluated for the presence of a KIF5B-RET protein fusion, such
as by immunohistochemistry, Western blot, immunoprecipitation, or
immunomagnetic bead assay.
[0282] Evaluation of a patient can also include a cytogenetic
assay, such as by fluorescence in situ hybridization (FISH), to
identify the chromosomal rearrangement resulting in the KIF5B-RET
fusion. For example, to perform FISH, at least a first probe tagged
with a first detectable label can be designed to target KIF5B, such
as in one or more of exons 1-15 of KIF5B (e.g., the exons
containing the part of the protein that includes the kinesin motor
domain and the oligomeric domain), and at least a second probe
tagged with a second detectable label can be designed to target
RET, such as in one or more of exons 12-25 of RET (e.g., the exons
containing the part of the protein that includes the tyrosine
kinase domain). The at least one first probe and the at least one
second probe will be closer together in patients who carry the
KIF5B-RET fusion than in patients who do not carry the KIF5B-RET
fusion.
[0283] Additional methods for KIF5B-RET fusion detection are
provided below.
[0284] In one aspect, the results of a clinical trial, e.g., a
successful or unsuccessful clinical trial, can be repurposed to
identify agents that target a KIF5B-RET fusion. By one exemplary
method, a candidate agent used in a clinical trial can be
reevaluated to determine if the agent in the trial targets a
KIF5B-RET fusion, or is effective to treat a tumor containing a
KIF5B-RET fusion. For example, subjects who participated in a
clinical trial for an agent, such as a kinase inhibitor, can be
identified. Patients who experienced an improvement in symptoms,
e.g., cancer (e.g., lung cancer) symptoms, such as decreased tumor
size, or decreased rate of tumor growth, can be evaluated for the
presence of a KIF5B-RET fusion. Patients who did not experience an
improvement in cancer symptoms can also be evaluated for the
presence of a KIF5B-RET fusion. Where patients carrying a KIF5B-RET
fusion are found to have been more likely to respond to the test
agent than patients who did not carry a KIF5B-RET fusion, then the
agent is determined to be an appropriate treatment option for a
patient carrying the KIF5B-RET fusion.
[0285] "Reevaluation" of patients can include, for example,
determining the genomic sequence of the patients, or a subset of
the clinical trial patients, e.g., by an NGS method. Alternatively,
or in addition, reevaluation of the patients can include directly
assaying for the presence of a KIF5B-RET fusion in the patient,
such as by an assay to detect a KIF5B-RET nucleic acid (e.g., RNA),
such as by RT-PCR, e.g., qRT-PCR. Alternatively, or in addition, a
patient can be evaluated for the presence of a KIF5B-RET protein
fusion, such as by immunohistochemistry, Western blot,
immunoprecipitation, or immunomagnetic bead assay.
[0286] Clinical trials suitable for repurposing as described above
include trials that tested RET inhibitors, tyrosine kinase
inhibitors, multikinase inhibitors, and drugs purported to act
upstream or downstream of RET in a pathway involving RET. Other
clinical trials suitable for repurposing as described above include
trials that tested KIF5B inhibitors, kinesin inhibitors, inhibitors
of cell trafficking and drugs purported to act upstream or
downstream of KIF5B in a pathway involving KIF5B.
Methods for Detection of KIF5B-RET Fusion Nucleic Acids and
Polypeptides
[0287] Methods for evaluating a KIF5B-RET gene, mutations and/or
gene products are known to those of skill in the art. In one
embodiment, the KIF5B-RET fusion is detected in a nucleic acid
molecule by a method chosen from one or more of: nucleic acid
hybridization assay, amplification-based assays (e.g., polymerase
chain reaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing,
screening analysis (including metaphase cytogenetic analysis by
standard karyotype methods, FISH (e.g., break away FISH), spectral
karyotyping or MFISH, comparative genomic hybridization), in situ
hybridization, SSP, HPLC or mass-spectrometric genotyping.
[0288] Additional exemplary methods include, traditional "direct
probe" methods such as Southern blots or in situ hybridization
(e.g., fluorescence in situ hybridization (FISH) and FISH plus
SKY), and "comparative probe" methods such as comparative genomic
hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH,
can be used. The methods can be used in a wide variety of formats
including, but not limited to, substrate (e.g., membrane or glass)
bound methods or array-based approaches.
[0289] In certain embodiments, the evaluation methods include the
probes/primers described herein.
[0290] In one embodiment, probes/primers can be designed to detect
a KIF5B/RET fusion. The KIF5B probes/primers can be from
nucleotides 1-1725 of SEQ ID NO:1 (e.g., can hybridize to the
nucleotides encoding exons 1-15 of the KIF5B protein). These
probes/primers are suitable, e.g., for FISH or PCR amplification.
The RET probes/primers can be from nucleotides 1726-2934 of SEQ ID
NO:1 (e.g., can hybridize to the nucleotides encoding exons 12-20
of the RET protein). These probes/primers are suitable, e.g., for
FISH or PCR amplification. For PCR, e.g., to amply the region
including the KIF5B/RET fusion junction, forward primers can be
designed to hybridize to KIF5B sequence from nucleotides 1-1725 of
SEQ ID NO:1, and reverse primers can be designed to hybridize from
nucleotides 1726-2934 of SEQ ID NO:1.
[0291] In another embodiment, probes/primers can be designed to
detect a RET/KIF5B fusion. The RET probes/primers can be from
nucleotides 1-2138 of SEQ ID NO:3 (i.e., can hybridize to the
nucleotides encoding exons 1-11 of the RET protein). These
probes/primers are suitable, e.g., for FISH or PCR amplification.
The KIF5B probes/primers can be from nucleotides 2139-3360 of SEQ
ID NO:3 (i.e., can hybridize to the nucleotides encoding exons
16-26 of the KIF5B protein). These probes/primers are suitable,
e.g., for FISH or PCR amplification. For PCR, e.g., to amply the
region including the RET/KIF5B fusion junction, forward primers can
be designed to hybridize to Ret sequence from nucleotides 1-2138 of
SEQ ID NO:3, and reverse primers can be designed to hybridize from
nucleotides 2139-3360 of SEQ ID NO:3.
[0292] In one embodiment, FISH analysis is used to identify the
chromosomal rearrangement resulting in the KIF5B-RET fusion as
described above. For example, to perform FISH, at least a first
probe tagged with a first detectable label can be designed to
target KIF5B, such as in one or more of exons 1-15 of KIF5B (e.g.,
the exons containing the part of the protein that includes the
kinesin motor domain and the oligomeric domain), and at least a
second probe tagged with a second detectable label can be designed
to target RET, such as in one or more of exons 12-25 of RET (e.g.,
the exons containing the part of the protein that includes the
tyrosine kinase domain). The at least one first probe and the at
least one second probe will be closer together in a subject who
carries the KIF5B-RET fusion compared to a subject who does not
carry the KIF5B-RET fusion.
[0293] In one approach, a variation of a FISH assay, e.g.,
"break-away FISH", is used to evaluate a patient. By this method,
at least one probe targeting the RET intron 11/RET exon 12 junction
and at least one probe targeting KIF5B, e.g., at one or more of
exons 1-15, and or introns 1-14, are utilized. In normal cells,
both probes with be observed (or a secondary color will be observed
due to the close proximity of the KIF5B and RET genes), and only
the KIF5B probe will be observed when the translocation occurs.
Other variations of the FISH method known in the art are suitable
for evaluating a patient.
[0294] Probes are used that contain DNA segments that are
essentially complementary to DNA base sequences existing in
different portions of chromosomes. Examples of probes useful
according to the invention, and labeling and hybridization of
probes to samples are described in two U.S. patents to Vysis, Inc.
U.S. Pat. Nos. 5,491,224 and 6,277,569 to Bittner, et al.
[0295] Additional protocols for FISH detection are described
below.
[0296] Chromosomal probes are typically about 50 to about 10.sup.5
nucleotides in length. Longer probes typically comprise smaller
fragments of about 100 to about 500 nucleotides in length. Probes
that hybridize with centromeric DNA and locus-specific DNA are
available commercially, for example, from Vysis, Inc. (Downers
Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.) or from
Cytocell (Oxfordshire, UK). Alternatively, probes can be made
non-commercially from chromosomal or genomic DNA through standard
techniques. For example, sources of DNA that can be used include
genomic DNA, cloned DNA sequences, somatic cell hybrids that
contain one, or a part of one, chromosome (e.g., human chromsome)
along with the normal chromosome complement of the host, and
chromosomes purified by flow cytometry or microdissection. The
region of interest can be isolated through cloning, or by
site-specific amplification via the polymerase chain reaction
(PCR). See, for example, Nath and Johnson, Biotechnic Histochem.,
1998, 73(1):6-22, Wheeless et al., Cytometry 1994, 17:319-326, and
U.S. Pat. No. 5,491,224.
[0297] The probes to be used hybridize to a specific region of a
chromosome to determine whether a cytogenetic abnormality is
present in this region. One type of cytogenetic abnormality is a
deletion. Although deletions can be of one or more entire
chromosomes, deletions normally involve loss of part of one or more
chromosomes. If the entire region of a chromosome that is contained
in a probe is deleted from a cell, hybridization of that probe to
the DNA from the cell will normally not occur and no signal will be
present on that chromosome. If the region of a chromosome that is
partially contained within a probe is deleted from a cell,
hybridization of that probe to the DNA from the cell can still
occur, but less of a signal can be present. For example, the loss
of a signal is compared to probe hybridization to DNA from control
cells that do not contain the genetic abnormalities which the
probes are intended to detect. In some embodiments, at least 1, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, or more cells are enumerated for presence
of the cytogenetic abnormality.
[0298] Cytogenetic abnormalities to be detected can include, but
are not limited to, non-reciprocal translocations,
intra-chromosomal inversions, point mutations, deletions, gene copy
number changes, gene expression level changes, and germ line
mutations. In particular, one type of cytogenetic abnormality is a
duplication. Duplications can be of entire chromosomes, or of
regions smaller than an entire chromosome. If the region of a
chromosome that is contained in a probe is duplicated in a cell,
hybridization of that probe to the DNA from the cell will normally
produce at least one additional signal as compared to the number of
signals present in control cells with no abnormality of the
chromosomal region contained in the probe.
[0299] Chromosomal probes are labeled so that the chromosomal
region to which they hybridize can be detected. Probes typically
are directly labeled with a fluorophore, an organic molecule that
fluoresces after absorbing light of lower wavelength/higher energy.
The fluorophore allows the probe to be visualized without a
secondary detection molecule. After covalently attaching a
fluorophore to a nucleotide, the nucleotide can be directly
incorporated into the probe with standard techniques such as nick
translation, random priming, and PCR labeling. Alternatively,
deoxycytidine nucleotides within the probe can be transaminated
with a linker. The fluorophore then is covalently attached to the
transaminated deoxycytidine nucleotides. See, U.S. Pat. No.
5,491,224.
[0300] U.S. Pat. No. 5,491,224 describes probe labeling as a number
of the cytosine residues having a fluorescent label covalently
bonded thereto. The number of fluorescently labeled cytosine bases
is sufficient to generate a detectable fluorescent signal while the
individual so labeled DNA segments essentially retain their
specific complementary binding (hybridizing) properties with
respect to the chromosome or chromosome region to be detected. Such
probes are made by taking the unlabeled DNA probe segment,
transaminating with a linking group a number of deoxycytidine
nucleotides in the segment, covalently bonding a fluorescent label
to at least a portion of the transaminated deoxycytidine bases.
[0301] Probes can also be labeled by nick translation, random
primer labeling or PCR labeling. Labeling is done using either
fluorescent (direct)- or haptene (indirect)-labeled nucleotides.
Representative, non-limiting examples of labels include:
AMCA-6-dUTP, CascadeBlue-4-dUTP, Fluorescein-12-dUTP,
Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP, Cy5-dUTP, Biotin
(BIO)-11-dUTP, Digoxygenin (DIG)-11-dUTP or Dinitrophenyl
(DNP)-11-dUTP.
[0302] Probes also can be indirectly labeled with biotin or
digoxygenin, or labeled with radioactive isotopes such as .sup.32p
and ..sup.3H, although secondary detection molecules or further
processing then is required to visualize the probes. For example, a
probe labeled with biotin can be detected by avidin conjugated to a
detectable marker. For example, avidin can be conjugated to an
enzymatic marker such as alkaline phosphatase or horseradish
peroxidase. Enzymatic markers can be detected in standard
colorimetric reactions using a substrate and/or a catalyst for the
enzyme. Catalysts for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
Diaminobenzoate can be used as a catalyst for horseradish
peroxidase.
[0303] Probes can also be prepared such that a fluorescent or other
label is not part of the DNA before or during the hybridization,
and is added after hybridization to detect the probe hybridized to
a chromosome. For example, probes can be used that have antigenic
molecules incorporated into the DNA. After hybridization, these
antigenic molecules are detected using specific antibodies reactive
with the antigenic molecules. Such antibodies can themselves
incorporate a fluorochrome, or can be detected using a second
antibody with a bound fluorochrome.
[0304] However treated or modified, the probe DNA is commonly
purified in order to remove unreacted, residual products (e.g.,
fluorochrome molecules not incorporated into the DNA) before use in
hybridization.
[0305] Prior to hybridization, chromosomal probes are denatured
according to methods well known in the art. Probes can be
hybridized or annealed to the chromosomal DNA under hybridizing
conditions. "Hybridizing conditions" are conditions that facilitate
annealing between a probe and target chromosomal DNA. Since
annealing of different probes will vary depending on probe length,
base concentration and the like, annealing is facilitated by
varying probe concentration, hybridization temperature, salt
concentration and other factors well known in the art.
[0306] Hybridization conditions are facilitated by varying the
concentrations, base compositions, complexities, and lengths of the
probes, as well as salt concentrations, temperatures, and length of
incubation. For example, in situ hybridizations are typically
performed in hybridization buffer containing 1-2.times.SSC, 50-65%
formamide and blocking DNA to suppress non-specific hybridization.
In general, hybridization conditions, as described above, include
temperatures of about 25.degree. C. to about 55.degree. C., and
incubation lengths of about 0.5 hours to about 96 hours.
[0307] Non-specific binding of chromosomal probes to DNA outside of
the target region can be removed by a series of washes. Temperature
and concentration of salt in each wash are varied to control
stringency of the washes. For example, for high stringency
conditions, washes can be carried out at about 65.degree. C. to
about 80.degree. C., using 0.2.times. to about 2.times.SSC, and
about 0.1% to about 1% of a non-ionic detergent such as Nonidet
P-40 (NP40). Stringency can be lowered by decreasing the
temperature of the washes or by increasing the concentration of
salt in the washes. In some applications it is necessary to block
the hybridization capacity of repetitive sequences. Thus, in some
embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block
non-specific hybridization. After washing, the slide is allowed to
drain and air dry, then mounting medium, a counterstain such as
DAPI, and a coverslip are applied to the slide. Slides can be
viewed immediately or stored at -20.degree. C. before
examination.
[0308] For fluorescent probes used in fluorescence in situ
hybridization (FISH) techniques, fluorescence can be viewed with a
fluorescence microscope equipped with an appropriate filter for
each fluorophore, or by using dual or triple band-pass filter sets
to observe multiple fluorophores. See, for example, U.S. Pat. No.
5,776,688. Alternatively, techniques such as flow cytometry can be
used to examine the hybridization pattern of the chromosomal
probes.
[0309] In CGH methods, a first collection of nucleic acids (e.g.,
from a sample, e.g., a possible tumor) is labeled with a first
label, while a second collection of nucleic acids (e.g., a control,
e.g., from a healthy cell/tissue) is labeled with a second label.
The ratio of hybridization of the nucleic acids is determined by
the ratio of the two (first and second) labels binding to each
fiber in the array. Where there are chromosomal deletions or
multiplications, differences in the ratio of the signals from the
two labels will be detected and the ratio will provide a measure of
the copy number. Array-based CGH can also be performed with
single-color labeling (as opposed to labeling the control and the
possible tumor sample with two different dyes and mixing them prior
to hybridization, which will yield a ratio due to competitive
hybridization of probes on the arrays). In single color CGH, the
control is labeled and hybridized to one array and absolute signals
are read, and the possible tumor sample is labeled and hybridized
to a second array (with identical content) and absolute signals are
read. Copy number difference is calculated based on absolute
signals from the two arrays.
[0310] Hybridization protocols suitable for use with the methods of
the invention are described, e.g., in Albertson (1984) EMBO J. 3:
1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142;
EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In
situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J.
(1994), etc. In one embodiment, the hybridization protocol of
Pinkel, et al. (1998) Nature Genetics 20: 207-211, or of
Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is
used. Array-based CGH is described in U.S. Pat. No. 6,455,258, the
contents of each of which are incorporated herein by reference.
[0311] In still another embodiment, amplification-based assays can
be used to measure presence/absence and copy number. In such
amplification-based assays, the nucleic acid sequences act as a
template in an amplification reaction (e.g., Polymerase Chain
Reaction (PCR). In a quantitative amplification, the amount of
amplification product will be proportional to the amount of
template in the original sample. Comparison to appropriate
controls, e.g., healthy tissue, provides a measure of the copy
number.
[0312] Methods of "quantitative" amplification are well known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that can
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided in Innis, et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.). Measurement of DNA copy number at microsatellite loci
using quantitative PCR analysis is described in Ginzonger, et al.
(2000) Cancer Research 60:5405-5409. The known nucleic acid
sequence for the genes is sufficient to enable one of skill in the
art to routinely select primers to amplify any portion of the gene.
Fluorogenic quantitative PCR can also be used in the methods of the
invention. In fluorogenic quantitative PCR, quantitation is based
on amount of fluorescence signals, e.g., TaqMan and sybr green.
[0313] Other suitable amplification methods include, but are not
limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4: 560, Landegren, et al. (1988) Science 241:1077, and
Barringer et al. (1990) Gene 89: 117), transcription amplification
(Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173),
self-sustained sequence replication (Guatelli, et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR,
etc.
Nucleic Acid Samples
[0314] A variety of tissue samples can be the source of the nucleic
acid samples used in the present methods. Genomic or subgenomic DNA
fragments can be isolated from a subject's sample (e.g., a tumor
sample, a normal adjacent tissue (NAT), a blood sample or any
normal control)). In certain embodiments, the tissue sample is
preserved as a frozen sample or as formaldehyde- or
paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation.
For example, the sample can be embedded in a matrix, e.g., an FFPE
block or a frozen sample. The isolating step can include
flow-sorting of individual chromosomes; and/or micro-dissecting a
subject's sample (e.g., a tumor sample, a NAT, a blood sample).
[0315] Protocols for DNA isolation from a tissue sample are known
in the art. Additional methods to isolate nucleic acids (e.g., DNA)
from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded
(FFPE) tissues are disclosed, e.g., in Cronin M. et al., (2004) Am
J Pathol. 164(1):35-42; Masuda N. et al., (1999) Nucleic Acids Res.
27(22):4436-4443; Specht K. et al., (2001) Am J Pathol.
158(2):419-429, Ambion RecoverAll.TM. Total Nucleic Acid Isolation
Protocol (Ambion, Cat. No. AM1975, September 2008), and QIAamp.RTM.
DNA FFPE Tissue Handbook (Qiagen, Cat. No. 37625, October 2007).
RecoverAll.TM. Total Nucleic Acid Isolation Kit uses xylene at
elevated temperatures to solubilize paraffin-embedded samples and a
glass-fiber filter to capture nucleic acids. QIAamp.RTM. DNA FFPE
Tissue Kit uses QIAamp.RTM. DNA Micro technology for purification
of genomic and mitochondrial DNA.
[0316] The isolated nucleic acid samples (e.g., genomic DNA
samples) can be fragmented or sheared by practicing routine
techniques. For example, genomic DNA can be fragmented by physical
shearing methods, enzymatic cleavage methods, chemical cleavage
methods, and other methods well known to those skilled in the art.
The nucleic acid library can contain all or substantially all of
the complexity of the genome. The term "substantially all" in this
context refers to the possibility that there can in practice be
some unwanted loss of genome complexity during the initial steps of
the procedure. The methods described herein also are useful in
cases where the nucleic acid library is a portion of the genome,
i.e., where the complexity of the genome is reduced by design. In
some embodiments, any selected portion of the genome can be used
with the methods described herein. In certain embodiments, the
entire exome or a subset thereof is isolated.
[0317] Methods can further include isolating a nucleic acid sample
to provide a library (e.g., a nucleic acid library). In certain
embodiments, the nucleic acid sample includes whole genomic,
subgenomic fragments, or both. The isolated nucleic acid samples
can be used to prepare nucleic acid libraries. Thus, in one
embodiment, the methods featured in the invention further include
isolating a nucleic acid sample to provide a library (e.g., a
nucleic acid library as described herein). Protocols for isolating
and preparing libraries from whole genomic or subgenomic fragments
are known in the art (e.g., Illumina's genomic DNA sample
preparation kit). In certain embodiments, the genomic or subgenomic
DNA fragment is isolated from a subject's sample (e.g., a tumor
sample, a normal adjacent tissue (NAT), a blood sample or any
normal control)). In one embodiment, the sample (e.g., the tumor or
NAT sample) is a preserved. For example, the sample is embedded in
a matrix, e.g., an FFPE block or a frozen sample. In certain
embodiments, the isolating step includes flow-sorting of individual
chromosomes; and/or microdissecting a subject's sample (e.g., a
tumor sample, a NAT, a blood sample). In certain embodiments, the
nucleic acid sample used to generate the nucleic acid library is
less than 5, less than 1 microgram, less than 500 ng, less than 200
ng, less than 100 ng, less than 50 ng or less than 20 ng (e.g., 10
ng or less).
[0318] In still other embodiments, the nucleic acid sample used to
generate the library includes RNA or cDNA derived from RNA. In some
embodiments, the RNA includes total cellular RNA. In other
embodiments, certain abundant RNA sequences (e.g., ribosomal RNAs)
have been depleted. In some embodiments, the poly(A)-tailed mRNA
fraction in the total RNA preparation has been enriched. In some
embodiments, the cDNA is produced by random-primed cDNA synthesis
methods. In other embodiments, the cDNA synthesis is initiated at
the poly(A) tail of mature mRNAs by priming by oligo(dT)-containing
oligonucleotides. Methods for depletion, poly(A) enrichment, and
cDNA synthesis are well known to those skilled in the art.
[0319] The method can further include amplifying the nucleic acid
sample (e.g., DNA or RNA sample) by specific or non-specific
nucleic acid amplification methods that are well known to those
skilled in the art. In some embodiments, certain embodiments, the
nucleic acid sample is amplified, e.g., by whole-genome
amplification methods such as random-primed strand-displacement
amplification.
[0320] In other embodiments, the nucleic acid sample is fragmented
or sheared by physical or enzymatic methods and ligated to
synthetic adapters, size-selected (e.g., by preparative gel
electrophoresis) and amplified (e.g., by PCR). In other
embodiments, the fragmented and adapter-ligated group of nucleic
acids is used without explicit size selection or amplification
prior to hybrid selection.
[0321] In other embodiments, the isolated DNA (e.g., the genomic
DNA) is fragmented or sheared. In some embodiments, the library
includes less than 50% of genomic DNA, such as a subfraction of
genomic DNA that is a reduced representation or a defined portion
of a genome, e.g., that has been subfractionated by other means. In
other embodiments, the library includes all or substantially all
genomic DNA.
[0322] In some embodiments, the library includes less than 50% of
genomic DNA, such as a subfraction of genomic DNA that is a reduced
representation or a defined portion of a genome, e.g., that has
been subfractionated by other means. In other embodiments, the
library includes all or substantially all genomic DNA. Protocols
for isolating and preparing libraries from whole genomic or
subgenomic fragments are known in the art (e.g., Illumina's genomic
DNA sample preparation kit). Alternative DNA shearing methods can
be more automatable and/or more efficient (e.g., with degraded FFPE
samples). Alternatives to DNA shearing methods can also be used to
avoid a ligation step during library preparation.
[0323] The methods described herein can be performed using a small
amount of nucleic acids, e.g., when the amount of source DNA is
limiting (e.g., even after whole-genome amplification). In one
embodiment, the nucleic acid comprises less than about 5 .mu.g, 4
.mu.g, 3 .mu.g, 2 .mu.g, 1 .mu.g, 0.8 .mu.g, 0.7 .mu.g, 0.6 .mu.g,
0.5 .mu.g, or 400 ng, 300 ng, 200 ng, 100 ng, 50 ng, or 20 ng or
less of nucleic acid sample. For example, to prepare 500 ng of
hybridization-ready nucleic acids, one typically begins with 3
.mu.g of genomic DNA. One can start with less, however, if one
amplifies the genomic DNA (e.g., using PCR) before the step of
solution hybridization. Thus it is possible, but not essential, to
amplify the genomic DNA before solution hybridization.
[0324] In some embodiments, a library is generated using DNA (e.g.,
genomic DNA) from a sample tissue, and a corresponding library is
generated with RNA (or cDNA) isolated from the same sample
tissue.
Design of Baits
[0325] A bait can be a nucleic acid molecule, e.g., a DNA or RNA
molecule, which can hybridize to (e.g., be complementary to), and
thereby allow capture of a target nucleic acid. In one embodiment,
a bait is an RNA molecule. In other embodiments, a bait includes a
binding entity, e.g., an affinity tag, that allows capture and
separation, e.g., by binding to a binding entity, of a hybrid
formed by a bait and a nucleic acid hybridized to the bait. In one
embodiment, a bait is suitable for solution phase
hybridization.
[0326] Baits can be produced and used by methods and hybridization
conditions as described in US 2010/0029498 and Gnirke, A. et al.
(2009) Nat Biotechnol. 27(2):182-189, and U.S. Ser. No. 61/428,568,
filed Dec. 30, 2010, incorporated herein by reference. For example,
biotinylated RNA baits can be produced by obtaining a pool of
synthetic long oligonucleotides, originally synthesized on a
microarray, and amplifying the oligonucleotides to produce the bait
sequences. In some embodiments, the baits are produced by adding an
RNA polymerase promoter sequence at one end of the bait sequences,
and synthesizing RNA sequences using RNA polymerase. In one
embodiment, libraries of synthetic oligodeoxynucleotides can be
obtained from commercial suppliers, such as Agilent Technologies,
Inc., and amplified using known nucleic acid amplification
methods.
[0327] Each bait sequence can include a target-specific (e.g., a
member-specific) bait sequence and universal tails on each end. As
used herein, the term "bait sequence" can refer to the
target-specific bait sequence or the entire oligonucleotide
including the target-specific "bait sequence" and other nucleotides
of the oligonucleotide. In one embodiment, a target-specific bait
hybridizes to a nucleic acid sequence comprising a nucleic acid
sequence in intron 15 of KIF5B, in intron 11 of RET, or a fusion
junction joining exon 15 of KIF5B and exon 12 of RET.
[0328] In one embodiment, the bait is an oligonucleotide about 200
nucleotides in length, of which 170 nucleotides are target-specific
"bait sequence". The other 30 nucleotides (e.g., 15 nucleotides on
each end) are universal arbitrary tails used for PCR amplification.
The tails can be any sequence selected by the user. For example,
the pool of synthetic oligonucleotides can include oligonucleotides
of the sequence of 5'-ATCGCACCAGCGTGTN.sub.170CACTGCGGCTCCTCA-3'
with N.sub.170 indicating the target-specific bait sequences.
[0329] The bait sequences described herein can be used for
selection of exons and short target sequences. In one embodiment,
the bait is between about 100 nucleotides and 300 nucleotides in
length. In another embodiment, the bait is between about 130
nucleotides and 230 nucleotides in length. In yet another
embodiment, the bait is between about 150 nucleotides and 200
nucleotides in length. The target-specific sequences in the baits,
e.g., for selection of exons and short target sequences, are
between about 40 nucleotides and 1000 nucleotides in length. In one
embodiment, the target-specific sequence is between about 70
nucleotides and 300 nucleotides in length. In another embodiment,
the target-specific sequence is between about 100 nucleotides and
200 nucleotides in length. In yet another embodiment, the
target-specific sequence is between about 120 nucleotides and 170
nucleotides in length.
Sequencing
[0330] The invention also includes methods of sequencing nucleic
acids. In one embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence at least a
portion of a KIF5B-RET fusion. In one embodiment, the KIF5B-RET
fusion sequence is compared to a corresponding reference (control)
sequence.
[0331] In one embodiment, the sequence of the KIF5B-RET fusion
nucleic acid molecule is determined by a method that includes one
or more of: hybridizing an oligonucleotide, e.g., an allele
specific oligonucleotide for one alteration described herein to
said nucleic acid; hybridizing a primer, or a primer set (e.g., a
primer pair), that amplifies a region comprising the mutation or a
fusion junction of the allele; amplifying, e.g., specifically
amplifying, a region comprising the mutation or a fusion junction
of the allele; attaching an adapter oligonucleotide to one end of a
nucleic acid that comprises the mutation or a fusion junction of
the allele; generating an optical, e.g., a colorimetric signal,
specific to the presence of the one of the mutation or fusion
junction; hybridizing a nucleic acid comprising the mutation or
fusion junction to a second nucleic acid, e.g., a second nucleic
acid attached to a substrate; generating a signal, e.g., an
electrical or fluorescent signal, specific to the presence of the
mutation or fusion junction; and incorporating a nucleotide into an
oligonucleotide that is hybridized to a nucleic acid that contains
the mutation or fusion junction.
[0332] In another embodiment, the sequence is determined by a
method that comprises one or more of: determining the nucleotide
sequence from an individual nucleic acid molecule, e.g., where a
signal corresponding to the sequence is derived from a single
molecule as opposed, e.g., from a sum of signals from a plurality
of clonally expanded molecules; determining the nucleotide sequence
of clonally expanded proxies for individual nucleic acid molecules;
massively parallel short-read sequencing; template-based
sequencing; pyrosequencing; real-time sequencing comprising imaging
the continuous incorporation of dye-labeling nucleotides during DNA
synthesis; nanopore sequencing; sequencing by hybridization;
nano-transistor array based sequencing; polony sequencing; scanning
tunneling microscopy (STM) based sequencing; or nanowire-molecule
sensor based sequencing.
[0333] Any method of sequencing known in the art can be used.
Exemplary sequencing reactions include those based on techniques
developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977)
74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci
74:5463). Any of a variety of automated sequencing procedures can
be utilized when performing the assays (Biotechniques (1995)
19:448), including sequencing by mass spectrometry (see, for
example, U.S. Pat. No. 5,547,835 and international patent
application Publication Number WO 94/16101, entitled DNA Sequencing
by Mass Spectrometry by H. Koster; U.S. Pat. No. 5,547,835 and
international patent application Publication Number WO 94/21822
entitled DNA Sequencing by Mass Spectrometry Via Exonuclease
Degradation by H. Koster), and U.S. Pat. No. 5,605,798 and
International Patent Application No. PCT/US96/03651 entitled DNA
Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et al.
(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl
Biochem Biotechnol 38:147-159).
[0334] Sequencing of nucleic acid molecules can also be carried out
using next-generation sequencing (NGS). Next-generation sequencing
includes any sequencing method that determines the nucleotide
sequence of either individual nucleic acid molecules or clonally
expanded proxies for individual nucleic acid molecules in a highly
parallel fashion (e.g., greater than 10.sup.5 molecules are
sequenced simultaneously). In one embodiment, the relative
abundance of the nucleic acid species in the library can be
estimated by counting the relative number of occurrences of their
cognate sequences in the data generated by the sequencing
experiment. Next generation sequencing methods are known in the
art, and are described, e.g., in Metzker, M. (2010) Nature
Biotechnology Reviews 11:31-46, incorporated herein by
reference.
[0335] In one embodiment, the next-generation sequencing allows for
the determination of the nucleotide sequence of an individual
nucleic acid molecule (e.g., Helicos BioSciences' HeliScope Gene
Sequencing system, and Pacific Biosciences' PacBio RS system). In
other embodiments, the sequencing method determines the nucleotide
sequence of clonally expanded proxies for individual nucleic acid
molecules (e.g., the Solexa sequencer, Illumina Inc., San Diego,
Calif.; 454 Life Sciences (Branford, Conn.), and Ion Torrent).
e.g., massively parallel short-read sequencing (e.g., the Solexa
sequencer, Illumina Inc., San Diego, Calif.), which generates more
bases of sequence per sequencing unit than other sequencing methods
that generate fewer but longer reads. Other methods or machines for
next-generation sequencing include, but are not limited to, the
sequencers provided by 454 Life Sciences (Branford, Conn.), Applied
Biosystems (Foster City, Calif.; SOLiD sequencer), and Helicos
BioSciences Corporation (Cambridge, Mass.).
[0336] Platforms for next-generation sequencing include, but are
not limited to, Roche/454's Genome Sequencer (GS) FLX System,
Illumina/Solexa's Genome Analyzer (GA), Life/APG's Support
Oligonucleotide Ligation Detection (SOLiD) system, Polonator's
G.007 system, Helicos BioSciences' HeliScope Gene Sequencing
system, and Pacific Biosciences' PacBio RS system.
[0337] NGS technologies can include one or more of steps, e.g.,
template preparation, sequencing and imaging, and data
analysis.
Template Preparation
[0338] Methods for template preparation can include steps such as
randomly breaking nucleic acids (e.g., genomic DNA or cDNA) into
smaller sizes and generating sequencing templates (e.g., fragment
templates or mate-pair templates). The spatially separated
templates can be attached or immobilized to a solid surface or
support, allowing massive amounts of sequencing reactions to be
performed simultaneously. Types of templates that can be used for
NGS reactions include, e.g., clonally amplified templates
originating from single DNA molecules, and single DNA molecule
templates.
[0339] Methods for preparing clonally amplified templates include,
e.g., emulsion PCR (emPCR) and solid-phase amplification.
[0340] EmPCR can be used to prepare templates for NGS. Typically, a
library of nucleic acid fragments is generated, and adapters
containing universal priming sites are ligated to the ends of the
fragment. The fragments are then denatured into single strands and
captured by beads. Each bead captures a single nucleic acid
molecule. After amplification and enrichment of emPCR beads, a
large amount of templates can be attached or immobilized in a
polyacrylamide gel on a standard microscope slide (e.g.,
Polonator), chemically crosslinked to an amino-coated glass surface
(e.g., Life/APG; Polonator), or deposited into individual
PicoTiterPlate (PTP) wells (e.g., Roche/454), in which the NGS
reaction can be performed.
[0341] Solid-phase amplification can also be used to produce
templates for NGS. Typically, forward and reverse primers are
covalently attached to a solid support. The surface density of the
amplified fragments is defined by the ratio of the primers to the
templates on the support. Solid-phase amplification can produce
hundreds of millions spatially separated template clusters (e.g.,
Illumina/Solexa). The ends of the template clusters can be
hybridized to universal sequencing primers for NGS reactions.
[0342] Other methods for preparing clonally amplified templates
also include, e.g., Multiple Displacement Amplification (MDA)
(Lasken R. S. Curr Opin Microbiol. 2007; 10(5):510-6). MDA is a
non-PCR based DNA amplification technique. The reaction involves
annealing random hexamer primers to the template and DNA synthesis
by high fidelity enzyme, typically .PHI.29 at a constant
temperature. MDA can generate large sized products with lower error
frequency.
[0343] Template amplification methods such as PCR can be coupled
with NGS platforms to target or enrich specific regions of the
genome (e.g., exons). Exemplary template enrichment methods
include, e.g., microdroplet PCR technology (Tewhey R. et al.,
Nature Biotech. 2009, 27:1025-1031), custom-designed
oligonucleotide microarrays (e.g., Roche/NimbleGen oligonucleotide
microarrays), and solution-based hybridization methods (e.g.,
molecular inversion probes (MIPs) (Porreca G. J. et al., Nature
Methods, 2007, 4:931-936; Krishnakumar S. et al., Proc. Natl. Acad.
Sci. USA, 2008, 105:9296-9310; Turner E. H. et al., Nature Methods,
2009, 6:315-316), and biotinylated RNA capture sequences (Gnirke A.
et al., Nat. Biotechnol. 2009; 27(2):182-9)
[0344] Single-molecule templates are another type of templates that
can be used for NGS reaction. Spatially separated single molecule
templates can be immobilized on solid supports by various methods.
In one approach, individual primer molecules are covalently
attached to the solid support. Adapters are added to the templates
and templates are then hybridized to the immobilized primers. In
another approach, single-molecule templates are covalently attached
to the solid support by priming and extending single-stranded,
single-molecule templates from immobilized primers. Universal
primers are then hybridized to the templates. In yet another
approach, single polymerase molecules are attached to the solid
support, to which primed templates are bound.
Sequencing and Imaging
[0345] Exemplary sequencing and imaging methods for NGS include,
but are not limited to, cyclic reversible termination (CRT),
sequencing by ligation (SBL), single-molecule addition
(pyrosequencing), and real-time sequencing.
[0346] CRT uses reversible terminators in a cyclic method that
minimally includes the steps of nucleotide incorporation,
fluorescence imaging, and cleavage. Typically, a DNA polymerase
incorporates a single fluorescently modified nucleotide
corresponding to the complementary nucleotide of the template base
to the primer. DNA synthesis is terminated after the addition of a
single nucleotide and the unincorporated nucleotides are washed
away. Imaging is performed to determine the identity of the
incorporated labeled nucleotide. Then in the cleavage step, the
terminating/inhibiting group and the fluorescent dye are removed.
Exemplary NGS platforms using the CRT method include, but are not
limited to, Illumina/Solexa Genome Analyzer (GA), which uses the
clonally amplified template method coupled with the four-color CRT
method detected by total internal reflection fluorescence (TIRF);
and Helicos BioSciences/HeliScope, which uses the single-molecule
template method coupled with the one-color CRT method detected by
TIRF.
[0347] SBL uses DNA ligase and either one-base-encoded probes or
two-base-encoded probes for sequencing. Typically, a fluorescently
labeled probe is hybridized to its complementary sequence adjacent
to the primed template. DNA ligase is used to ligate the
dye-labeled probe to the primer. Fluorescence imaging is performed
to determine the identity of the ligated probe after non-ligated
probes are washed away. The fluorescent dye can be removed by using
cleavable probes to regenerate a 5'-PO.sub.4 group for subsequent
ligation cycles. Alternatively, a new primer can be hybridized to
the template after the old primer is removed. Exemplary SBL
platforms include, but are not limited to, Life/APG/SOLiD (support
oligonucleotide ligation detection), which uses two-base-encoded
probes.
[0348] Pyrosequencing method is based on detecting the activity of
DNA polymerase with another chemiluminescent enzyme. Typically, the
method allows sequencing of a single strand of DNA by synthesizing
the complementary strand along it, one base pair at a time, and
detecting which base was actually added at each step. The template
DNA is immobile, and solutions of A, C, G, and T nucleotides are
sequentially added and removed from the reaction. Light is produced
only when the nucleotide solution complements the first unpaired
base of the template. The sequence of solutions which produce
chemiluminescent signals allows the determination of the sequence
of the template. Exemplary pyrosequencing platforms include, but
are not limited to, Roche/454, which uses DNA templates prepared by
emPCR with 1-2 million beads deposited into PTP wells.
[0349] Real-time sequencing involves imaging the continuous
incorporation of dye-labeled nucleotides during DNA synthesis.
Exemplary real-time sequencing platforms include, but are not
limited to, Pacific Biosciences platform, which uses DNA polymerase
molecules attached to the surface of individual zero-mode waveguide
(ZMW) detectors to obtain sequence information when phospholinked
nucleotides are being incorporated into the growing primer strand;
Life/VisiGen platform, which uses an engineered DNA polymerase with
an attached fluorescent dye to generate an enhanced signal after
nucleotide incorporation by fluorescence resonance energy transfer
(FRET); and LI-COR Biosciences platform, which uses dye-quencher
nucleotides in the sequencing reaction.
[0350] Other sequencing methods for NGS include, but are not
limited to, nanopore sequencing, sequencing by hybridization,
nano-transistor array based sequencing, polony sequencing, scanning
tunneling microscopy (STM) based sequencing, and nanowire-molecule
sensor based sequencing.
[0351] Nanopore sequencing involves electrophoresis of nucleic acid
molecules in solution through a nano-scale pore which provides a
highly confined space within which single-nucleic acid polymers can
be analyzed. Exemplary methods of nanopore sequencing are
described, e.g., in Branton D. et al., Nat Biotechnol. 2008;
26(10):1146-53.
[0352] Sequencing by hybridization is a non-enzymatic method that
uses a DNA microarray. Typically, a single pool of DNA is
fluorescently labeled and hybridized to an array containing known
sequences. Hybridization signals from a given spot on the array can
identify the DNA sequence. The binding of one strand of DNA to its
complementary strand in the DNA double-helix is sensitive to even
single-base mismatches when the hybrid region is short or is
specialized mismatch detection proteins are present. Exemplary
methods of sequencing by hybridization are described, e.g., in
Hanna G. J. et al., J. Clin. Microbiol. 2000; 38 (7): 2715-21; and
Edwards J. R. et al., Mut. Res. 2005; 573 (1-2): 3-12.
[0353] Polony sequencing is based on polony amplification and
sequencing-by-synthesis via multiple single-base-extensions
(FISSEQ). Polony amplification is a method to amplify DNA in situ
on a polyacrylamide film. Exemplary polony sequencing methods are
described, e.g., in US Patent Application Publication No.
2007/0087362.
[0354] Nano-transistor array based devices, such as Carbon NanoTube
Field Effect Transistor (CNTFET), can also be used for NGS. For
example, DNA molecules are stretched and driven over nanotubes by
micro-fabricated electrodes. DNA molecules sequentially come into
contact with the carbon nanotube surface, and the difference in
current flow from each base is produced due to charge transfer
between the DNA molecule and the nanotubes. DNA is sequenced by
recording these differences. Exemplary Nano-transistor array based
sequencing methods are described, e.g., in U.S. Patent Application
Publication No. 2006/0246497.
[0355] Scanning tunneling microscopy (STM) can also be used for
NGS. STM uses a piezo-electric-controlled probe that performs a
raster scan of a specimen to form images of its surface. STM can be
used to image the physical properties of single DNA molecules,
e.g., generating coherent electron tunneling imaging and
spectroscopy by integrating scanning tunneling microscope with an
actuator-driven flexible gap. Exemplary sequencing methods using
STM are described, e.g., in U.S. Patent Application Publication No.
2007/0194225.
[0356] A molecular-analysis device which is comprised of a
nanowire-molecule sensor can also be used for NGS. Such device can
detect the interactions of the nitrogenous material disposed on the
nanowires and nucleic acid molecules such as DNA. A molecule guide
is configured for guiding a molecule near the molecule sensor,
allowing an interaction and subsequent detection. Exemplary
sequencing methods using nanowire-molecule sensor are described,
e.g., in U.S. Patent Application Publication No. 2006/0275779.
[0357] Double ended sequencing methods can be used for NGS. Double
ended sequencing uses blocked and unblocked primers to sequence
both the sense and antisense strands of DNA. Typically, these
methods include the steps of annealing an unblocked primer to a
first strand of nucleic acid; annealing a second blocked primer to
a second strand of nucleic acid; elongating the nucleic acid along
the first strand with a polymerase; terminating the first
sequencing primer; deblocking the second primer; and elongating the
nucleic acid along the second strand. Exemplary double ended
sequencing methods are described, e.g., in U.S. Pat. No.
7,244,567.
Data Analysis
[0358] After NGS reads have been generated, they can be aligned to
a known reference sequence or assembled de novo.
[0359] For example, identifying genetic variations such as
single-nucleotide polymorphism and structural variants in a sample
(e.g., a tumor sample) can be accomplished by aligning NGS reads to
a reference sequence (e.g., a wild-type sequence). Methods of
sequence alignment for NGS are described e.g., in Trapnell C. and
Salzberg S. L. Nature Biotech., 2009, 27:455-457.
[0360] Examples of de novo assemblies are described, e.g., in
Warren R. et al., Bioinformatics, 2007, 23:500-501; Butler J. et
al., Genome Res., 2008, 18:810-820; and Zerbino D. R. and Birney
E., Genome Res., 2008, 18:821-829.
[0361] Sequence alignment or assembly can be performed using read
data from one or more NGS platforms, e.g., mixing Roche/454 and
Illumina/Solexa read data.
[0362] Algorithms and methods for data analysis are described in
U.S. Ser. No. 61/428,568, filed Dec. 30, 2010, incorporated herein
by reference.
KIF5B-RET Fusion Expression Level
[0363] In certain embodiments, KIF5B-RET fusion expression level
can also be assayed. KIF5B-RET fusion expression can be assessed by
any of a wide variety of methods for detecting expression of a
transcribed molecule or protein. Non-limiting examples of such
methods include immunological methods for detection of secreted,
cell-surface, cytoplasmic, or nuclear proteins, protein
purification methods, protein function or activity assays, nucleic
acid hybridization methods, nucleic acid reverse transcription
methods, and nucleic acid amplification methods.
[0364] In certain embodiments, activity of a particular gene is
characterized by a measure of gene transcript (e.g., mRNA), by a
measure of the quantity of translated protein, or by a measure of
gene product activity. KIF5B-RET fusion expression can be monitored
in a variety of ways, including by detecting mRNA levels, protein
levels, or protein activity, any of which can be measured using
standard techniques. Detection can involve quantification of the
level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein,
or enzyme activity), or, alternatively, can be a qualitative
assessment of the level of gene expression, in particular in
comparison with a control level. The type of level being detected
will be clear from the context.
[0365] Methods of detecting and/or quantifying the KIF5B-RET gene
transcript (mRNA or cDNA made therefrom) using nucleic acid
hybridization techniques are known to those of skill in the art
(see Sambrook et al. supra). For example, one method for evaluating
the presence, absence, or quantity of cDNA involves a Southern
transfer as described above. Briefly, the mRNA is isolated (e.g.,
using an acid guanidinium-phenol-chloroform extraction method,
Sambrook et al. supra.) and reverse transcribed to produce cDNA.
The cDNA is then optionally digested and run on a gel in buffer and
transferred to membranes. Hybridization is then carried out using
the nucleic acid probes specific for the KIF5B-RET fusion cDNA,
e.g., using the probes and primers described herein.
[0366] In other embodiments, KIF5B-RET expression is assessed by
preparing genomic DNA or mRNA/cDNA (i.e., a transcribed
polynucleotide) from cells in a subject sample, and by hybridizing
the genomic DNA or mRNA/cDNA with a reference polynucleotide which
is a complement of a polynucleotide comprising the KIF5B-RET
fusion, and fragments thereof. cDNA can, optionally, be amplified
using any of a variety of polymerase chain reaction methods prior
to hybridization with the reference polynucleotide. Expression of
KIF5B-RET fusion can likewise be detected using quantitative PCR
(QPCR) to assess the level of KIF5B-RET expression.
Detection of KIF5B-RET Fusion Polypeptide
[0367] The activity or level of a KIF5B-RET fusion polypeptide can
also be detected and/or quantified by detecting or quantifying the
expressed polypeptide. The KIF5B-RET fusion polypeptide can be
detected and quantified by any of a number of means known to those
of skill in the art. These can include analytic biochemical methods
such as electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, or various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassay (RIA), enzyme-linked immunosorbent assays
(ELISAs), immunofluorescent assays, Western blotting,
immunohistochemistry (IHC) and the like. A skilled artisan can
adapt known protein/antibody detection methods.
[0368] Another agent for detecting a KIF5B-RET fusion polypeptide
is an antibody molecule capable of binding to a polypeptide
corresponding to a marker of the invention, e.g., an antibody with
a detectable label. Techniques for generating antibodies are
described herein. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin.
[0369] In another embodiment, the antibody is labeled, e.g., a
radio-labeled, chromophore-labeled, fluorophore-labeled, or
enzyme-labeled antibody. In another embodiment, an antibody
derivative (e.g., an antibody conjugated with a substrate or with
the protein or ligand of a protein-ligand pair {e.g.,
biotin-streptavidin}), or an antibody fragment (e.g., a
single-chain antibody, an isolated antibody hypervariable domain,
etc.) which binds specifically with a KIF5B-RET fusion protein, is
used.
[0370] KIF5B-RET fusion polypeptides from cells can be isolated
using techniques that are known to those of skill in the art. The
protein isolation methods employed can, for example, be such as
those described in Harlow and Lane (Harlow and Lane, 1988,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).
[0371] Means of detecting proteins using electrophoretic techniques
are well known to those of skill in the art (see generally, R.
Scopes (1982) Protein Purification, Springer-Verlag, N.Y.;
Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein
Purification, Academic Press, Inc., N.Y.).
[0372] In another embodiment, Western blot (immunoblot) analysis is
used to detect and quantify the presence of a polypeptide in the
sample.
[0373] In another embodiment, the polypeptide is detected using an
immunoassay. As used herein, an immunoassay is an assay that
utilizes an antibody to specifically bind to the analyte. The
immunoassay is thus characterized by detection of specific binding
of a polypeptide to an anti-antibody as opposed to the use of other
physical or chemical properties to isolate, target, and quantify
the analyte.
[0374] The KIF5B-RET fusion polypeptide is detected and/or
quantified using any of a number of immunological binding assays
(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and
4,837,168). For a review of the general immunoassays, see also Asai
(1993) Methods in Cell Biology Volume 37: Antibodies in Cell
Biology, Academic Press, Inc. New York; Stites & Terr (1991)
Basic and Clinical Immunology 7th Edition.
Kits
[0375] In one aspect, the invention features, a kit, e.g.,
containing an oligonucleotide having a mutation described herein,
e.g., a KIT5B-RET fusion. Optionally, the kit can also contain an
oligonucleotide that is the wildtype counterpart of the mutant
oligonucleotide.
[0376] A kit featured in the invention can include a carrier, e.g.,
a means being compartmentalized to receive in close confinement one
or more container means. In one embodiment the container contains
an oligonucleotide, e.g., a primer or probe as described above. The
components of the kit are useful, for example, to diagnose identify
a mutation in a tumor sample in a patient, and to accordingly
identify an appropriate therapeutic agent to treat the cancer. The
probe or primer of the kit can be used in any sequencing or
nucleotide detection assay known in the art, e.g., a sequencing
assay, e.g., an NGS method, RT-PCR, or in situ hybridization.
[0377] A kit featured in the invention can include, e.g., assay
positive and negative controls, nucleotides, enzymes (e.g., RNA or
DNA polymerase or ligase), solvents or buffers, a stabilizer, a
preservative, a secondary antibody, e.g., an anti-HRP antibody
(IgG) and a detection reagent.
[0378] An oligonucleotide can be provided in any form, e.g.,
liquid, dried, semi-dried, or lyophilized, or in a form for storage
in a frozen condition.
[0379] Typically, an oligonucleotide, and other components in a kit
are provided in a form that is sterile. When an oligonucleotide,
e.g., an oligonucleotide that contains a RET mutation, e.g., a
KIF5B-RET fusion, described herein, or an oligonucleotide
complementary to a mutation RET mutation described herein, is
provided in a liquid solution, the liquid solution generally is an
aqueous solution, e.g., a sterile aqueous solution. When the
oligonucleotide is provided as a dried form, reconstitution
generally is accomplished by the addition of a suitable solvent.
The solvent, e.g., sterile buffer, can optionally be provided in
the kit.
[0380] The kit can include one or more containers for the
composition containing an oligonucleotide in a concentration
suitable for use in the assay or with instructions for dilution for
use in the assay. In some embodiments, the kit contains separate
containers, dividers or compartments for the oligonucleotide and
assay components, and the informational material. For example, the
oligonucleotides can be contained in a bottle or vial, and the
informational material can be contained in a plastic sleeve or
packet. In other embodiments, the separate elements of the kit are
contained within a single, undivided container. For example, an
oligonucleotide composition is contained in a bottle or vial that
has attached thereto the informational material in the form of a
label. In some embodiments, the kit includes a plurality (e.g., a
pack) of individual containers, each containing one or more unit
forms (e.g., for use with one assay) of an oligonucleotide. For
example, the kit includes a plurality of ampoules, foil packets, or
blister packs, each containing a single unit of oligonucleotide for
use in a sequencing or detecting a mutation in a tumor sample. The
containers of the kits can be air tight and/or waterproof. The
container can be labeled for use.
[0381] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
KIF5B-RET fusion polypeptide; and, optionally, (2) a second,
different antibody which binds to either the polypeptide or the
first antibody and is conjugated to a detectable agent.
[0382] In one embodiment, the kit can include informational
material for performing and interpreting the sequencing or
diagnostic. In another embodiment, the kit can provide guidance as
to where to report the results of the assay, e.g., to a treatment
center or healthcare provider. The kit can include forms for
reporting the results of a sequencing or diagnostic assay described
herein, and address and contact information regarding where to send
such forms or other related information; or a URL (Uniform Resource
Locator) address for reporting the results in an online database or
an online application (e.g., an app). In another embodiment, the
informational material can include guidance regarding whether a
patient should receive treatment with a particular chemotherapeutic
drug, depending on the results of the assay.
[0383] The informational material of the kits is not limited in its
form. In many cases, the informational material, e.g.,
instructions, is provided in printed matter, e.g., a printed text,
drawings, and/or photographs, e.g., a label or printed sheet.
However, the informational material can also be provided in other
formats, such as computer readable material, video recording, or
audio recording. In another embodiment, the informational material
of the kit is contact information, e.g., a physical address, email
address, website, or telephone number, where a user of the kit can
obtain substantive information about the sequencing or diagnostic
assay and/or its use in the methods described herein. The
informational material can also be provided in any combination of
formats.
[0384] In some embodiments, a biological sample is provided to an
assay provider, e.g., a service provider (such as a third party
facility) or a healthcare provider, who evaluates the sample in an
assay and provides a read out. For example, in one embodiment, an
assay provider receives a biological sample from a subject, such as
a blood or tissue sample, e.g., a biopsy sample, and evaluates the
sample using an assay described herein, e.g., a sequencing assay or
in situ hybridization assay, and determines that the sample
contains a nucleic acid containing a mutation described in the
Example. The assay provider, e.g., a service provider or healthcare
provider, can then conclude that the subject is, or is not, a
candidate for a particular drug or a particular cancer treatment
regimen.
[0385] The assay provider can provide the results of the
evaluation, and optionally, conclusions regarding one or more of
diagnosis, prognosis, or appropriate therapy options to, for
example, a healthcare provider, or patient, or an insurance
company, in any suitable format, such as by mail or electronically,
or through an online database. The information collected and
provided by the assay provider can be stored in a database.
[0386] The invention is further illustrated by the following
example, which should not be construed as further limiting.
EXAMPLES
Example 1: Massively Parallel Sequencing Assays to Identify Novel
Alterations
[0387] The following exemplifies the use of massively parallel
sequencing assays to identify novel alterations, such as KIF5B-RET
fusions. Based on the results shown herein, additional alterations,
e.g., RET translocations, can be screened using, e.g., either
qRT-PCR analysis of cDNA prepared from a pre-selected tumor
sample.
Introduction
[0388] A pan-cancer diagnostic assay based on massively parallel
sequencing technology was developed to interrogate 2574 coding
exons representing 145 cancer relevant genes (associated with
cancer-related pathways, targeted-therapy or prognosis), plus 37
introns from 14 genes frequently rearranged in cancer, using
minimal DNA from formalin fixed paraffin embedded (FFPE) tumor
specimens. This assay can identify all classes of DNA alterations
(e.g., base substitutions, insertions and deletions, copy number
alterations and rearrangements) in a single diagnostic test. In a
cohort of 40 colorectal cancer (CRC) and 24 non-small cell lung
cancer (NSCLC) specimens, 175 alterations were identified in 33
cancer genes. 38 samples (59%) harbored at least one alteration
that could be linked to a clinical treatment option or clinical
trial of novel targeted therapies, including a novel KIF5B-RET gene
fusion in NSCLC (the KIF5B-RET gene fusions is described in Example
2 herein).
Results
[0389] An evaluation of assays using genomic DNA derived from 40
CRC and 24 NSCLC FFPE tissue samples was performed (Supplementary
Table 1). In total, 2574 coding exons representing 145 cancer
genes, plus 37 introns from 15 frequently rearranged cancer genes
(Supplementary Table 2) were selected using solution phase hybrid
capture and sequenced on the Illumina HiSeq2000 platform (Illumina,
Inc., San Diego, Calif.) to an average coverage of 253X.175
alterations in 33 cancer genes were identified of which 116 were
single base substitutions (75 non synonymous, 41 nonsense), 46 were
small insertions or deletions (indels) (44 frameshift, 2 in-frame),
10 were copy number alterations (9 amplifications, 1 homozygous
deletion) and 3 were rearrangements (FIG. 5, Supplementary Table
2).
[0390] The percentage distribution of DNA alterations in 40 CRCs is
roughly as follows: about 58% are base substitutions, 28% are
insertions and deletions, 12% are copy number alterations, about 1%
are gene fusions.
[0391] The percentage distribution of DNA alterations in 24 NSCLCs
is roughly as follows: about 72% are base substitutions, 14% are
insertions and deletions, 12% are copy number alterations, about 2%
are gene fusions.
Mutations Identified in CRC Cases
[0392] Among 40 CRCs, 125 alterations were identified in 21 genes.
39 tumors carried at least one mutation (range 1-9) and 25 tumors
(62.5%) contained at least two different classes of DNA alteration
(FIG. 5). TP53 and APC were the most frequently altered genes
(32/40 (80%) and 27/40 (67.5%), respectively), with both mutated at
higher frequencies than reported in COSMIC (on the worldwide web at
sanger.ac.uk/genetics/CGP/cosmic/). 41/42 (98%) of the APC
mutations in the cohort were truncating (21 nonsense, 17 frameshift
indel), an observation unique to CRC tumors which indicates the
specificity of the assay. 15 samples had bi-allelic inactivation
and 12 a single truncating APC mutation and LOH.
[0393] In addition, 11 known cancer genes were mutated, amplified
or rearranged in multiple CRC cases: KRAS (10), BRAF (6), FBXW7
(5), ATM (2), BCL2L1 (2), BRCA2 (2), CDH1 (2), ERBB3 (2), GNAS (2),
PIK3CA (2) and SMAD4 (2), ALK, CDK8, LRP1B, MYC, MSH6, RICTOR,
SMAD2 and STK11 were each altered in a single case (FIG. 5,
Supplementary Table 1). Notably, 21 CRCs (52.5%) harbored at least
one alteration that could be linked to a clinical treatment option
or clinical trial of novel targeted therapies. Examples include
mutations in KRAS and BRAF (resistance to cetuximab (Lievre et al.,
Cancer Res 66:3992-3995, 2006; Di Nicolantonio et al., J Clin
Oncol. 26:5705-5712, 2008) or panitumumab (Di Nicolantonio et al.,
supra; Lievre et al., Bull Cancer 95:133-140, 2008), FBXW7
(resistance to anti-tubulins (Wertz, et al., Nature 471:110-114,
2011)), BRCA2 (clinical trials of PARP inhibitors (Turner et al.,
Curr Opin Pharmacol 5:388-393, 2005), GNAS (clinical trials of MEK
or ERK inhibitors), PIK3CA (clinical trials of PI3 kinase/mTOR
inhibitors), and CDK8 (clinical trials of CDK inhibitors ref)
(Supplementary Table 3).
[0394] Mutations Identified in NSCLC Cases
[0395] Among 24 NSCLCs, 50 mutations were identified in 21 genes.
20 of 24 tumors harbored at least one mutation (range 1-7)
(Supplementary FIGS. 4 and 5, Supplementary Table 2). Twelve genes
were altered in multiple tumors: KRAS (10), TP53 (7), STK11 (4),
LRP1B (3), JAK2 (3), EGFR (2), BRAF (2), CDKN2A (2), CTNNB1 (2),
MDM2 (2), PIK3CA (2) and ATM (2). APC, CCNE1, CDK4, MLH1, MSH6,
NF1, RB1, RET and TSC1 were each mutated in a single case. In
addition, 3 patients had a truncating mutation in the putative
tumor suppressor gene LRP1B (Liu et al., Genomics 69:271-274,
2000), further supporting the role of inactivation of this gene in
oncogenesis. In 72% (36/50) of NSCLCs at least one alteration was
associated with a current clinical treatment or targeted therapy
trial, including mutations in KRAS (resistance to EGFR kinase
inhibitors (Pao et al., PLoS Med 2:e17, 2005); clinical trials of
PI3K and MEK inhibitors) and BRAF (clinical trials of BRAF
inhibitors including vemurafenibref and GSK 2118436 ref), EGFR
(sensitivity to gefitinib or erlotinib ref), MDM2 (clinical trials
of nutlins (Vassilev et al., Science 303:844-848, 2004), CDKN2A,
CCNE1 and CDK4 (clinical trials of CDK4 inhibitors (Finn et al.,
Breast Cancer Res. 11:R77, 2009; Toogood et al., J Med Chem
48:2388-2406, 2005; Konecny et al., Clin Cancer Res 17:1591-1602,
2011), and PIK3CA (clinical trials of PI3 kinase/mTOR inhibitors
ref) (Supplementary Table 4). Although the test detects the
EML4-ALK inversions in cell lines, EML4-ALK was not identified in
this patient cohort (Soda et al., Nature 448:561-566, 2007).
[0396] Surprisingly, three patients were found to have a
c.1849G>Tp.V617F mutation in JAK, which is commonly observed
myelodysplastic syndromes (MDS) but has not been identified in
solid tumors (James et al., Nature 434:1144-1148, 2005) (see online
at sanger.ac.uk/genetics/CGP/cosmic/), although one patient had a
history of polycythemia vera with a bone marrow positive for the
JAK2 mutation. Sequencing a larger series of NSCLC specimens can be
conducted to further characterize JAK2 mutations in NSCLC, and
further studies can be conducted to assess whether these predict
clinical sensitivity to JAK2 inhibitors.
Example 2A: Novel KIF5B-RET Fusions
[0397] A novel inversion event was identified in a lung
adenocarcinoma using NGS (Next Generation) sequencing. The
adenocarcinoma was from a 44-year old, male Caucasian never-smoker.
A genomic sequence suggestive of a novel chromosome 10
rearrangement, specifically an 11 MB pericentric inversion with
breakpoints in intron 15 of KIF5B and intron 11 of RET, was
observed. The breakpoints were determined to be at
Chr10:32,316,376-32,316,416 within intron 15 of KIF5B and Chr
10:43,611,042-43,611,118 within intron 11 of RET (see FIG. 1). More
specifically, an 11,294,741 bp pericentric inversion generates the
RET gene fusion joining exons 1-15 of KIF5B to exon 12-20 of RET.
FIG. 6A provides a schematic representation of the 11,294,741 bp
inversion in NSCLC that generates an in-frame KIF5B-RET gene fusion
(not to scale). The inverted region of chromosome 10 starts at
32,316,377 bps (within KIF5B intron 15) and ends at 43,611,118 bps
(within RET intron 11). FIG. 6D is a summary of the exons present
or absent in the KIF5B-RET fusion.
[0398] The inversion results in an in-frame fusion of the 5'end of
KIF5B and the 3'end of RET, which fusion is expressed to generate a
fusion protein that includes a tyrosine kinase domain in the RET
protein. More specifically, the protein fusion results in an
in-frame fusion of exon 15 of KIF5B with exon 12 of RET. See FIGS.
2B and 3A-3D. In FIGS. 3A-3D, SEQ ID NO:1 is the cDNA sequence of
the KIF5B-RET fusion (i.e., 5'-KIF5B-3'-RET fusion) and SEQ ID NO:2
is the amino acid sequence of the KIF5B-RET fusion protein. The
underlined sequence in FIGS. 3A-3D represents KIF5B cDNA sequence
(nucleotides 471-2195 of RefSeq No. NM_004521.2 (GenBank Record
Aug. 14, 2011)) and KIF5B protein sequence (amino acids 1-575 of
RefSeq No. NP_004512 (GenBank Record Aug. 14, 2011)). The sequence
not underlined in FIGS. 3A-3D represents RET cDNA sequence
(nucleotides 2327-3535 of RefSeq No. NM_020975 (GenBank Record Aug.
14, 2011) and RET protein sequence (amino acids 713-1114 of RefSeq
No. NP_066124.1 (GenBank Record Aug. 14, 2011)). The RET protein
sequence in the KIF5B-RET fusion maintains the intact tyrosine
kinase domain of RET, and this domain is indicated by a gray box in
FIG. 3C. The KIF5B protein sequence in the fusion maintains the
intact kinesin motor domain, and this domain is indicated by a
double-underlining in FIGS. 3A-3B. KIF5B exons 1 to 15 also contain
the coiled-coil domain that mediates homodimerization. At least
because the KIF5B-RET fusion is an in-frame fusion that maintains
the intact tyrosine kinase domain of RET, and the kinesin motor
domain and coiled-coil domain of KIF5B, this fusion protein is
likely to contribute to the oncogenic phenotype of the lung tumor
sample.
[0399] FIG. 7A provides another schematic representation of the
KIF5B-RET fusion. This variant was identified in 10 CRC cases,
where KIF5B exon 15 is fused in-frame to RET exon 12. The predicted
full length fusion protein is 977 amino acids in length, with amino
acids 1-575 derived from KIF5B and amino acids 576-977 derived from
RET (shown above). Capillary sequence confirmation of the exon
junction boundaries derived from cDNA is shown below. FIG. 7B is
another representation of the predicted KIF5B-RET variant amino
acid sequence Amino acids derived from KIF5B (normal text) Amino
acids derived from RET (italics, underlined).
[0400] KIF5B exons 1-15 comprise a kinesin motor domain and a
coiled-coil domain that directly mediates homodimerization. KIF5B
exon 15 is a known fusion point in NSCLC patients with KIF5B-ALK
fusions (Wong et al., Cancer, 2011; Takeuchi et al., Clin Cancer
Res 15:3143-3149, 2009).
[0401] FIG. 6B is a schematic of the protein domain structure of
the RET-KIF5B and KIF5B-RET gene fusions. The cadherin domain of
RET is included in the predicted RET-KIF5B protein. The kinesin and
coiled coil domains of KIF5B and the tyrosine kinase domain of RET
are included in the KIF5B-RET fusion protein. RT-PCR of primer
designed to RET exon 11 and KIF5B exon 16 yielded no product.
RT-PCR of primer designed to KIF5B exon 15 and RET exon 12 yielded
a strong product. Using cDNA sequencing, a 7.3 fold RET expression
increase beginning at exon 12 relative to exons 1 to 11, suggesting
the KIF5B-RET fusion transcript results in RET kinase domain
overexpression. In addition, cDNA sequencing revealed that 490
unique read pairs spanned the fusion junction.
[0402] IHC demonstrated focal moderate cytoplasmic immunoreactivity
for RET protein expression (FIG. 6C). FIG. 6C is a photograph
depicting the distribution of RET expression in the case of NSCLC
with confirmed RET fusion on DNA sequencing by NGS. Note focal
moderate cytoplasmic immunoreactivity for RET protein expression
(avidin-biotin perxodase.times.200). High magnification detail of
cytoplasmic immunostaining for RET is shown in the insert at the
lower right.
[0403] Previous RET fusion proteins have been identified in cancers
other than lung cancer. For example, the RET oncogene has been
shown to be activated through somatic rearrangements in papillary
thyroid carcinomas. RET exons 12-20 comprise the RET tyrosine
kinase domain which commonly forms the 3' portion of the PTC/RET
fusions observed in .about.35% of papillary thyroid carcinomas.
KIF5B-ALK fusions have been previously identified in ALK-positive
lung cancer. To our knowledge, this is the first demonstration of a
RET fusion cDNA in a lung cancer, and also the first demonstration
of a KIF5B-RET fusion.
[0404] Thyroid cancer and cell lines harboring oncogenic PTC/RET
translocations, which overexpress the RET kinase domain, are
sensitive to RET inhibitor sorafenib suggesting that this KIF5B-RET
gene fusion may identify a new druggable subset of NSCLC tumors
(Henderson et al., Clin Cancer Res 14:4908-4914, 2008; Carlomagno
et al., J Natl Cancer Inst 98:326-334, 2006). Other RET activating
mutations have been shown to be sensitive to treatment with the
multikinase inhibitor sorafenib (e.g., Plaza-Menacho et al., Jour.
Biol. Chem. 282:29230-29240, 2007). It is, therefore, expected that
cancers carrying a KIF5B-RET fusion as described herein is likely
to be susceptible to treatment with multikinase inhibitors, such as
sorafenib and sunitinib.
[0405] Together these results suggest druggable gene fusions can
occur broadly in cancer at low frequency and are more effectively
identified with a sequencing-based diagnostic such as the one
described herein.
RET Fusion Recurrence
[0406] To determine whether the RET fusion is recurrently expressed
in NSCLC, a series of 117 NSCLCs (92 Caucasian, 5 African-American,
20 ethnicity unknown) were screened for RET by IHC; 22/117 cases
showed moderate to intense staining. RT-PCR and cDNA sequencing of
RNA from 15 IHC-positive tumors identified one additional KIF5B-RET
fusion in a male Caucasian smoking patient (Table 2).
Example 2B: Additional KIF5B-RET Fusions
[0407] The chromosome 10 inversion that creates the KIF5B-RET
fusion, can also create a reciprocal RET-KIF5B fusion (i.e.,
5'-RET-3'-KIF5B fusion). See FIGS. 2A and 4A-4D. The predicted
RET-KIF5B protein fusion results in a fusion of exon 11 of RET with
exon 16 of KIF5B. See FIGS. 2A and 4A-4D. In FIGS. 4A-4D, SEQ ID
NO:3 is the cDNA sequence of the RET-KIF5B fusion and SEQ ID NO:4
is the amino acid sequence of the predicted RET-KIF5B fusion
protein. The underlined sequence in FIGS. 4A-4D represents KIF5B
cDNA sequence (nucleotides 2196-3362 of RefSeq No. NM_004521.2
(GenBank Record Aug. 14, 2011)) and KIF5B protein sequence (amino
acids 576-963 of RefSeq No. NP_004512 (GenBank Record Aug. 14,
2011)). The sequence not underlined in FIGS. 4A-4D represents RET
cDNA sequence (nucleotides 190-2326 of RefSeq No. NM_020975
(GenBank Record Aug. 14, 2011) and RET protein sequence (amino
acids 1-712 of RefSeq No. NP_066124.1 (GenBank Record Aug. 14,
2011)). The predicted RET protein sequence in the RET-KIF5B fusion
does not include the tyrosine kinase domain. RT-PCR studies
designed to RET exon 11 and KIF5B exon 16 yielded no product. Thus,
this orientation of the fusion is not expressed.
[0408] Further screening of tumor samples has identified additional
KIF5B-RET gene fusions that contain a RET catalytic domain.
[0409] Thyroid cancers and cell lines harboring PTC-RET
translocations are sensitive to sorafenib which inhibits RET,
suggesting the KIF5B-RET gene fusion in NSCLC may be druggable.
KIF5B-RET expression in Ba/F3 cells led to oncogenic transformation
as determined by IL-3 independent growth. These cells were
sensitive to sunitinib, sorafenib and vandetinib, multi-targeted
kinase inhibitors that inhibit RET, but not gefitinib, an EGFR
kinase inhibitor (FIG. 8). Sunitinib, but not gefitinib, inhibited
RET phosphorylation in KIF5B-RET Ba/F3 cells (FIG. 9). These
findings suggest RET kinase inhibitors should be tested in
prospective clinical trials for therapeutic benefit in NSCLC
patients bearing KIF5B-RET rearrangements.
Discussion
[0410] In the current study, a comprehensive genomic profiling
assay that robustly identifies base substitutions, insertions,
deletions, copy number alterations and genomic rearrangements from
FFPE tumor tissue is described. Results show that genomic
alterations with direct clinical therapeutic relevance are detected
in a large fraction of CRCs and NSCLCs demonstrating the value of
systematically testing for such alterations in cancer patients. Due
to the wide range of targeted therapies already approved or under
clinical development, performing simultaneous, comprehensive
querying for a wide breadth of genomic alterations will speed
identification of appropriate patient subsets for these therapeutic
approaches.
[0411] The method additionally identified two previously unknown
genomic rearrangements, each of which could potentially lead to a
therapeutic intervention. The first is an ALK rearrangement similar
to those previously detected in anaplastic lymphoma (Morris et al.,
Science 263:1281-1284, 1994), NSCLC (Soda et al., Nature
448:561-566, 2007, supra) and inflammatory myofibroblastic tumor
(Lawrence et al., Am J Pathol 157: 377-384, 2000) that are
associated with clinical sensitivity to the ALK kinase inhibitor
crizotinib (Kwak et al., N Engl J Med 363:1693-1703, 2010;
Butrynski et al., N Engl J Med 363:1727-1733, 2010). The current
findings suggest that other cancers, including CRC, can contain
additional previously undetected ALK rearrangements that could be
drug sensitive.
[0412] More importantly, applying comprehensive genomic profiling
to tumor specimens identified recurrent novel KIF5B-RET inversions
in NSCLC patients. This fusion gene contains the entire kinase
domain of RET and results in increased RET kinase domain
expression. Oncogenic PTC/RET translocations detected in thyroid
cancer and cell lines are sensitive in vitro and in vivo to RET
inhibitors (Henderson et al., Clin Cancer Res 14:4908-4914, 2008;
Carlomagno et al., J Natl Cancer Inst 98:326-334, 2006; Kim et al.,
J Clin Endocrinol Metab 91:4070-4076, 2006; Dawson et al.,
Anticancer Drugs 19:547-552, 2008). It is possible that sunitinib
or other RET tyrosine kinase inhibitors, including vandetinib or
sorafenib (Henderson et al., supra), can be clinically effective in
KIF5B-RET NSCLC. Identification of even a small subpopulation of
NSCLC patients responsive to a molecular targeted therapy can have
large clinical impact as highlighted by the recent FDA approval of
crizotinib for ALK-rearranged NSCLC. It is clear is that harnessing
the power of massively parallel sequencing to generate
comprehensive genomic profiles for patient tumors will be
increasingly clinically informative and eventually critical to
effective therapeutic management in the oncology clinic.
Example 3: Methods
[0413] The following exemplifies certain embodiments of the methods
and experimental conditions used to identify the KIF5B-RET fusion
according to Examples 1-2. Additional RET translocation screening
can be done using, e.g., either qRT-PCR analysis of cDNA prepared
from a pre-selected tumor sample.
[0414] Massively parallel DNA sequencing was done on hybridization
captured, adaptor ligation-based libraries using DNA isolated from
archived fixed paraffin-embedded tissue. A combination of analysis
tools were used to analyze the data and assign DNA alteration
calls. Additional RET translocation screening was done using either
qRT-PCR analysis of cDNA prepared from frozen tumors or RET IHC
assessment of archived FFPE specimens. Massively parallel cDNA
sequencing was performed to confirm expression of both novel
translocations using RNA isolated from FFPE tissue. Matched normal
reference genomic DNA from blood was sequenced for the index
KIF5B-RET NSCLC patient to confirm the somatic origin of the
rearrangement.
Genomic DNA Sequencing
[0415] Sequencing of 2574 exons of 145 cancer genes was performed
using DNA from archived formalin fixed paraffin embedded (FFPE)
tumor specimens; 24 from NSCLC patients and 40 from colorectal
(CRC) pateints. Sequencing libraries of the 606,675 bp content were
constructed by the adapter ligation method using genomic DNA
followed by solution phase hybridization selection with optimized
RNA hybridization capture probes (Agilent SureSelect custom kit).
Sequencing on the HiSeq2000 instrument (Illumina) was performed
using 36.times.36 paired reads to an average coverage of 229.times.
with 84% exons at .gtoreq.100.times. (Supplementary Tables 2A and
2B). Data processing and mutation assignments for base
substitutions, indels, copy number alterations and genomic
rearrangements was performed using a combination of tools optimized
for mutation calling from tumor tissue. To maximize mutation
detection sensitivity in heterogeneous cancer biopsies, the test
was validated to detect base substitutions at .gtoreq.10% mutant
allele frequency with .gtoreq.99% sensitivity and indels at
.gtoreq.20% mutant allele frequency with .gtoreq.95% sensitivity,
with a false discovery rate of <1%.
cDNA Sequencing
[0416] cDNA was generated from total RNA extracted from a single
5-10 um FFPE tissue section using the Roche High Pure kit and
reverse transcribed to cDNA with random hexamer primers by the
SuperScript.RTM. III First-Strand Synthesis System (Invitrogen).
Double stranded cDNA was made with the NEBNext.RTM. mRNA Second
Strand Synthesis Module (New England Biolabs) and used as input to
library construction, hybrid capture and sequencing as for FFPE DNA
samples. Analysis of expression levels was done with a combination
of analysis tools.
RET Protein Immunohistochemistry
[0417] NSCLC were immunostained after microwave-based epitope
retrieval by automated methods (Ventana Medical Systems, Tucson,
Ariz.) for RET expression using the mouse monoclonal RET
Oncoprotein clone 3F8 (Vector Laboratories, Burlingame, Calif.).
Slides were scored semi-quantitatively for tumor cell staining
intensity and distribution using the H Score system.
TABLE-US-00001 TABLE 1 Distribution of 125 CRC mutations across 21
mutated cancer genes Mu- tated Total Non INDEL Ampli- Rear- Sam-
Muta- Synony- Non- frame- fica- range- Gene ples tions mous sense
shift tion ment TP53 32 34 23 3 8 -- -- APC 27 42 1 24 17 -- --
KRAS 10 10 10 -- -- -- -- BRAF 6 6 6 -- -- -- -- FBXW7 5 5 -- 2 3
-- -- ATM 2 2 -- 1 1 -- -- BCL2L1 2 2 -- -- -- 2 -- BRCA2 2 2 -- --
2 -- -- CDH1 2 4 -- 1 3 -- -- ERBB3 2 2 2 -- -- -- -- GNAS 2 2 2 --
-- -- -- PIK3CA 2 2 2 -- -- -- -- SMAD4 2 2 1 -- 1 -- -- ALK 1 1 --
-- -- -- 1 CDK8 1 1 -- -- -- 1 -- LRP1B 1 3 -- -- 3 -- -- MYC 1 1
-- -- -- 1 -- MSH6 1 1 -- -- 1 -- -- RICTOR 1 1 -- -- -- -- 1 SMAD2
1 1 -- 1 -- -- -- STK11 1 1 -- 1 -- -- --
TABLE-US-00002 TABLE 2 Summary of non-small cell lung cancer
patients analyzed by RET immunohistochemistry Characteristic No. of
Patients (n = 117) Gender Male 62 Female 55 Histology
Adenocarcinoma 83 Squamous cell Carcinoma 26 Carcinoid 8 Other
Smoking Never 5 Limited former 53 Current 34 Unknown 25 Stage I 77
II 16 III 13 IV 8 N/A 3 RET IHC 0 78 1+/2+ 17 3+/4+ 22 No of
KIF5B-RET fusions: 1 Frequency in all patients: 1/117: 0.85%
Frequency Adenocarcinoma: 1/89: 1.1%
TABLE-US-00003 SUPPLEMENTARY TABLE 1a 145 genes sequenced across
entire coding sequence Gene RefSeq ABL1 NM_007313 AKT1 NM_005163
AKT2 NM_001626 AKT3 NM_181690 ALK NM_004304 APC NM_000038 AR
NM_000044 BRAF NM_004333 CCND1 NM_053056 CDK4 NM_000075 CDKN2A
NM_000077 CEBPA NM_004364 CTNNB1 NM_001904 EGFR NM_005228 ERBB2
NM_004448 ESR1 NM_000125 FGFR1 NM_015850 FGFR2 NM_000141 FGFR3
NM_000142 FLT3 NM_004119 HRAS NM_005343 JAK2 NM_004972 KIT
NM_000222 KRAS NM_004985 MAP2K1 NM_002755 MAP2K2 NM_030662 MET
NM_000245 MLL NM_005933 MYC NM_002467 NF1 NM_000267 NOTCH1
NM_017617 NPM1 NM_002520 NRAS NM_002524 NTRK3 NM_002530 PDGFRA
NM_006206 PIK3CA NM_006218 PIK3R1 NM_0181523 PTCH1 NM_000264 PTEN
NM_000314 RB1 NM_000321 RET NM_020630 SMO NM_005631 STK11 NM_000455
TP53 NM_000546 ABL2 NM_005158 ATM NM_000051 AURKA NM_003600 AURKB
NM_004217 BCL2 NM_000633 BCL2L1 NM_001191 BCL2L2 NM_004050 BCL6
NM_001706 BRCA1 NM_007294 BRCA2 NM_000059 CBL NM_005188 CCNE1
NM_001238 CDH1 NM_004360 CDH2 NM_001792 CDK6 NM_001259 CDK8
NM_001260 CHEK1 NM_001274 CHEK2 NM_007194 CRKL NM_005207 EPHA6
NM_173655 EPHB4 NM_004444 EPHB6 NM_004445 ERBB3 NM_001982 ERBB4
NM_005235 FBXW7 NM_018315 FGFR4 NM_002011 FLT1 NM_002019 FLT4
NM_182925 GATA1 NM_002049 GNAS NM_016592 HOXA3 NM_030661 HSP90AA1
NM_005348 IDH1 NM_005896 IDH2 NM_002168 IGF1R NM_000875 IGF2R
NM_000876 IKBKE NM_014002 INHBA NM_002192 IRS2 NM_003749 JAK3
NM_000215 KDR NM_002253 MAP2K4 NM_003010 MCL1 NM_021960 MDM2
NM_002392 MDM4 NM_002393 MEN1 NM_000244 MITF NM_198159 MLH1
NM_000249 MPL NM_005373 MRE11A NM_005590 MSH2 NM_000251 MSH6
NM_000179 MTOR NM_004958 MYCL1 NM_005376 MYCN NM_005378 NF2
NM_000268 NKX2-1 NM_003317 NTRK1 NM_002529 PAX5 NM_016734 PDGFRB
NM_002609 PKHD1 NM_138694 PRKDC NM_006904 PTPN11 NM_002834 RAF1
NM_002880 RARA NM_000964 RICTOR NM_152756 RPTOR NM_020761 RUNX1
NM_001754 SMAD2 NM_005901 SMAD3 NM_005902 SMAD4 NM_005359 SMARCA4
NM_003072 SMARCB1 NM_003073 SOX10 NM_006941 SOX2 NM_003106 SRC
NM_005417 TET2 NM_017628 TGFBR2 NM_003242 TOP1 NM_003286 TSC1
NM_000368 TSC2 NM_000548 VHL NM_000551 WT1 NM_000378 ARFRP1
NM_003224 BCL2A1 NM_004049 CDH20 NM_031891 CDH5 NM_001795 EPHA3
NM_005233 EPHA5 NM_004439 EPHA7 NM_004440 EPHB1 NM_004441 FOXP4
NM_138457 GPR124 NM_032777 GUCY1A2 NM_000855 LRP1B NM_018557 LTK
NM_002344 PAK3 NM_002578 PLCG1 NM_002660 PTPRD NM_002839 TBX22
NM_016954 USP9X NM_001039590 Gene Group # markers RefSeq Exons 45
CDKN2A Del. A 52 NM_000077 Introns -2 MAP2K4 Del. B 118 NM_003010
PGx -2 PTEN Del. A 97 NM_000314 Amp/Del 4 RB1 Del. A 105 NM_000321
Exons + Intros 152 WT1 Del. B 94 NM_000378 Ex + Int + PGx 184
TABLE-US-00004 SUPPLEMENTARY TABLE 1b genes sequenced across
selected introns (n = 14) Gene RefSeq Introns sequenced ALK
NM_004304 19 BCR NM_004327 8, 13, 14 BRAF NM_004333 7, 8, 9, 10
EGFR NM_005228 7 ETV1 NM_004956 3, 4 ETV4 NM_001986 8 ETV5
NM_004454 6, 7 ETV6 NM_001987 5, 6 EWSR1 NM_005243 8, 9, 10, 11,
12, 13 MLL NM_005933 6, 7, 8, 9 RAF1 NM_002880 5, 6, 7, 8, 9 RARA
NM_000964 2 RET NM_020630 9, 10, 11 TMPRSS2 NM_005656 1, 2
TABLE-US-00005 SUPPLEMENTARY TABLE 2a Alterations in 40 CRC cases
Total Copy alter- Substitu- number Rearrange- Sample ations tions
INDELs changes ment APC TP53 KRAS BRAF FBXW7 S1346* SM77E 3 3 R213*
R213* SM1B 3 3 D13941s*21 R243W SM41E 3 2 1 DEL G245S G12A SMB8E 8
3 3 DEL R273C R181C V600E DEL SM75 3 1 2 DEL DEL C242F SM16 2 1 1
E130012*4 R306* E1353* SM21 4 4 E1374* R175H E1544* SM0 3 2 1 DEL
V272M G1288* SM7 3 2 1 DEL R248W SM42E 2 2 K1370* G12V SM83 2 2
K534* C238Y L14881A*19 SM20 4 3 1 R213* G12A P1324K*91 SM32 3 2 1
Q1406* K132R P1373K*42 SM40 3 1 2 DEL C176Y SM4 3 3 Q1294* G12D
R278* Q1406* SM6 4 3 1 L1120S R2BOT SM2B 3 3 Q1429* G12C SM23 4 4
Q160V* V600E Q789* SM81 3 2 1 DEL L194F SM8B 3 2 1 R139918*9 E258G
R858* R1450* SM8E 9 3 8 DEL R196* V600E SM45 3 3 R213* R262W G12V
R216* SM100E 3 2 1 C1252* V122fs*26 R232* SM67 3 2 1 Q1131* SM110E
3 2 1 R564* F212fs*3 G12V R584* SM3 4 3 1 E1322* R282W S1465fs*3
SM64 5 1 4 INS DEL V600E INS SM29 3 1 1 1 C135F SM14 2 1 1 DEL G12V
SM13E 1 1 DEL SM34 1 1 E204fs*43 SM19 4 2 2 E258A V600E S688fs*28
SM36E 3 0 2 1 INS SM12 7 3 4 P260H V600E SM11 2 2 R248W G12V SM30 3
2 1 R273C DEL SM73 2 2 S240R SM74NE 2 2 V173M G12C SM61 1 1 SM78E 0
Total 125 80 39 4 2 42 34 10 6 5 Sample CDH1 MYC ATM BCL2L1 BRCA2
ERBB3 GNAS PIK3CA SMAD4 ALK SM77E SM1B S434* SM41E SMB8E DEL SM75
SM16 SM21 G284R SM0 SM7 SM42E SM83 SM20 E645K SM32 SM40 SM4 SM6
SM2B E545K SM23 V104M R201H SM81 SM8B SM8E P1281s*39 DEL SM45
SM100E SM67 Amp SM110E SM3 Amp SM64 SM29 Amp Rearranged SM14 SM13E
SM34 SM19 DEL SM36E S1982fs*22 R74* P201fs*14 SM12 P126fs*89 R1876*
SM11 SM30 A118V SM73 SM74NE SM61 201H SM78E Total 4 3 2 2 2 2 2 2 2
1 Sample CDK8 LRP1B MSH6 RICTOR SMAD2 STK11 SM77E SM1B S434* SM41E
SMB8E SM75 SM16 SM21 SM0 SM7 SM42E SM83 SM20 SM32 SM40 SM4 SM6 Amp
SM2B SM23 SM81 SM8B DEL, DEL SM8E INS SM45 SM100E SM67 SM110E SM3
SM64 SM29 SM14 SM13E SM34 SM19 SM36E Rearranged SM12 P1087fs*6 SM11
SM30 SM73 Q214R SM74NE SM61 SM78E Total 1 1 1 1 1 1
TABLE-US-00006 SUPPLEMENTARY TABLE 2b Alterations in 24 NSCLC cases
Total Sub- Copy alter- stitu- Total number Gene Sample ations tions
INDELs changes fusion KRAS TP63 STK11 LAP1B JAK2 CTNNB1 RET EGFR
SM109 1 1 D1NJC SM86 4 4 G12C V617F 135S SM51 3 3 G12C E165* SM71E
1 1 G12C SM89 2 1 1 G12F SM90 2 2 G12V M2371 SM96 3 3 G12V Q2940*
SM44 3 2 1 G12V DEL S37Y SM7DE 1 1 G12V SM107 1 1 G12A 5M91A3 7 6 1
C229fs*10 D194Y K4112* V617F SM93 2 2 C242F SM63 1 1 G2455 SM48 3 2
1 K132* E3058* SM114 3 1 1 1 R24BL Hom del FUSION SM53 3 1 1 1
Y163C SM92 8 4 2 V617F SM87 2 2 AMP D770_M771 SM64E 1 1 insSVD
SM113 1 1 SM46 0 SM112 0 SM4DA5E 0 SM55 0 Total 50 36 7 6 1 10 7 4
3 3 2 1 2 Sample BRAF CDKN2A MDN2 PIK3CA ATM TSC1 CCNE1 NF1 RB1 APC
MLH1 MSH6 CCK4 SM109 SM86 E645K SM51 G468A SM71E SM89 DEL SM90 SM96
H83Y SM44 SM7DE SM107 SM91A3 Y1635* E290 L11295 SM93 E13* SM63 SM48
DEL SM114 SM53 INS AMP SM92 G458V AMP N345K V5ORA AMP SM87 AMP
SM64E SM113 DEL SM46 SM112 SM4DA5E SM55 Total 2 2 2 2 2 1 1 1 1 1 1
1 1
TABLE-US-00007 SUPPLEMENTARY TABLE 3 alterations that could be
linked to a clinical treatment option or clinical trial of novel
targeted therapies Mutated Gene Samples Potential therapeutic
treatment or clinical trial TP53 32 Presently unknown APC 27
Presently unknown KRAS 10 Resistance to cetuximab and panitumumab
BRAF 6 Resistance to cetuximab and panitumumab FBXW7 5 Potential
resistance to tubulins ATM 2 PARP inhibitors BCL2L1 2 Presently
unknown BRCA2 2 PARP inhibitors CDH1 2 Presently unknown ERBB3 2
Presently unknown GNAS 2 MEK or ERK inhibitors PIK3CA 2 PI3
kinase/mTOR inhibitors SMAD4 2 prognostic poor ALK 1 ALK inhibitors
e.g. Crizotinib CDK8 1 CDK inhibitors e.g. PD0332991 LRP1B 1
Presently unknown MYC 1 Presently unknown MSH6 1 Prognostic factor
RICTOR 1 Presently unknown SMAD2 1 Presently unknown STK11 1
Presently unknown
TABLE-US-00008 SUPPLEMENTARY TABLE 4 Distribution of 51 mutations
across 21 mutated NSCLC genes Mu- Homozy- tated Total Non INDEL
Ampli- gous Sam- Muta- Synony- Non- frame- fica- Dele- Gene Gene
ples tions mous sense shift tion tion Fusion KRAS 10 10 10 -- -- --
-- -- TP53 7 7 5 1 1 -- -- -- STK11 4 4 1 1 1 -- 1 -- LRP1B 3 3 --
3 -- -- -- -- JAK2 3 3 3 -- -- -- -- -- EGFR 2 2 -- -- -- 1 -- --
BRAF 2 2 2 -- -- -- -- -- CDKN2A 2 2 1 -- -- -- -- -- RET 1 1 -- --
-- -- -- 1 CTNNB1 2 2 2 -- -- -- -- -- MDM2 2 2 -- -- -- 2 -- --
PIK3CA 2 2 2 -- -- -- -- -- ATM 2 2 -- -- 2 -- -- -- TSCI 1 1 -- --
1 -- -- -- CCNE1 1 1 -- -- -- 1 -- -- NF1 1 1 -- 1 -- -- -- -- RB1
1 1 -- 1 -- -- -- -- APC 1 1 1 -- -- -- -- -- MLH1 1 1 -- 1 -- --
-- -- MSH6 1 1 1 -- -- -- -- -- CDK4 1 1 -- -- -- 1 -- --
TABLE-US-00009 SUPPLEMENTARY TABLE 5 NSCLC alteration that could be
linked to a clinical treatment option or clinical trial of novel
targeted therapies Total Gene Mutations Potential therapeutic
treatment or clinical trial KRAS 10 Resistance to EGFR kinase
inhibitors, clinical trials of PI3K and MEK inhibitors STK11 4
Presently unknown JAK2 3 JAK2 inhibitors EGFR 2 Erlotinib or
gefitinib BRAF 2 Vemurafenib and GSK 2118436 CDKN2A 2 CDK
inhibitors e.g., PD0332991 RET 2 RET inhibitors e.g., Sorafanib or
sunitinib CTNNB1 2 Presently unknown MDM2 2 Nutlins PIK3CA 2 PI3
kinase/mTOR inhibitors ATM 2 PARP inhibitors TSC1 1 mTOR inhibitors
CCNE1 1 CDK4 inhibitors e.g., PD0332991 NF1 1 Presently unknown RB1
1 Presently unknown MLH1 1 Presently unknown MSH6 1 Presently
unknown CDK4 1 CDK4 inhibitors e.g., PD0332991
ADDITIONAL REFERENCES
[0418] James et al., Nucl. Acids Res. 16:1999-2014, 1988. [0419]
Levin et al., Genome Biol. 10:R115, 2009. [0420] Li and Durbin,
Bioinformatics 26:589-95, 2010. [0421] Li et al., Bioinformatics
25:2078-9, 2009. [0422] online at picard.sourceforge.net [0423]
McKenna et al., Genome Res. 20:1297-303, 2010. [0424] Forbes et al,
Nucl. Acids Res. 39 (suppl 1): D945-D950, 2011. [0425] Smigielski
et al., Nucl. Acids Res. 28:352-355, 2000.
INCORPORATION BY REFERENCE
[0426] All publications, patents, and patent applications mentioned
herein are hereby incorporated by reference in their entirety as if
each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference. In case of conflict, the present application, including
any definitions herein, will control.
[0427] Also incorporated by reference in their entirety are any
polynucleotide and polypeptide sequences which reference an
accession number correlating to an entry in a public database, such
as those maintained by the COSMIC database, available on the
worldwide web at sanger.ac.uk/genetics/CGP/cosmic/; and the
Institute for Genomic Research (TIGR) on the world wide web at
tigr.org and/or the National Center for Biotechnology Information
(NCBI) on the world wide web at ncbi.nlm.nih.gov.
EQUIVALENTS
[0428] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments described herein. Such
equivalents are intended to be encompassed by the following claims.
Sequence CWU 1
1
912934DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotideCDS(1)..(2931) 1atg gcg gac ctg gcc gag tgc
aac atc aaa gtg atg tgt cgc ttc aga 48Met Ala Asp Leu Ala Glu Cys
Asn Ile Lys Val Met Cys Arg Phe Arg 1 5 10 15 cct ctc aac gag tct
gaa gtg aac cgc ggc gac aag tac atc gcc aag 96Pro Leu Asn Glu Ser
Glu Val Asn Arg Gly Asp Lys Tyr Ile Ala Lys 20 25 30 ttt cag gga
gaa gac acg gtc gtg atc gcg tcc aag cct tat gca ttt 144Phe Gln Gly
Glu Asp Thr Val Val Ile Ala Ser Lys Pro Tyr Ala Phe 35 40 45 gat
cgg gtg ttc cag tca agc aca tct caa gag caa gtg tat aat gac 192Asp
Arg Val Phe Gln Ser Ser Thr Ser Gln Glu Gln Val Tyr Asn Asp 50 55
60 tgt gca aag aag att gtt aaa gat gta ctt gaa gga tat aat gga aca
240Cys Ala Lys Lys Ile Val Lys Asp Val Leu Glu Gly Tyr Asn Gly Thr
65 70 75 80 ata ttt gca tat gga caa aca tcc tct ggg aag aca cac aca
atg gag 288Ile Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr His Thr
Met Glu 85 90 95 ggt aaa ctt cat gat cca gaa ggc atg gga att att
cca aga ata gtg 336Gly Lys Leu His Asp Pro Glu Gly Met Gly Ile Ile
Pro Arg Ile Val 100 105 110 caa gat att ttt aat tat att tac tcc atg
gat gaa aat ttg gaa ttt 384Gln Asp Ile Phe Asn Tyr Ile Tyr Ser Met
Asp Glu Asn Leu Glu Phe 115 120 125 cat att aag gtt tca tat ttt gaa
ata tat ttg gat aag ata agg gac 432His Ile Lys Val Ser Tyr Phe Glu
Ile Tyr Leu Asp Lys Ile Arg Asp 130 135 140 ctg tta gat gtt tca aag
acc aac ctt tca gtt cat gaa gac aaa aac 480Leu Leu Asp Val Ser Lys
Thr Asn Leu Ser Val His Glu Asp Lys Asn 145 150 155 160 cga gtt ccc
tat gta aag ggg tgc aca gag cgt ttt gta tgt agt cca 528Arg Val Pro
Tyr Val Lys Gly Cys Thr Glu Arg Phe Val Cys Ser Pro 165 170 175 gat
gaa gtt atg gat acc ata gat gaa gga aaa tcc aac aga cat gta 576Asp
Glu Val Met Asp Thr Ile Asp Glu Gly Lys Ser Asn Arg His Val 180 185
190 gca gtt aca aat atg aat gaa cat agc tct agg agt cac agt ata ttt
624Ala Val Thr Asn Met Asn Glu His Ser Ser Arg Ser His Ser Ile Phe
195 200 205 ctt att aat gtc aaa caa gag aac aca caa acg gaa caa aag
ctg agt 672Leu Ile Asn Val Lys Gln Glu Asn Thr Gln Thr Glu Gln Lys
Leu Ser 210 215 220 gga aaa ctt tat ctg gtt gat tta gct ggt agt gaa
aag gtt agt aaa 720Gly Lys Leu Tyr Leu Val Asp Leu Ala Gly Ser Glu
Lys Val Ser Lys 225 230 235 240 act gga gct gaa ggt gct gtg ctg gat
gaa gct aaa aac atc aac aag 768Thr Gly Ala Glu Gly Ala Val Leu Asp
Glu Ala Lys Asn Ile Asn Lys 245 250 255 tca ctt tct gct ctt gga aat
gtt att tct gct ttg gct gag ggt agt 816Ser Leu Ser Ala Leu Gly Asn
Val Ile Ser Ala Leu Ala Glu Gly Ser 260 265 270 aca tat gtt cca tat
cga gat agt aaa atg aca aga atc ctt caa gat 864Thr Tyr Val Pro Tyr
Arg Asp Ser Lys Met Thr Arg Ile Leu Gln Asp 275 280 285 tca tta ggt
ggc aac tgt aga acc act att gta att tgc tgc tct cca 912Ser Leu Gly
Gly Asn Cys Arg Thr Thr Ile Val Ile Cys Cys Ser Pro 290 295 300 tca
tca tac aat gag tct gaa aca aaa tct aca ctc tta ttt ggc caa 960Ser
Ser Tyr Asn Glu Ser Glu Thr Lys Ser Thr Leu Leu Phe Gly Gln 305 310
315 320 agg gcc aaa aca att aag aac aca gtt tgt gtc aat gtg gag tta
act 1008Arg Ala Lys Thr Ile Lys Asn Thr Val Cys Val Asn Val Glu Leu
Thr 325 330 335 gca gaa cag tgg aaa aag aag tat gaa aaa gaa aaa gaa
aaa aat aag 1056Ala Glu Gln Trp Lys Lys Lys Tyr Glu Lys Glu Lys Glu
Lys Asn Lys 340 345 350 atc ctg cgg aac act att cag tgg ctt gaa aat
gag ctc aac aga tgg 1104Ile Leu Arg Asn Thr Ile Gln Trp Leu Glu Asn
Glu Leu Asn Arg Trp 355 360 365 cgt aat ggg gag acg gtg cct att gat
gaa cag ttt gac aaa gag aaa 1152Arg Asn Gly Glu Thr Val Pro Ile Asp
Glu Gln Phe Asp Lys Glu Lys 370 375 380 gcc aac ttg gaa gct ttc aca
gtg gat aaa gat att act ctt acc aat 1200Ala Asn Leu Glu Ala Phe Thr
Val Asp Lys Asp Ile Thr Leu Thr Asn 385 390 395 400 gat aaa cca gca
acc gca att gga gtt ata gga aat ttt act gat gct 1248Asp Lys Pro Ala
Thr Ala Ile Gly Val Ile Gly Asn Phe Thr Asp Ala 405 410 415 gaa aga
aga aag tgt gaa gaa gaa att gct aaa tta tac aaa cag ctt 1296Glu Arg
Arg Lys Cys Glu Glu Glu Ile Ala Lys Leu Tyr Lys Gln Leu 420 425 430
gat gac aag gat gaa gaa att aac cag caa agt caa ctg gta gag aaa
1344Asp Asp Lys Asp Glu Glu Ile Asn Gln Gln Ser Gln Leu Val Glu Lys
435 440 445 ctg aag acg caa atg ttg gat cag gag gag ctt ttg gca tct
acc aga 1392Leu Lys Thr Gln Met Leu Asp Gln Glu Glu Leu Leu Ala Ser
Thr Arg 450 455 460 agg gat caa gac aat atg caa gct gag ctg aat cgc
ctt caa gca gaa 1440Arg Asp Gln Asp Asn Met Gln Ala Glu Leu Asn Arg
Leu Gln Ala Glu 465 470 475 480 aat gat gcc tct aaa gaa gaa gtg aaa
gaa gtt tta cag gcc cta gaa 1488Asn Asp Ala Ser Lys Glu Glu Val Lys
Glu Val Leu Gln Ala Leu Glu 485 490 495 gaa ctt gct gtc aat tat gat
cag aag tct cag gaa gtt gaa gac aaa 1536Glu Leu Ala Val Asn Tyr Asp
Gln Lys Ser Gln Glu Val Glu Asp Lys 500 505 510 act aag gaa tat gaa
ttg ctt agt gat gaa ttg aat cag aaa tcg gca 1584Thr Lys Glu Tyr Glu
Leu Leu Ser Asp Glu Leu Asn Gln Lys Ser Ala 515 520 525 act tta gcg
agt ata gat gct gag ctt cag aaa ctt aag gaa atg acc 1632Thr Leu Ala
Ser Ile Asp Ala Glu Leu Gln Lys Leu Lys Glu Met Thr 530 535 540 aac
cac cag aaa aaa cga gca gct gag atg atg gca tct tta cta aaa 1680Asn
His Gln Lys Lys Arg Ala Ala Glu Met Met Ala Ser Leu Leu Lys 545 550
555 560 gac ctt gca gaa ata gga att gct gtg gga aat aat gat gta aag
gag 1728Asp Leu Ala Glu Ile Gly Ile Ala Val Gly Asn Asn Asp Val Lys
Glu 565 570 575 gat cca aag tgg gaa ttc cct cgg aag aac ttg gtt ctt
gga aaa act 1776Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu
Gly Lys Thr 580 585 590 cta gga gaa ggc gaa ttt gga aaa gtg gtc aag
gca acg gcc ttc cat 1824Leu Gly Glu Gly Glu Phe Gly Lys Val Val Lys
Ala Thr Ala Phe His 595 600 605 ctg aaa ggc aga gca ggg tac acc acg
gtg gcc gtg aag atg ctg aaa 1872Leu Lys Gly Arg Ala Gly Tyr Thr Thr
Val Ala Val Lys Met Leu Lys 610 615 620 gag aac gcc tcc ccg agt gag
ctg cga gac ctg ctg tca gag ttc aac 1920Glu Asn Ala Ser Pro Ser Glu
Leu Arg Asp Leu Leu Ser Glu Phe Asn 625 630 635 640 gtc ctg aag cag
gtc aac cac cca cat gtc atc aaa ttg tat ggg gcc 1968Val Leu Lys Gln
Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala 645 650 655 tgc agc
cag gat ggc ccg ctc ctc ctc atc gtg gag tac gcc aaa tac 2016Cys Ser
Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr 660 665 670
ggc tcc ctg cgg ggc ttc ctc cgc gag agc cgc aaa gtg ggg cct ggc
2064Gly Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly
675 680 685 tac ctg ggc agt gga ggc agc cgc aac tcc agc tcc ctg gac
cac ccg 2112Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp
His Pro 690 695 700 gat gag cgg gcc ctc acc atg ggc gac ctc atc tca
ttt gcc tgg cag 2160Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser
Phe Ala Trp Gln 705 710 715 720 atc tca cag ggg atg cag tat ctg gcc
gag atg aag ctc gtt cat cgg 2208Ile Ser Gln Gly Met Gln Tyr Leu Ala
Glu Met Lys Leu Val His Arg 725 730 735 gac ttg gca gcc aga aac atc
ctg gta gct gag ggg cgg aag atg aag 2256Asp Leu Ala Ala Arg Asn Ile
Leu Val Ala Glu Gly Arg Lys Met Lys 740 745 750 att tcg gat ttc ggc
ttg tcc cga gat gtt tat gaa gag gat tcc tac 2304Ile Ser Asp Phe Gly
Leu Ser Arg Asp Val Tyr Glu Glu Asp Ser Tyr 755 760 765 gtg aag agg
agc cag ggt cgg att cca gtt aaa tgg atg gca att gaa 2352Val Lys Arg
Ser Gln Gly Arg Ile Pro Val Lys Trp Met Ala Ile Glu 770 775 780 tcc
ctt ttt gat cat atc tac acc acg caa agt gat gta tgg tct ttt 2400Ser
Leu Phe Asp His Ile Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe 785 790
795 800 ggt gtc ctg ctg tgg gag atc gtg acc cta ggg gga aac ccc tat
cct 2448Gly Val Leu Leu Trp Glu Ile Val Thr Leu Gly Gly Asn Pro Tyr
Pro 805 810 815 ggg att cct cct gag cgg ctc ttc aac ctt ctg aag acc
ggc cac cgg 2496Gly Ile Pro Pro Glu Arg Leu Phe Asn Leu Leu Lys Thr
Gly His Arg 820 825 830 atg gag agg cca gac aac tgc agc gag gag atg
tac cgc ctg atg ctg 2544Met Glu Arg Pro Asp Asn Cys Ser Glu Glu Met
Tyr Arg Leu Met Leu 835 840 845 caa tgc tgg aag cag gag ccg gac aaa
agg ccg gtg ttt gcg gac atc 2592Gln Cys Trp Lys Gln Glu Pro Asp Lys
Arg Pro Val Phe Ala Asp Ile 850 855 860 agc aaa gac ctg gag aag atg
atg gtt aag agg aga gac tac ttg gac 2640Ser Lys Asp Leu Glu Lys Met
Met Val Lys Arg Arg Asp Tyr Leu Asp 865 870 875 880 ctt gcg gcg tcc
act cca tct gac tcc ctg att tat gac gac ggc ctc 2688Leu Ala Ala Ser
Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu 885 890 895 tca gag
gag gag aca ccg ctg gtg gac tgt aat aat gcc ccc ctc cct 2736Ser Glu
Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro 900 905 910
cga gcc ctc cct tcc aca tgg att gaa aac aaa ctc tat ggc atg tca
2784Arg Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser
915 920 925 gac ccg aac tgg cct gga gag agt cct gta cca ctc acg aga
gct gat 2832Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg
Ala Asp 930 935 940 ggc act aac act ggg ttt cca aga tat cca aat gat
agt gta tat gct 2880Gly Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp
Ser Val Tyr Ala 945 950 955 960 aac tgg atg ctt tca ccc tca gcg gca
aaa tta atg gac acg ttt gat 2928Asn Trp Met Leu Ser Pro Ser Ala Ala
Lys Leu Met Asp Thr Phe Asp 965 970 975 agt taa 2934Ser
2977PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Met Ala Asp Leu Ala Glu Cys Asn Ile Lys Val
Met Cys Arg Phe Arg 1 5 10 15 Pro Leu Asn Glu Ser Glu Val Asn Arg
Gly Asp Lys Tyr Ile Ala Lys 20 25 30 Phe Gln Gly Glu Asp Thr Val
Val Ile Ala Ser Lys Pro Tyr Ala Phe 35 40 45 Asp Arg Val Phe Gln
Ser Ser Thr Ser Gln Glu Gln Val Tyr Asn Asp 50 55 60 Cys Ala Lys
Lys Ile Val Lys Asp Val Leu Glu Gly Tyr Asn Gly Thr 65 70 75 80 Ile
Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr His Thr Met Glu 85 90
95 Gly Lys Leu His Asp Pro Glu Gly Met Gly Ile Ile Pro Arg Ile Val
100 105 110 Gln Asp Ile Phe Asn Tyr Ile Tyr Ser Met Asp Glu Asn Leu
Glu Phe 115 120 125 His Ile Lys Val Ser Tyr Phe Glu Ile Tyr Leu Asp
Lys Ile Arg Asp 130 135 140 Leu Leu Asp Val Ser Lys Thr Asn Leu Ser
Val His Glu Asp Lys Asn 145 150 155 160 Arg Val Pro Tyr Val Lys Gly
Cys Thr Glu Arg Phe Val Cys Ser Pro 165 170 175 Asp Glu Val Met Asp
Thr Ile Asp Glu Gly Lys Ser Asn Arg His Val 180 185 190 Ala Val Thr
Asn Met Asn Glu His Ser Ser Arg Ser His Ser Ile Phe 195 200 205 Leu
Ile Asn Val Lys Gln Glu Asn Thr Gln Thr Glu Gln Lys Leu Ser 210 215
220 Gly Lys Leu Tyr Leu Val Asp Leu Ala Gly Ser Glu Lys Val Ser Lys
225 230 235 240 Thr Gly Ala Glu Gly Ala Val Leu Asp Glu Ala Lys Asn
Ile Asn Lys 245 250 255 Ser Leu Ser Ala Leu Gly Asn Val Ile Ser Ala
Leu Ala Glu Gly Ser 260 265 270 Thr Tyr Val Pro Tyr Arg Asp Ser Lys
Met Thr Arg Ile Leu Gln Asp 275 280 285 Ser Leu Gly Gly Asn Cys Arg
Thr Thr Ile Val Ile Cys Cys Ser Pro 290 295 300 Ser Ser Tyr Asn Glu
Ser Glu Thr Lys Ser Thr Leu Leu Phe Gly Gln 305 310 315 320 Arg Ala
Lys Thr Ile Lys Asn Thr Val Cys Val Asn Val Glu Leu Thr 325 330 335
Ala Glu Gln Trp Lys Lys Lys Tyr Glu Lys Glu Lys Glu Lys Asn Lys 340
345 350 Ile Leu Arg Asn Thr Ile Gln Trp Leu Glu Asn Glu Leu Asn Arg
Trp 355 360 365 Arg Asn Gly Glu Thr Val Pro Ile Asp Glu Gln Phe Asp
Lys Glu Lys 370 375 380 Ala Asn Leu Glu Ala Phe Thr Val Asp Lys Asp
Ile Thr Leu Thr Asn 385 390 395 400 Asp Lys Pro Ala Thr Ala Ile Gly
Val Ile Gly Asn Phe Thr Asp Ala 405 410 415 Glu Arg Arg Lys Cys Glu
Glu Glu Ile Ala Lys Leu Tyr Lys Gln Leu 420 425 430 Asp Asp Lys Asp
Glu Glu Ile Asn Gln Gln Ser Gln Leu Val Glu Lys 435 440 445 Leu Lys
Thr Gln Met Leu Asp Gln Glu Glu Leu Leu Ala Ser Thr Arg 450 455 460
Arg Asp Gln Asp Asn Met Gln Ala Glu Leu Asn Arg Leu Gln Ala Glu 465
470 475 480 Asn Asp Ala Ser Lys Glu Glu Val Lys Glu Val Leu Gln Ala
Leu Glu 485 490 495 Glu Leu Ala Val Asn Tyr Asp Gln Lys Ser Gln Glu
Val Glu Asp Lys 500 505 510 Thr Lys Glu Tyr Glu Leu Leu Ser Asp Glu
Leu Asn Gln Lys Ser Ala 515 520 525 Thr Leu Ala Ser Ile Asp Ala Glu
Leu Gln Lys Leu Lys Glu Met Thr 530 535 540 Asn His Gln Lys Lys Arg
Ala Ala Glu Met Met Ala Ser Leu Leu Lys 545 550 555 560 Asp Leu Ala
Glu Ile Gly Ile Ala Val Gly Asn Asn Asp Val Lys Glu 565 570 575 Asp
Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly Lys Thr 580
585 590 Leu Gly Glu Gly Glu Phe Gly Lys Val Val Lys Ala Thr Ala Phe
His 595 600 605 Leu Lys Gly Arg Ala Gly Tyr Thr Thr Val Ala Val Lys
Met Leu Lys 610 615 620 Glu Asn Ala Ser Pro Ser Glu Leu Arg Asp Leu
Leu Ser Glu Phe Asn 625 630 635 640 Val Leu Lys Gln Val Asn His Pro
His Val Ile Lys Leu Tyr Gly Ala 645 650 655 Cys Ser Gln Asp Gly Pro
Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr 660 665 670 Gly Ser Leu Arg
Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly 675 680 685 Tyr Leu
Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His Pro 690 695 700
Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala Trp Gln 705
710 715 720 Ile Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu Val
His Arg 725 730 735 Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly
Arg Lys Met Lys 740 745 750 Ile Ser Asp Phe Gly Leu Ser Arg Asp Val
Tyr Glu Glu Asp Ser Tyr 755 760 765 Val Lys Arg Ser Gln Gly Arg Ile
Pro Val Lys Trp Met Ala Ile Glu 770 775 780 Ser Leu Phe Asp His Ile
Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe 785 790 795 800 Gly Val Leu
Leu Trp Glu Ile Val Thr Leu Gly Gly Asn Pro Tyr Pro 805 810 815 Gly
Ile Pro Pro Glu Arg Leu Phe Asn Leu Leu Lys Thr Gly His Arg 820 825
830 Met Glu Arg Pro Asp Asn Cys Ser Glu Glu Met Tyr Arg Leu Met Leu
835 840 845 Gln Cys Trp Lys Gln Glu Pro Asp Lys Arg Pro Val Phe Ala
Asp Ile 850 855 860 Ser Lys Asp Leu Glu Lys Met Met Val Lys Arg Arg
Asp Tyr Leu Asp 865 870 875 880 Leu Ala Ala Ser Thr Pro Ser Asp Ser
Leu Ile Tyr Asp Asp Gly Leu 885 890 895 Ser Glu Glu Glu Thr Pro Leu
Val Asp Cys Asn Asn Ala Pro Leu Pro 900 905 910 Arg Ala Leu Pro Ser
Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser 915 920 925 Asp Pro Asn
Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg Ala Asp 930 935 940 Gly
Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val Tyr Ala 945 950
955 960 Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp Thr Phe
Asp 965 970 975 Ser 33303DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotideCDS(1)..(3300) 3atg gcg
aag gcg acg tcc ggt gcc gcg ggg ctg cgt ctg ctg ttg ctg 48Met Ala
Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu Leu 1 5 10 15
ctg ctg ctg ccg ctg cta ggc aaa gtg gca ttg ggc ctc tac ttc tcg
96Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr Phe Ser
20 25 30 agg gat gct tac tgg gag aag ctg tat gtg gac cag gca gcc
ggc acg 144Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala Ala
Gly Thr 35 40 45 ccc ttg ctg tac gtc cat gcc ctg cgg gac gcc cct
gag gag gtg ccc 192Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro
Glu Glu Val Pro 50 55 60 agc ttc cgc ctg ggc cag cat ctc tac ggc
acg tac cgc aca cgg ctg 240Ser Phe Arg Leu Gly Gln His Leu Tyr Gly
Thr Tyr Arg Thr Arg Leu 65 70 75 80 cat gag aac aac tgg atc tgc atc
cag gag gac acc ggc ctc ctc tac 288His Glu Asn Asn Trp Ile Cys Ile
Gln Glu Asp Thr Gly Leu Leu Tyr 85 90 95 ctt aac cgg agc ctg gac
cat agc tcc tgg gag aag ctc agt gtc cgc 336Leu Asn Arg Ser Leu Asp
His Ser Ser Trp Glu Lys Leu Ser Val Arg 100 105 110 aac cgc ggc ttt
ccc ctg ctc acc gtc tac ctc aag gtc ttc ctg tca 384Asn Arg Gly Phe
Pro Leu Leu Thr Val Tyr Leu Lys Val Phe Leu Ser 115 120 125 ccc aca
tcc ctt cgt gag ggc gag tgc cag tgg cca ggc tgt gcc cgc 432Pro Thr
Ser Leu Arg Glu Gly Glu Cys Gln Trp Pro Gly Cys Ala Arg 130 135 140
gta tac ttc tcc ttc ttc aac acc tcc ttt cca gcc tgc agc tcc ctc
480Val Tyr Phe Ser Phe Phe Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu
145 150 155 160 aag ccc cgg gag ctc tgc ttc cca gag aca agg ccc tcc
ttc cgc att 528Lys Pro Arg Glu Leu Cys Phe Pro Glu Thr Arg Pro Ser
Phe Arg Ile 165 170 175 cgg gag aac cga ccc cca ggc acc ttc cac cag
ttc cgc ctg ctg cct 576Arg Glu Asn Arg Pro Pro Gly Thr Phe His Gln
Phe Arg Leu Leu Pro 180 185 190 gtg cag ttc ttg tgc ccc aac atc agc
gtg gcc tac agg ctc ctg gag 624Val Gln Phe Leu Cys Pro Asn Ile Ser
Val Ala Tyr Arg Leu Leu Glu 195 200 205 ggt gag ggt ctg ccc ttc cgc
tgc gcc ccg gac agc ctg gag gtg agc 672Gly Glu Gly Leu Pro Phe Arg
Cys Ala Pro Asp Ser Leu Glu Val Ser 210 215 220 acg cgc tgg gcc ctg
gac cgc gag cag cgg gag aag tac gag ctg gtg 720Thr Arg Trp Ala Leu
Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val 225 230 235 240 gcc gtg
tgc acc gtg cac gcc ggc gcg cgc gag gag gtg gtg atg gtg 768Ala Val
Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met Val 245 250 255
ccc ttc ccg gtg acc gtg tac gac gag gac gac tcg gcg ccc acc ttc
816Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala Pro Thr Phe
260 265 270 ccc gcg ggc gtc gac acc gcc agc gcc gtg gtg gag ttc aag
cgg aag 864Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe Lys
Arg Lys 275 280 285 gag gac acc gtg gtg gcc acg ctg cgt gtc ttc gat
gca gac gtg gta 912Glu Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp
Ala Asp Val Val 290 295 300 cct gca tca ggg gag ctg gtg agg cgg tac
aca agc acg ctg ctc ccc 960Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr
Thr Ser Thr Leu Leu Pro 305 310 315 320 ggg gac acc tgg gcc cag cag
acc ttc cgg gtg gaa cac tgg ccc aac 1008Gly Asp Thr Trp Ala Gln Gln
Thr Phe Arg Val Glu His Trp Pro Asn 325 330 335 gag acc tcg gtc cag
gcc aac ggc agc ttc gtg cgg gcg acc gta cat 1056Glu Thr Ser Val Gln
Ala Asn Gly Ser Phe Val Arg Ala Thr Val His 340 345 350 gac tat agg
ctg gtt ctc aac cgg aac ctc tcc atc tcg gag aac cgc 1104Asp Tyr Arg
Leu Val Leu Asn Arg Asn Leu Ser Ile Ser Glu Asn Arg 355 360 365 acc
atg cag ctg gcg gtg ctg gtc aat gac tca gac ttc cag ggc cca 1152Thr
Met Gln Leu Ala Val Leu Val Asn Asp Ser Asp Phe Gln Gly Pro 370 375
380 gga gcg ggc gtc ctc ttg ctc cac ttc aac gtg tcg gtg ctg ccg gtc
1200Gly Ala Gly Val Leu Leu Leu His Phe Asn Val Ser Val Leu Pro Val
385 390 395 400 agc ctg cac ctg ccc agt acc tac tcc ctc tcc gtg agc
agg agg gct 1248Ser Leu His Leu Pro Ser Thr Tyr Ser Leu Ser Val Ser
Arg Arg Ala 405 410 415 cgc cga ttt gcc cag atc ggg aaa gtc tgt gtg
gaa aac tgc cag gca 1296Arg Arg Phe Ala Gln Ile Gly Lys Val Cys Val
Glu Asn Cys Gln Ala 420 425 430 ttc agt ggc atc aac gtc cag tac aag
ctg cat tcc tct ggt gcc aac 1344Phe Ser Gly Ile Asn Val Gln Tyr Lys
Leu His Ser Ser Gly Ala Asn 435 440 445 tgc agc acg cta ggg gtg gtc
acc tca gcc gag gac acc tcg ggg atc 1392Cys Ser Thr Leu Gly Val Val
Thr Ser Ala Glu Asp Thr Ser Gly Ile 450 455 460 ctg ttt gtg aat gac
acc aag gcc ctg cgg cgg ccc aag tgt gcc gaa 1440Leu Phe Val Asn Asp
Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu 465 470 475 480 ctt cac
tac atg gtg gtg gcc acc gac cag cag acc tct agg cag gcc 1488Leu His
Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln Ala 485 490 495
cag gcc cag ctg ctt gta aca gtg gag ggg tca tat gtg gcc gag gag
1536Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala Glu Glu
500 505 510 gcg ggc tgc ccc ctg tcc tgt gca gtc agc aag aga cgg ctg
gag tgt 1584Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg Arg Leu
Glu Cys 515 520 525 gag gag tgt ggc ggc ctg ggc tcc cca aca ggc agg
tgt gag tgg agg 1632Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg
Cys Glu Trp Arg 530 535 540 caa gga gat ggc aaa ggg atc acc agg aac
ttc tcc acc tgc tct ccc 1680Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn
Phe Ser Thr Cys Ser Pro 545 550 555 560 agc acc aag acc tgc ccc gac
ggc cac tgc gat gtt gtg gag acc caa 1728Ser Thr Lys Thr Cys Pro Asp
Gly His Cys Asp Val Val Glu Thr Gln 565 570 575 gac atc aac att tgc
cct cag gac tgc ctc cgg ggc agc att gtt ggg 1776Asp Ile Asn Ile Cys
Pro Gln Asp Cys Leu Arg Gly Ser Ile Val Gly 580 585 590 gga cac gag
cct ggg gag ccc cgg ggg att aaa gct ggc tat ggc acc 1824Gly His Glu
Pro Gly Glu Pro Arg Gly Ile Lys Ala Gly Tyr Gly Thr 595 600 605 tgc
aac tgc ttc cct gag gag gag aag tgc ttc tgc gag ccc gaa gac 1872Cys
Asn Cys Phe Pro Glu Glu Glu Lys Cys Phe Cys Glu Pro Glu Asp 610 615
620 atc cag gat cca ctg tgc gac gag ctg tgc cgc acg gtg atc gca gcc
1920Ile Gln Asp Pro Leu Cys Asp Glu Leu Cys Arg Thr Val Ile Ala Ala
625 630 635 640 gct gtc ctc ttc tcc ttc atc gtc tcg gtg ctg ctg tct
gcc ttc tgc 1968Ala Val Leu Phe Ser Phe Ile Val Ser Val Leu Leu Ser
Ala Phe Cys 645 650 655 atc cac tgc tac cac aag ttt gcc cac aag cca
ccc atc tcc tca gct 2016Ile His Cys Tyr His Lys Phe Ala His Lys Pro
Pro Ile Ser Ser Ala 660 665 670 gag atg acc ttc cgg agg ccc gcc cag
gcc ttc ccg gtc agc tac tcc 2064Glu Met Thr Phe Arg Arg Pro Ala Gln
Ala Phe Pro Val Ser Tyr Ser 675 680 685 tct tcc ggt gcc cgc cgg ccc
tcg ctg gac tcc atg gag aac cag gtc 2112Ser Ser Gly Ala Arg Arg Pro
Ser Leu Asp Ser Met Glu Asn Gln Val 690 695 700 tcc gtg gat gcc ttc
aag atc ctg cag cct gag gga act ggc atg ata 2160Ser Val Asp Ala Phe
Lys Ile Leu Gln Pro Glu Gly Thr Gly Met Ile 705 710 715 720 gat gaa
gag ttc act gtt gca aga ctc tac att agc aaa atg aag tca 2208Asp Glu
Glu Phe Thr Val Ala Arg Leu Tyr Ile Ser Lys Met Lys Ser 725 730 735
gaa gta aaa acc atg gtg aaa cgt tgc aag cag tta gaa agc aca caa
2256Glu Val Lys Thr Met Val Lys Arg Cys Lys Gln Leu Glu Ser Thr Gln
740 745 750 act gag agc aac aaa aaa atg gaa gaa aat gaa aag gag tta
gca gca 2304Thr Glu Ser Asn Lys Lys Met Glu Glu Asn Glu Lys Glu Leu
Ala Ala 755 760 765 tgt cag ctt cgt atc tct caa cat gaa gcc aaa atc
aag tca ttg act 2352Cys Gln Leu Arg Ile Ser Gln His Glu Ala Lys Ile
Lys Ser Leu Thr 770 775 780 gaa tac ctt caa aat gtg gaa caa aag aaa
aga cag ttg gag gaa tct 2400Glu Tyr Leu Gln Asn Val Glu Gln Lys Lys
Arg Gln Leu Glu Glu Ser 785 790 795 800 gtc gat gcc ctc agt gaa gaa
cta gtc cag ctt cga gca caa gag aaa 2448Val Asp Ala Leu Ser Glu Glu
Leu Val Gln Leu Arg Ala Gln Glu Lys 805 810 815 gtc cat gaa atg gaa
aag gag cac tta aat aag gtt cag act gca aat 2496Val His Glu Met Glu
Lys Glu His Leu Asn Lys Val Gln Thr Ala Asn 820 825 830 gaa gtt aag
caa gct gtt gaa cag cag atc cag agc cat aga gaa act 2544Glu Val Lys
Gln Ala Val Glu Gln Gln Ile Gln Ser His Arg Glu Thr 835 840 845 cat
caa aaa cag atc agt agt ttg aga gat gaa gta gaa gca aaa gca 2592His
Gln Lys Gln Ile Ser Ser Leu Arg Asp Glu Val Glu Ala Lys Ala 850 855
860 aaa ctt att act gat ctt caa gac caa aac cag aaa atg atg tta gag
2640Lys Leu Ile Thr Asp Leu Gln Asp Gln Asn Gln Lys Met Met Leu Glu
865 870 875 880 cag gaa cgt cta aga gta gaa cat gag aag ttg aaa gcc
aca gat cag 2688Gln Glu Arg Leu Arg Val Glu His Glu Lys Leu Lys Ala
Thr Asp Gln 885 890 895 gaa aag agc aga aaa cta cat gaa ctt acg gtt
atg caa gat aga cga 2736Glu Lys Ser Arg Lys Leu His Glu Leu Thr Val
Met Gln Asp Arg Arg 900 905 910 gaa caa gca aga caa gac ttg aag ggt
ttg gaa gag aca gtg gca aaa 2784Glu Gln Ala Arg Gln Asp Leu Lys Gly
Leu Glu Glu Thr Val Ala Lys 915 920 925 gaa ctt cag act tta cac aac
ctg cgc aaa ctc ttt gtt cag gac ctg 2832Glu Leu Gln Thr Leu His Asn
Leu Arg Lys Leu Phe Val Gln Asp Leu 930 935 940 gct aca aga gtt aaa
aag agt gct gag att gat tct gat gac acc gga 2880Ala Thr Arg Val Lys
Lys Ser Ala Glu Ile Asp Ser Asp Asp Thr Gly 945 950 955 960 ggc agc
gct gct cag aag caa aaa atc tcc ttt ctt gaa aat aat ctt 2928Gly Ser
Ala Ala Gln Lys Gln Lys Ile Ser Phe Leu Glu Asn Asn Leu 965 970 975
gaa cag ctc act aaa gtg cac aaa cag ttg gta cgt gat aat gca gat
2976Glu Gln Leu Thr Lys Val His Lys Gln Leu Val Arg Asp Asn Ala Asp
980 985 990 ctc cgc tgt gaa ctt cct aag ttg gaa aag cga ctt cga gct
aca gct 3024Leu Arg Cys Glu Leu Pro Lys Leu Glu Lys Arg Leu Arg Ala
Thr Ala 995 1000 1005 gag aga gtg aaa gct ttg gaa tca gca ctg aaa
gaa gct aaa gaa 3069Glu Arg Val Lys Ala Leu Glu Ser Ala Leu Lys Glu
Ala Lys Glu 1010 1015 1020 aat gca tct cgt gat cgc aaa cgc tat cag
caa gaa gta gat cgc 3114Asn Ala Ser Arg Asp Arg Lys Arg Tyr Gln Gln
Glu Val Asp Arg 1025 1030 1035 ata aag gaa gca gtc agg tca aag aat
atg gcc aga aga ggg cat 3159Ile Lys Glu Ala Val Arg Ser Lys Asn Met
Ala Arg Arg Gly His 1040 1045 1050 tct gca cag att gct aaa cct att
cgt ccc ggg caa cat cca gca 3204Ser Ala Gln Ile Ala Lys Pro Ile Arg
Pro Gly Gln His Pro Ala 1055 1060 1065 gct tct cca act cac cca agt
gca att cgt gga gga ggt gca ttt 3249Ala Ser Pro Thr His Pro Ser Ala
Ile Arg Gly Gly Gly Ala Phe 1070 1075 1080 gtt cag aac agc cag cca
gtg gca gtg cga ggt gga gga ggc aaa 3294Val Gln Asn Ser Gln Pro Val
Ala Val Arg Gly Gly Gly Gly Lys 1085 1090 1095
caa gtg taa 3303Gln Val 1100 41100PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 4Met Ala Lys Ala Thr
Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu
Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr Phe Ser 20 25 30 Arg
Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala Ala Gly Thr 35 40
45 Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro Glu Glu Val Pro
50 55 60 Ser Phe Arg Leu Gly Gln His Leu Tyr Gly Thr Tyr Arg Thr
Arg Leu 65 70 75 80 His Glu Asn Asn Trp Ile Cys Ile Gln Glu Asp Thr
Gly Leu Leu Tyr 85 90 95 Leu Asn Arg Ser Leu Asp His Ser Ser Trp
Glu Lys Leu Ser Val Arg 100 105 110 Asn Arg Gly Phe Pro Leu Leu Thr
Val Tyr Leu Lys Val Phe Leu Ser 115 120 125 Pro Thr Ser Leu Arg Glu
Gly Glu Cys Gln Trp Pro Gly Cys Ala Arg 130 135 140 Val Tyr Phe Ser
Phe Phe Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu 145 150 155 160 Lys
Pro Arg Glu Leu Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile 165 170
175 Arg Glu Asn Arg Pro Pro Gly Thr Phe His Gln Phe Arg Leu Leu Pro
180 185 190 Val Gln Phe Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu
Leu Glu 195 200 205 Gly Glu Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser
Leu Glu Val Ser 210 215 220 Thr Arg Trp Ala Leu Asp Arg Glu Gln Arg
Glu Lys Tyr Glu Leu Val 225 230 235 240 Ala Val Cys Thr Val His Ala
Gly Ala Arg Glu Glu Val Val Met Val 245 250 255 Pro Phe Pro Val Thr
Val Tyr Asp Glu Asp Asp Ser Ala Pro Thr Phe 260 265 270 Pro Ala Gly
Val Asp Thr Ala Ser Ala Val Val Glu Phe Lys Arg Lys 275 280 285 Glu
Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp Ala Asp Val Val 290 295
300 Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr Ser Thr Leu Leu Pro
305 310 315 320 Gly Asp Thr Trp Ala Gln Gln Thr Phe Arg Val Glu His
Trp Pro Asn 325 330 335 Glu Thr Ser Val Gln Ala Asn Gly Ser Phe Val
Arg Ala Thr Val His 340 345 350 Asp Tyr Arg Leu Val Leu Asn Arg Asn
Leu Ser Ile Ser Glu Asn Arg 355 360 365 Thr Met Gln Leu Ala Val Leu
Val Asn Asp Ser Asp Phe Gln Gly Pro 370 375 380 Gly Ala Gly Val Leu
Leu Leu His Phe Asn Val Ser Val Leu Pro Val 385 390 395 400 Ser Leu
His Leu Pro Ser Thr Tyr Ser Leu Ser Val Ser Arg Arg Ala 405 410 415
Arg Arg Phe Ala Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala 420
425 430 Phe Ser Gly Ile Asn Val Gln Tyr Lys Leu His Ser Ser Gly Ala
Asn 435 440 445 Cys Ser Thr Leu Gly Val Val Thr Ser Ala Glu Asp Thr
Ser Gly Ile 450 455 460 Leu Phe Val Asn Asp Thr Lys Ala Leu Arg Arg
Pro Lys Cys Ala Glu 465 470 475 480 Leu His Tyr Met Val Val Ala Thr
Asp Gln Gln Thr Ser Arg Gln Ala 485 490 495 Gln Ala Gln Leu Leu Val
Thr Val Glu Gly Ser Tyr Val Ala Glu Glu 500 505 510 Ala Gly Cys Pro
Leu Ser Cys Ala Val Ser Lys Arg Arg Leu Glu Cys 515 520 525 Glu Glu
Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg Cys Glu Trp Arg 530 535 540
Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn Phe Ser Thr Cys Ser Pro 545
550 555 560 Ser Thr Lys Thr Cys Pro Asp Gly His Cys Asp Val Val Glu
Thr Gln 565 570 575 Asp Ile Asn Ile Cys Pro Gln Asp Cys Leu Arg Gly
Ser Ile Val Gly 580 585 590 Gly His Glu Pro Gly Glu Pro Arg Gly Ile
Lys Ala Gly Tyr Gly Thr 595 600 605 Cys Asn Cys Phe Pro Glu Glu Glu
Lys Cys Phe Cys Glu Pro Glu Asp 610 615 620 Ile Gln Asp Pro Leu Cys
Asp Glu Leu Cys Arg Thr Val Ile Ala Ala 625 630 635 640 Ala Val Leu
Phe Ser Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys 645 650 655 Ile
His Cys Tyr His Lys Phe Ala His Lys Pro Pro Ile Ser Ser Ala 660 665
670 Glu Met Thr Phe Arg Arg Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser
675 680 685 Ser Ser Gly Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn
Gln Val 690 695 700 Ser Val Asp Ala Phe Lys Ile Leu Gln Pro Glu Gly
Thr Gly Met Ile 705 710 715 720 Asp Glu Glu Phe Thr Val Ala Arg Leu
Tyr Ile Ser Lys Met Lys Ser 725 730 735 Glu Val Lys Thr Met Val Lys
Arg Cys Lys Gln Leu Glu Ser Thr Gln 740 745 750 Thr Glu Ser Asn Lys
Lys Met Glu Glu Asn Glu Lys Glu Leu Ala Ala 755 760 765 Cys Gln Leu
Arg Ile Ser Gln His Glu Ala Lys Ile Lys Ser Leu Thr 770 775 780 Glu
Tyr Leu Gln Asn Val Glu Gln Lys Lys Arg Gln Leu Glu Glu Ser 785 790
795 800 Val Asp Ala Leu Ser Glu Glu Leu Val Gln Leu Arg Ala Gln Glu
Lys 805 810 815 Val His Glu Met Glu Lys Glu His Leu Asn Lys Val Gln
Thr Ala Asn 820 825 830 Glu Val Lys Gln Ala Val Glu Gln Gln Ile Gln
Ser His Arg Glu Thr 835 840 845 His Gln Lys Gln Ile Ser Ser Leu Arg
Asp Glu Val Glu Ala Lys Ala 850 855 860 Lys Leu Ile Thr Asp Leu Gln
Asp Gln Asn Gln Lys Met Met Leu Glu 865 870 875 880 Gln Glu Arg Leu
Arg Val Glu His Glu Lys Leu Lys Ala Thr Asp Gln 885 890 895 Glu Lys
Ser Arg Lys Leu His Glu Leu Thr Val Met Gln Asp Arg Arg 900 905 910
Glu Gln Ala Arg Gln Asp Leu Lys Gly Leu Glu Glu Thr Val Ala Lys 915
920 925 Glu Leu Gln Thr Leu His Asn Leu Arg Lys Leu Phe Val Gln Asp
Leu 930 935 940 Ala Thr Arg Val Lys Lys Ser Ala Glu Ile Asp Ser Asp
Asp Thr Gly 945 950 955 960 Gly Ser Ala Ala Gln Lys Gln Lys Ile Ser
Phe Leu Glu Asn Asn Leu 965 970 975 Glu Gln Leu Thr Lys Val His Lys
Gln Leu Val Arg Asp Asn Ala Asp 980 985 990 Leu Arg Cys Glu Leu Pro
Lys Leu Glu Lys Arg Leu Arg Ala Thr Ala 995 1000 1005 Glu Arg Val
Lys Ala Leu Glu Ser Ala Leu Lys Glu Ala Lys Glu 1010 1015 1020 Asn
Ala Ser Arg Asp Arg Lys Arg Tyr Gln Gln Glu Val Asp Arg 1025 1030
1035 Ile Lys Glu Ala Val Arg Ser Lys Asn Met Ala Arg Arg Gly His
1040 1045 1050 Ser Ala Gln Ile Ala Lys Pro Ile Arg Pro Gly Gln His
Pro Ala 1055 1060 1065 Ala Ser Pro Thr His Pro Ser Ala Ile Arg Gly
Gly Gly Ala Phe 1070 1075 1080 Val Gln Asn Ser Gln Pro Val Ala Val
Arg Gly Gly Gly Gly Lys 1085 1090 1095 Gln Val 1100
5200DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(16)..(185)a, c, t, g, unknown
or other 5atcgcaccag cgtgtnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 180nnnnncactg cggctcctca 200616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Lys
Val Asp Asn Asn Gly Val Ala Ile Gly Ile Glu Ala Leu Asp Lys 1 5 10
15 717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Glu Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu
Val Leu Gly Lys 1 5 10 15 Thr 814DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 8tgtaaaggag gatc
149976PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Met Ala Asp Leu Ala Glu Cys Asn Ile Lys Val
Met Cys Arg Phe Arg 1 5 10 15 Pro Leu Asn Glu Ser Glu Val Asn Arg
Gly Asp Lys Tyr Ile Ala Lys 20 25 30 Phe Gln Gly Glu Asp Thr Val
Val Ile Ala Ser Lys Pro Tyr Ala Phe 35 40 45 Asp Arg Val Phe Gln
Ser Ser Thr Ser Gln Glu Gln Val Tyr Asn Asp 50 55 60 Cys Ala Lys
Lys Ile Val Lys Asp Val Leu Glu Gly Tyr Asn Gly Thr 65 70 75 80 Ile
Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr His Thr Met Glu 85 90
95 Gly Lys Leu His Asp Pro Glu Gly Met Gly Ile Ile Pro Arg Ile Val
100 105 110 Gln Asp Ile Phe Asn Tyr Ile Tyr Ser Met Asp Glu Asn Leu
Glu Phe 115 120 125 His Ile Lys Val Ser Tyr Phe Glu Ile Tyr Leu Asp
Lys Ile Arg Asp 130 135 140 Leu Leu Asp Val Ser Lys Thr Asn Leu Ser
Val His Glu Asp Lys Asn 145 150 155 160 Arg Val Pro Tyr Val Lys Gly
Cys Thr Glu Arg Phe Val Cys Ser Pro 165 170 175 Asp Glu Val Met Asp
Thr Ile Asp Glu Gly Lys Ser Asn Arg His Val 180 185 190 Ala Val Thr
Asn Met Asn Glu His Ser Ser Arg Ser His Ser Ile Phe 195 200 205 Leu
Ile Asn Val Lys Gln Glu Asn Thr Gln Thr Glu Gln Lys Leu Ser 210 215
220 Gly Lys Leu Tyr Leu Val Asp Leu Ala Gly Ser Glu Lys Val Ser Lys
225 230 235 240 Thr Gly Ala Glu Gly Ala Val Leu Asp Glu Ala Lys Asn
Ile Asn Lys 245 250 255 Ser Leu Ser Ala Leu Gly Asn Val Ile Ser Ala
Leu Ala Glu Gly Ser 260 265 270 Thr Tyr Val Pro Tyr Arg Asp Ser Lys
Met Thr Arg Ile Leu Gln Asp 275 280 285 Ser Leu Gly Gly Asn Cys Arg
Thr Thr Ile Ile Cys Cys Ser Pro Ser 290 295 300 Ser Tyr Asn Glu Ser
Glu Thr Lys Ser Thr Leu Leu Phe Gly Gln Arg 305 310 315 320 Ala Lys
Thr Ile Lys Asn Thr Val Cys Val Asn Val Glu Leu Thr Ala 325 330 335
Glu Gln Trp Lys Lys Lys Tyr Glu Lys Glu Lys Glu Lys Asn Lys Ile 340
345 350 Leu Arg Asn Thr Ile Gln Trp Leu Glu Asn Glu Leu Asn Arg Trp
Arg 355 360 365 Asn Gly Glu Thr Val Pro Ile Asp Glu Gln Phe Asp Lys
Glu Lys Ala 370 375 380 Asn Leu Glu Ala Phe Thr Val Asp Lys Asp Ile
Thr Leu Thr Asn Asp 385 390 395 400 Lys Pro Ala Thr Ala Ile Gly Val
Ile Gly Asn Phe Thr Asp Ala Glu 405 410 415 Arg Arg Lys Cys Glu Glu
Glu Ile Ala Lys Leu Tyr Lys Gln Leu Asp 420 425 430 Asp Lys Asp Glu
Glu Ile Asn Gln Gln Ser Gln Leu Val Glu Lys Leu 435 440 445 Lys Thr
Gln Met Leu Asp Gln Glu Glu Leu Leu Ala Ser Thr Arg Arg 450 455 460
Asp Gln Asp Asn Met Gln Ala Glu Leu Asn Arg Leu Gln Ala Glu Asn 465
470 475 480 Asp Ala Ser Lys Glu Glu Val Lys Glu Val Leu Gln Ala Leu
Glu Glu 485 490 495 Leu Ala Val Asn Tyr Asp Gln Lys Ser Gln Glu Val
Glu Asp Lys Thr 500 505 510 Lys Glu Tyr Glu Leu Leu Ser Asp Glu Leu
Asn Gln Lys Ser Ala Thr 515 520 525 Leu Ala Ser Ile Asp Ala Glu Leu
Gln Lys Leu Lys Glu Met Thr Asn 530 535 540 His Gln Lys Lys Arg Ala
Ala Glu Met Met Ala Ser Leu Leu Lys Asp 545 550 555 560 Leu Ala Glu
Ile Gly Ile Ala Val Gly Asn Asn Asp Val Lys Glu Asp 565 570 575 Pro
Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly Lys Thr Leu 580 585
590 Gly Glu Gly Glu Phe Gly Lys Val Val Lys Ala Thr Ala Phe His Leu
595 600 605 Lys Gly Arg Ala Gly Tyr Thr Thr Val Ala Val Lys Met Leu
Lys Glu 610 615 620 Asn Ala Ser Pro Ser Glu Leu Arg Asp Leu Leu Ser
Glu Phe Asn Val 625 630 635 640 Leu Lys Gln Val Asn His Pro His Val
Ile Lys Leu Tyr Gly Ala Cys 645 650 655 Ser Gln Asp Gly Pro Leu Leu
Leu Ile Val Glu Tyr Ala Lys Tyr Gly 660 665 670 Ser Leu Arg Gly Phe
Leu Arg Glu Ser Arg Lys Val Gly Pro Gly Tyr 675 680 685 Leu Gly Ser
Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His Pro Asp 690 695 700 Glu
Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala Trp Gln Ile 705 710
715 720 Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu Val His Arg
Asp 725 730 735 Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly Arg Lys
Met Lys Ile 740 745 750 Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr Glu
Glu Asp Ser Tyr Val 755 760 765 Lys Arg Ser Gln Gly Arg Ile Pro Val
Lys Trp Met Ala Ile Glu Ser 770 775 780 Leu Phe Asp His Ile Tyr Thr
Thr Gln Ser Asp Val Trp Ser Phe Gly 785 790 795 800 Val Leu Leu Trp
Glu Ile Val Thr Leu Gly Gly Asn Pro Tyr Pro Gly 805 810 815 Ile Pro
Pro Glu Arg Leu Phe Asn Leu Leu Lys Thr Gly His Arg Met 820 825 830
Glu Arg Pro Asp Asn Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln 835
840 845 Cys Trp Lys Gln Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile
Ser 850 855 860 Lys Asp Leu Glu Lys Met Met Val Lys Arg Arg Asp Tyr
Leu Asp Leu 865 870 875 880 Ala Ala Ser Thr Pro Ser Asp Ser Leu Ile
Tyr Asp Asp Gly Leu Ser 885 890 895 Glu Glu Glu Thr Pro Leu Val Asp
Cys Asn Asn Ala Pro Leu Pro Arg 900 905 910 Ala Leu Pro Ser Thr Trp
Ile Glu Asn Lys Leu Tyr Gly Met Ser Asp 915 920 925 Pro Asn Trp Pro
Gly Glu Ser Pro Val Pro Leu Thr Arg Ala Asp Gly 930 935 940 Thr Asn
Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val Tyr Ala Asn 945 950 955
960 Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp Thr Phe Asp Ser
965 970 975
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