U.S. patent application number 15/446341 was filed with the patent office on 2017-07-13 for methods and compositions for determining resistance to androgen receptor therapy.
The applicant listed for this patent is ARAGON PHARMACEUTICALS, INC.. Invention is credited to JEFFREY H. HAGER, JAMES DAVID JOSEPH, NHIN LU, JING QIAN, JOHN LEE SENSINTAFFAR, NICHOLAS D. SMITH.
Application Number | 20170196840 15/446341 |
Document ID | / |
Family ID | 48948536 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170196840 |
Kind Code |
A1 |
JOSEPH; JAMES DAVID ; et
al. |
July 13, 2017 |
METHODS AND COMPOSITIONS FOR DETERMINING RESISTANCE TO ANDROGEN
RECEPTOR THERAPY
Abstract
Described herein are modified androgen receptor polypeptides
that are resistant to inhibition by an androgen receptor inhibitor.
Described herein are compositions, combinations, and kits
containing the modified androgen receptor polypeptides and methods
of using the modified androgen receptor polypeptides. Also
described herein are methods of using the modified androgen
receptor polypeptides as screening agents for the identification
and design of third-generation androgen receptor modulators. Also
described herein are third-generation androgen receptor modulators
that inhibit the activity of the modified androgen receptor
polypeptides. Also described are pharmaceutical compositions and
medicaments that include the compounds described herein, as well as
methods of using such androgen receptor modulators, alone and in
combination with other compounds, for treating diseases or
conditions, including cancers, such as castration resistant
prostate cancers, that are mediated or dependent upon androgen
receptors.
Inventors: |
JOSEPH; JAMES DAVID; (SAN
DIEGO, CA) ; HAGER; JEFFREY H.; (SAN DIEGO, CA)
; SENSINTAFFAR; JOHN LEE; (SAN DIEGO, CA) ; LU;
NHIN; (SAN DIEGO, CA) ; QIAN; JING; (SAN
DIEGO, CA) ; SMITH; NICHOLAS D.; (SAN DIEGO,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARAGON PHARMACEUTICALS, INC. |
SAN DIEGO |
CA |
US |
|
|
Family ID: |
48948536 |
Appl. No.: |
15/446341 |
Filed: |
March 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14417515 |
Jan 26, 2015 |
9617602 |
|
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PCT/US13/52395 |
Jul 26, 2013 |
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15446341 |
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61676842 |
Jul 27, 2012 |
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61783763 |
Mar 14, 2013 |
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61829123 |
May 30, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/4184 20130101; A61P 35/00 20180101; A61P 15/00 20180101;
C12Q 2600/158 20130101; C12Q 1/6897 20130101; C12Q 2600/136
20130101; G01N 2333/723 20130101; C07K 14/721 20130101; C12Q 1/6886
20130101; C12Q 2600/106 20130101; A61P 13/08 20180101; A61P 13/10
20180101; A61K 45/06 20130101; G01N 33/57492 20130101; A61K 38/09
20130101; A61P 29/00 20180101; A61K 31/4439 20130101; C12Q 2600/156
20130101; C12Q 2600/16 20130101; A61P 1/16 20180101; A61K 31/4166
20130101; C07K 16/2869 20130101; A61K 31/4166 20130101; A61K
2300/00 20130101; A61K 31/4439 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/4184 20060101
A61K031/4184; C12Q 1/68 20060101 C12Q001/68; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method for determining whether a subject is or will become
less responsive to therapy with a first- or second-generation
androgen receptor (AR) antagonist, comprising: (a) testing a sample
containing a nucleic acid molecule encoding an AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1;
and (b) characterizing the subject as resistant or will become
resistant to therapy with a first- or second-generation AR
antagonist if the subject has the modification.
2. A method for optimizing the therapy of a subject receiving a
first- or second-generation AR antagonist for treatment of a
cancer, comprising: (a) testing a sample containing a nucleic acid
molecule encoding an AR polypeptide from the subject to determine
whether the encoded AR polypeptide is modified at an amino acid
position corresponding to amino acid position 876 of the amino acid
sequence set forth in SEQ ID NO: 1; and (b) if the subject has the
modification, discontinuing treatment with the first- or
second-generation AR antagonist or; and (c) if the subject does not
have the modification, then (i) continuing treatment with a first-
or second-generation AR antagonist; or (ii) administering a
third-generation AR antagonist that inhibits the modified receptor;
or (iii) both discontinuing treatment with a first- or
second-generation AR antagonist and administering a
third-generation AR antagonist that inhibits the modified
receptor.
3. A method for screening compounds that antagonize a modified AR,
comprising: (a) expressing a modified AR in a cell, wherein the
modified AR is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1; (b) contacting the cell with a test compound being
screened; and (c) detecting the level of AR activity in the cell by
analyzing the expression of the reporter gene, the cell comprising
the reporter gene operably linked to an androgen responsive
promoter.
4. The method of claim 3, wherein the test compound (a) exhibits
full antagonist activity toward the modified AR receptor; (b) does
not exhibit agonist activity toward the modified AR receptor; or
(c) both exhibits full antagonist activity toward the modified AR
receptor and does not exhibit agonist activity toward the modified
AR receptor.
5. The method of claim 3, wherein the modification is a
substitution or deletion of the amino acid at position 876 of the
AR polypeptide.
6. The method of claim 5, wherein the modification is a
substitution of phenylalanine at position 876 of the AR
polypeptide, the phenylalanine being replaced by leucine,
isoleucine, valine, alanine, glycine, methionine, serine,
threonine, cysteine, tryptophan, lysine, arginine, histidine,
proline, tyrosine, asparagine, glutamine, aspartic acid, or
glutamic acid.
7. The method of claim 6, wherein the phenylalanine at position 876
of the AR polypeptide is replaced by glycine, alanine, valine,
leucine, or isoleucine.
8. The method of claim 7, wherein the phenylalanine at position 876
of the AR polypeptide is replaced by leucine.
9. The method of claim 3, wherein the cell is deficient for the
expression of wild-type AR, expresses a low level of wild-type AR,
or expresses a modified AR receptor.
10. The method of claim 3, wherein the cell is a HeLa, CV1, COS7,
HepG2, HEK-293, DU145, PC3, TSY-PR1, LNCaP, CWR, VCaP or LAPC4
cell.
11. The method of claim 3, wherein the promoter comprises an
androgen response element.
12. The method of claim 11, wherein the androgen response element
is 4X ARE or a probasin element.
13. The method of claim 3, wherein the promoter is a probasin, a
prostate specific antigen, MMTV LTR, FASN, STEAP4, TMPRSS2, ORM1,
or NKX3.1 promoter.
14. The method of claim 3, wherein the reporter gene encodes a
protein that is a luciferase, a fluorescent protein, a
bioluminescent protein, or an enzyme.
15. A method of treating cancer comprising administering to a
subject in need of such treatment a therapeutically effective
amount of a third-generation AR antagonist identified by the method
of claim 3.
16. The method of claim 15, wherein the cancer is a prostate
cancer, a breast cancer, a bladder cancer or a hepatocellular
cancer.
17. The method of claim 16, wherein the cancer is a castration
resistant prostate cancer.
18. The method of claim 15, wherein the subject expresses a mutant
AR, wherein the mutant AR comprises a substitution or a deletion of
the amino acid at amino acid position 876 in the AR
polypeptide.
19. The method of claim 18, wherein the mutant AR comprises a
substitution at amino acid position 876 in the AR polypeptide and
the substitution is F876L.
20. The method of claim 15, wherein the third-generation AR
antagonist is administered with an additional therapeutic
agent.
21. The method of claim 20, wherein the third-generation AR
antagonist and the additional therapeutic agent are administered
sequentially, simultaneously or intermittently.
22. The method of claim 20, wherein the additional therapeutic
agent is a hormone, hormone receptor agonist or antagonist,
corticosteroid, anti-emetic agent, analgesic, anti-cancer agent,
anti-inflammatory agent, kinase inhibitor, HSP90 inhibitor, or
histone deacetylase (HDAC) inhibitor.
23. The method of claim 20, wherein the additional therapeutic
agent is a gonadotropin-releasing hormone (GnRH) agonist or
antagonist.
24. The method of claim 23, wherein the GnRH agonist is leuprolide,
bruserelin or goserelin.
25. A method of maintenance therapy in a patient having a cancer,
comprising: (a) administering to the patient a maintenance therapy
regimen comprising administering a therapeutically effective dose
of first- or second-generation AR antagonist; and (b) monitoring
the patient at predetermined intervals of time over the course of
the maintenance therapy regimen to determine whether the subject
has mutation in an endogenous gene encoding AR that results in a
modification at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO:
1.
26. The method of claim 25, wherein monitoring comprises: testing a
sample containing a nucleic acid molecule encoding a AR polypeptide
from the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO:
1.
27. The method of claim 25, wherein the method further comprises
discontinuing maintenance therapy regimen if the subject has the
mutation or continuing maintenance therapy regimen if the subject
does not have the modification.
28. The method of claim 25, wherein the method further comprises
administering third-generation AR antagonist that inhibits the
modified AR if the subject has the modification.
29. The method of claim 25, wherein the modification in the AR
polypeptide is F876L.
30. The method of claim 25, wherein the first- or second-generation
AR antagonist inhibits a wild-type AR polypeptide by competitive
antagonism.
31. The method of claim 25, wherein the second-generation AR
antagonist is selected from among ARN-509, enzalutamide (MDV3100),
and RD162.
32. The method of claim 25, wherein the cancer is a prostate
cancer, a breast cancer, a bladder cancer, or a hepatocellular
cancer.
33. The method of claim 32, wherein the cancer is prostate
cancer.
34. The method any claim 33, wherein the cancer is a castration
resistant prostate cancer.
35. The method claim 25, wherein the predetermined interval of time
is every week, every month, every 2 months, every 3 months, every 4
months, every 5 months, every 6 months, every 7 months, every 8
months, every 9 months, every 10 months, every 11 months, or every
year.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application to U.S. patent
application Ser. No. 14/417,515, filed Jan. 26, 2015, which is a
national stage application of Patent Application No.
PCT/US2013/052395, filed Jul. 26, 2013, which claims the benefit of
U.S. Provisional Application Nos. 61/676,842, filed Jul. 27, 2012,
61/783,763, filed on Mar. 14, 2013 and 61/829,123, filed on May 30,
2013, each of which is incorporated herein by reference in its
entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED AS A TEXT
FILE VIA EFS-WEB
[0002] The instant application contains a Sequence Listing, which
has been submitted as a computer readable text file in ASCII format
via EFS-Web and is hereby incorporated in its entirety by reference
herein. The text file, created date of Feb. 27, 2017, is named
103693_000574_SL.txt and is 136.663 bytes in size.
BACKGROUND OF THE INVENTION
[0003] The androgen receptor ("AR") is a ligand-activated
transcriptional regulatory protein that mediates induction of a
variety of biological effects through its interaction with
endogenous androgens. Endogenous androgens include steroids such as
testosterone and dihydrotestosterone. Testosterone is converted to
dihydrotestosterone by the enzyme 5 alpha-reductase in many
tissues.
[0004] The actions of androgens with androgen receptors have been
implicated in a number of diseases or conditions, such as androgen
dependent cancers, virilization in women, and acne, among others.
Compounds that diminish the effects of androgen signaling via the
androgen receptor and/or lower the concentrations of androgen
receptors find use in the treatment of diseases or conditions in
which androgen receptors play a role.
SUMMARY OF THE INVENTION
[0005] Described herein is the identification of modifications in
the androgen receptor (AR) that confer resistance of patients to
treatment with a first- or second-generation androgen receptor
antagonist. In some embodiments, the modification is a substitution
of phenylalanine at position 876 of an AR polypeptide. In some
embodiments, the modification is F876L. In some embodiments, the
modified AR polypeptide is resistant to inhibition by a
second-generation androgen receptor antagonist is selected from
among ARN-509, enzalutamide (MDV3100), and RD162. In some
embodiments, the modified AR polypeptide is resistant to inhibition
by a first-generation androgen receptor antagonist selected from
among bicalutamide, flutamide, hydroxyflutamide or nilutamide. In
some embodiments, the patient has a cancer. In some embodiments,
the cancer is a prostate cancer, breast cancer, liver (i.e.
hepatocellular) cancer, or bladder cancer. In some embodiments, the
cancer is a castration-resistant prostate cancer. In some
embodiments, the modified AR polypeptide exhibits one or more
activities of a wildtype AR receptor, including but not limited to,
co-activator binding, DNA binding, ligand binding, or nuclear
translocation. In some embodiments, the first- or second
generation-antagonist exhibits decreased antagonistic activity
against a modified AR polypeptide compared to a wild type AR
polypeptide.
[0006] Described herein, in certain embodiments, are methods for
determining whether a subject is or will become resistant to
therapy with a first- or second-generation androgen receptor
antagonist, comprising, consisting of, and/or consisting
essentially of: (a) testing a sample that contains a nucleic acid
molecule encoding an androgen receptor polypeptide from the subject
to determine whether the encoded androgen receptor polypeptide is
modified at amino acid position 876 of the amino acid sequence set
forth in SEQ ID NO: 1; and (b) characterizing the subject as
resistant or will become resistant to therapy with a first- or
second-generation androgen receptor antagonist if the subject has
the modification. In some embodiments, the methods further comprise
a step of obtaining the sample from the subject. In some
embodiments, the subject has been administered a first- or
second-generation AR antagonist for the treatment of a cancer. In
some embodiments, the cancer is a prostate cancer, breast cancer,
liver (i.e. hepatocellular) cancer, or bladder cancer. In some
embodiments, the cancer is prostate cancer. In some embodiments,
the cancer is a castration-resistant prostate cancer. In some
embodiments, the methods further comprise, consists of, and/or
consists essentially of discontinuing treatment with the first- or
second-generation androgen receptor antagonist if the sample from
the subject has the modification at position 876 of SEQ ID NO: 1.
In some embodiments, the method further comprises, consists of,
and/or consists essentially of continuing treatment with a first-
or second-generation androgen receptor antagonist if the sample
from the subject does not have the modification at position 876 of
SEQ ID NO: 1. In some embodiments, the method further comprises,
consists of, and/or consists essentially of administering a
third-generation androgen receptor antagonist that inhibits the
modified AR if the sample from subject has the modification at
position 876 of SEQ ID NO: 1. In some embodiments, the
second-generation androgen receptor antagonist is selected from
among ARN-509, enzalutamide (MDV3100), and RD162. In some
embodiments, the first-generation androgen receptor antagonist is
selected from among bicalutamide, flutamide, hydroxyflutamide or
nilutamide.
[0007] Described herein, in certain embodiments, are methods for
selecting a subject for therapy with a third-generation androgen
receptor antagonist, comprising, consisting of, and/or consisting
essentially of: (a) testing a sample that contains a nucleic acid
molecule encoding an androgen receptor polypeptide from the subject
to determine whether the encoded androgen receptor polypeptide is
modified at amino acid position 876 of the amino acid sequence set
forth in SEQ ID NO: 1; and (b) characterizing the subject as a
candidate for therapy with a third-generation androgen receptor
antagonist if the subject has the modification. In some
embodiments, the methods further comprise a step of obtaining the
sample from the subject. In some embodiments, the methods further
comprise, consists of, and/or consists essentially of discontinuing
treatment with the first- or second-generation androgen receptor
antagonist if the sample from the subject has the modification at
position 876 of SEQ ID NO: 1. In some embodiments, the method
further comprises, consists of, and/or consists essentially of
continuing treatment with a first- or second-generation androgen
receptor antagonist if the sample from the subject does not have
the modification at position 876 of SEQ ID NO: 1. In some
embodiments, the method further comprises, consists of, and/or
consists essentially of administering a third-generation androgen
receptor antagonist that inhibits the modified AR if the sample
from subject has the modification at position 876 of SEQ ID NO: 1.
In some embodiments, the second-generation androgen receptor
antagonist is selected from among ARN-509, enzalutamide (MDV3100),
and RD162. In some embodiments, the first-generation androgen
receptor antagonist is selected from among bicalutamide, flutamide,
hydroxyflutamide or nilutamide.
[0008] Described herein, in certain embodiments, are methods for
characterizing a subject's androgen receptor to determine whether
such subject may be resistant to inhibition with a first- or
second-generation androgen receptor antagonist, comprising,
consisting of, and/or consisting essentially of: (a) testing a
sample that contains a nucleic acid molecule encoding an androgen
receptor polypeptide from the subject to determine whether the
encoded androgen receptor polypeptide is modified at amino acid
position 876 of SEQ ID NO: 1; and (b) if the subject has the
modification at position 876 of SEQ ID NO: 1, characterizing the
androgen receptor of the subject as being resistant to inhibition
with a first- or second-generation androgen receptor antagonist. In
some embodiments, the methods further comprise a step of obtaining
the sample from the subject. In some embodiments, the first- or
second generation-antagonist exhibits decreased antagonistic
activity against a modified AR polypeptide compared to a wild type
AR polypeptide. In some embodiments, the methods further comprise,
consists of, and/or consists essentially of discontinuing treatment
with the first- or second-generation androgen receptor antagonist
if the sample from the subject has the modification at position 876
of SEQ ID NO: 1. In some embodiments, the method further comprises,
consists of, and/or consists essentially of continuing treatment
with a first- or second-generation androgen receptor antagonist if
the sample from the subject does not have the modification at
position 876 of SEQ ID NO: 1. In some embodiments, the method
further comprises, consists of, and/or consists essentially of
administering a third-generation androgen receptor antagonist that
inhibits the modified AR if the sample from subject has the
modification at position 876 of SEQ ID NO: 1. In some embodiments,
the second-generation androgen receptor antagonist is selected from
among ARN-509, enzalutamide (MDV3100), and RD162. In some
embodiments, the first-generation androgen receptor antagonist is
selected from among bicalutamide, flutamide, hydroxyflutamide or
nilutamide.
[0009] Described herein, in certain embodiments, are methods for
monitoring whether a subject receiving a first- or
second-generation androgen receptor antagonist for treatment of a
cancer has developed or will develop resistance to the therapy,
comprising: (a) testing a sample that contains a nucleic acid
molecule encoding an androgen receptor polypeptide from the subject
to determine whether the encoded androgen receptor polypeptide is
modified at amino acid position 876 of SEQ ID NO: 1; and (b)
characterizing the subject as resistant or will become resistant to
therapy with a first- or second-generation androgen receptor
antagonist if the subject has the modification. In some
embodiments, the methods further comprise a step of obtaining the
sample from the subject. In some embodiments, the methods further
comprise, consists of, and/or consists essentially of discontinuing
treatment with the first- or second-generation androgen receptor
antagonist if the sample from the subject has the modification at
position 876 of SEQ ID NO: 1. In some embodiments, the method
further comprises, consists of, and/or consists essentially of
continuing treatment with a first- or second-generation androgen
receptor antagonist if the sample from the subject does not have
the modification at position 876 of SEQ ID NO: 1. In some
embodiments, the method further comprises, consists of, and/or
consists essentially of administering a third-generation androgen
receptor antagonist that inhibits the modified AR if the sample
from subject has the modification at position 876 of SEQ ID NO: 1.
In some embodiments, the second-generation androgen receptor
antagonist is selected from among ARN-509, enzalutamide (MDV3100),
and RD162. In some embodiments, the first-generation androgen
receptor antagonist is selected from among bicalutamide, flutamide,
hydroxyflutamide or nilutamide.
[0010] Described herein, in certain embodiments, are methods for
optimizing the therapy of a subject receiving a first- or
second-generation androgen receptor antagonist for treatment of a
cancer, comprising, consisting of, and/or consisting essentially
of: (a) testing a sample that contains a nucleic acid molecule
encoding an androgen receptor polypeptide from the subject to
determine whether the encoded androgen receptor polypeptide is
modified at amino acid position 876 of SEQ ID NO: 1; and (c)(i) if
the sample has the modification, discontinuing treatment of the
subject with the first- or second-generation androgen receptor
antagonist or (ii) if the sample does not have the modification,
continuing treatment of the subject with the first- or
second-generation androgen receptor antagonist if the subject does
not have the modification. In some embodiments, the methods further
comprise a step of obtaining the sample from the subject. In some
embodiments, the methods further comprise, consists of, and/or
consists essentially of discontinuing treatment with the first- or
second-generation androgen receptor antagonist if the sample from
the subject has the modification at position 876 of SEQ ID NO: 1.
In some embodiments, the method further comprises, consists of,
and/or consists essentially of continuing treatment with a first-
or second-generation androgen receptor antagonist if the sample
from the subject does not have the modification at position 876 of
SEQ ID NO: 1. In some embodiments, the method further comprises,
consists of, and/or consists essentially of administering a
third-generation androgen receptor antagonist that inhibits the
modified AR if the sample from subject has the modification at
position 876 of SEQ ID NO: 1. In some embodiments, the
second-generation androgen receptor antagonist is selected from
among ARN-509, enzalutamide (MDV3100), and RD162. In some
embodiments, the first-generation androgen receptor antagonist is
selected from among bicalutamide, flutamide, hydroxyflutamide or
nilutamide.
[0011] In some embodiments, the modified AR polypeptide comprises,
consists of, and or consists essentially of a substitution or a
deletion of the amino acid at position 876 of SEQ ID NO: 1. In some
embodiments, the modified AR polypeptide comprises, consists,
and/or consists essentially of a substitution of phenylalanine to
an amino acid selected from the group consisting of leucine,
isoleucine, valine, alanine, glycine, methionine, serine,
threonine, cysteine, tryptophan, lysine, arginine, histidine,
proline, tyrosine, asparagine, glutamine, aspartic acid and
glutamic acid at position 876 of SEQ ID NO: 1. In some embodiments,
the modified AR polypeptide comprises, consists of, and/or consists
essentially of a substitution of phenylalanine to an amino acid
selected from among glycine, alanine, valine, leucine, and
isoleucine at position 876 of SEQ ID NO: 1. In some embodiments,
the modified AR polypeptide comprises, consists of, and/or consists
essentially of a substitution of phenylalanine to leucine at
position 876 of SEQ ID NO: 1. In some embodiments, the modified AR
polypeptide comprises, consists of, and/or consists essentially of
a deletion of nucleic acid encoding amino acid position 876 of SEQ
ID NO: 1.
[0012] In some embodiments, the nucleic acid encoding the modified
AR polypeptide has (i) a mutation of thymine (t) to cytosine (c) at
a nucleic acid position corresponding to nucleic acid position 2626
in the sequence of nucleotides set forth in SEQ ID NO: 18; (ii) a
mutation of cytosine (c) to adenine (a) at nucleic acid position
corresponding to nucleic acid position 2628 in the sequence of
nucleotides set forth in SEQ ID NO: 18; or (ii) a mutation of
cytosine (c) to guanine (g) at nucleic acid position corresponding
to nucleic acid position 2628 in the sequence of nucleotides set
forth in SEQ ID NO: 18.
[0013] In some embodiments of the method, the nucleic acid sample
is RNA or DNA. In some embodiments of the method, the nucleic acid
sample is genomic DNA. In some embodiments, the method further
comprises, consists of, and/or consists essentially of isolating
mRNA from the RNA sample. In some embodiments, the nucleic acid is
isolated from a tumor cell sample obtained from the subject. In
some embodiments, the sample is a tumor biopsy sample, a blood
sample, a serum sample, a lymph sample, or a bone marrow aspirate
obtained from the subject. In some embodiments, the sample contains
circulating tumor cells. In some embodiments, the sample contains
disseminated tumor cells. In some embodiments, the nucleic acid is
purified from the sample prior to testing. In some embodiments, the
nucleic acid is amplified prior to testing.
[0014] In some embodiments of the method, testing comprises,
consists of, and/or consists essentially performing polymerase
chain reaction (PCR) amplification of a nucleic acid sample
encoding position 876 of SEQ ID NO: 1. In some embodiments, PCR
amplification comprises, consists of, and/or consists essentially
of using a pair of oligonucleotide primers that flank the region
encoding amino acid position 876 of SEQ ID NO: 1. In some
embodiments, the method further comprises sequencing the PCR
amplified nucleic acid using techniques known to those skilled in
the art.
[0015] In some embodiments, testing comprises, consists of, and/or
consists essentially of contacting the nucleic acid with a sequence
specific nucleic acid probe, wherein the sequence specific nucleic
acid probe: (a) binds to nucleic acid encoding a modified receptor
that is modified at amino acid position 876; and (b) does not bind
to nucleic acid encoding the wild-type receptor having
phenylalanine at position 876 of SEQ ID NO: 1.
[0016] In some embodiments, the first- or second-generation
androgen receptor antagonist inhibits a wild-type androgen receptor
polypeptide by competitive antagonism. In some embodiments, the
second-generation androgen receptor antagonist is selected from
among ARN-509, enzalutamide (MDV3100), or RD162.
[0017] In some embodiments, the subject has a disease or disorder
selected from among cancer, an inflammatory disorder or a
proliferative disorder. In some embodiments, the subject has
cancer. In some embodiments, the subject has an AR-mediated cancer.
In some embodiments, the cancer is selected from a prostate cancer,
a breast cancer, liver (i.e., hepatocellular) cancer, or a bladder
cancer. In some embodiments, the cancer is prostate cancer.
[0018] In some embodiments, the subject has a castration resistant
prostate cancer. In some embodiments, the subject has a solid
tumor.
[0019] In some embodiments, the subject is treated with the first-
or second-generation androgen receptor antagonist prior to
obtaining the sample from the subject. In some embodiments, the
subject is responsive the treatment with the first- or
second-generation androgen receptor antagonist when it is first
administered. In some embodiments, the sample for use in the
methods is a sample obtained at 1 week, 2 weeks, 3 weeks, 1 month,
2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8
months, 9 months, 10 months, 11 months, 12 months, 14 months, 16
months, 18 months, 20 months, 22 months, or 24 months following the
first administration of with the first- or second-generation AR
antagonist. In some embodiments, the sample is obtained 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 times over the course of treatment with the
first- or second-generation AR antagonist. In some embodiments, the
subject is responsive the treatment with the first- or
second-generation AR antagonist when it is first administered.
[0020] Described herein, in certain embodiments, are methods for
screening compounds that antagonize a modified androgen receptor,
comprising, consisting of and/or consisting essentially of: (a)
expressing a modified androgen receptor in a cell, wherein the
modified androgen receptor is modified at an amino acid position
corresponding to position 876 of SEQ ID NO: 1; (b) contacting the
cell with a test compound; and (c) detecting the level of androgen
receptor activity in the cell, wherein a decrease in activity
expressed indicates that the compound antagonizes the modified AR.
In some embodiments, the test compound exhibits full antagonist
activity toward the modified AR. In some embodiments, the test
compound does not exhibit agonist activity toward the modified AR.
Described herein, in certain embodiments, is a third-generation
androgen receptor inhibitor identified by the methods described
herein. In some embodiments, the modification of the AR polypeptide
used in the method is a substitution or deletion of the amino acid
at position 876 of the androgen receptor polypeptide. In some
embodiments, the modification of the AR polypeptide used in the
method is a substitution of phenylalanine to an amino acid selected
from among leucine, isoleucine, valine, alanine, glycine,
methionine, serine, threonine, cysteine, tryptophan, lysine,
arginine, histidine, proline, tyrosine, asparagine, glutamine,
aspartic acid and glutamic acid at amino acid position 876 of the
androgen receptor polypeptide. In some embodiments, the
modification of the AR polypeptide used in the method is a
substitution of phenylalanine to an amino acid selected from among
glycine, alanine, valine, leucine, and isoleucine at amino acid
position 876 of the androgen receptor polypeptide. In some
embodiments, the modification of the AR polypeptide used in the
method is a substitution of phenylalanine to leucine at amino acid
position 876 of the androgen receptor polypeptide.
[0021] In some embodiments, the cell employed in the method is
deficient for the expression of wild-type androgen receptor,
expresses a low level of wild-type androgen receptor, or expresses
a modified AR receptor. In some embodiments, the cell is a selected
from among HeLa. CV1, COS7, HepG2, HEK-293, DU145, PC3, TSY-PR1,
LNCaP, CWR, VCaP and LAPC4. In some embodiments, the cell comprises
a reporter gene operably linked to an androgen responsive promoter.
In some embodiments, the activity of the AR polypeptide is
determined by analyzing the expression of the reporter gene. In
some embodiments, the promoter comprises androgen response element.
In some embodiments, the androgen response element is 4XARE or a
probasin element. In some embodiments, the promoter is a probasin,
a prostate specific antigen, MMTV LTR, FASN, STEAP4, TMPRSS2, ORM1,
or NKX3.1 promoter. In some embodiments, the reporter gene encodes
a protein selected from among a luciferase, a fluorescent protein,
a bioluminescent protein, or an enzyme.
[0022] Described herein, in certain embodiments, is an isolated
androgen receptor polypeptide or a variant thereof having androgen
receptor activity comprising a modification at an amino acid
position corresponding to amino acid position 876 of the amino acid
sequence set forth in SEQ ID NO: 1, wherein the modification
confers resistance to an androgen receptor antagonist on the
modified androgen receptor polypeptide or variant. In some
embodiments, the modification comprises substitution of the amino
acid at position 876 compared to a wild-type androgen receptor set
forth in SEQ ID NO: 1. In some embodiments, the substitution is
F876L. In some embodiments, the modification comprises a deletion
of amino acid position 876. In some embodiments, the modified AR
polypeptide has the sequence of amino acids set forth in SEQ ID NO:
5. In some embodiments, the modified AR polypeptide is a
recombinant polypeptide. In some embodiments, the modified AR
polypeptide comprises a substitution of the amino acid at position
876 compared to a wild-type androgen receptor set forth in SEQ ID
NO: 1 and one or more additional amino acid substitutions. In some
embodiments, the modified AR polypeptide comprises a substitution
at amino acid at position 876 that is F876L. In some embodiments,
the modified AR polypeptide comprises one or more additional amino
acid substitutions selected from among one or more amino acid
substitutions associated with castration resistant prostate cancer.
In some embodiments, the one or more additional amino acid
substitutions is selected from among one or more substitutions at
amino acid positions 701, 741, 874 and 877 compared to a wild-type
androgen receptor set forth in SEQ ID NO: 1. In some embodiments,
the one or more additional amino acid substitutions is selected
from among T877A, W741C, W741L. W741R, L701H and H874Y. In some
embodiments, the modified AR polypeptide is a recombinant
polypeptide. In some embodiments, the modified AR polypeptide
comprises a sequence of amino acids set forth in SEQ ID NO: 5 or a
variant that has at least or at least about 60%, 70%, 75%, 80%,
85%, 90%, 95%, 96/%, 97%, 98%, 99% or more sequence identity with
the polypeptide having the sequence set forth in SEQ ID NO: 5,
wherein the amino acid at position 876 is not phenylalanine.
[0023] Described herein, in certain embodiments, is an isolated
nucleic acid molecule encoding the modified androgen receptor
polypeptide provided herein. In some embodiments, the nucleic acid
is a DNA or an RNA molecule. In some embodiments, the nucleic acid
is a cDNA molecule. In some embodiments, the nucleic acid is a PCR
amplification product. In some embodiments, the nucleic acid is a
recombinant molecule. In some embodiments, the nucleic acid is a
synthetic molecule. In some embodiments, the nucleic acid comprises
the sequence of nucleic acids set forth in SEQ ID NO: 19 or a
variant that has at least or at least about 60%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with
the polypeptide having the sequence set forth in SEQ ID NO: 19,
wherein the nucleic acid codon encoding amino acid at position 876
does not encode phenylalanine.
[0024] Described herein, in certain embodiments, is a vector,
comprising a nucleic acid molecule encoding the modified androgen
receptor polypeptide provided herein. In some embodiments, the
vector is a viral or plasmid vector. In some embodiments, the
nucleic acid is operably linked to a promoter. In some embodiments,
the promoter is a constitutive or an inducible promoter. Described
herein, in certain embodiments, is a host cell, comprising the
vector. In some embodiments, the cell is a prokaryotic cell or a
eukaryotic cell. Described herein, in certain embodiments, is a
mutant AR polypeptide expressed by the host cell.
[0025] Described herein, in certain embodiments, is a
pharmaceutical composition comprising a third-generation androgen
receptor inhibitor identified by the methods provided herein and a
pharmaceutically acceptable excipient. Described herein, in certain
embodiments, are uses of a third-generation androgen receptor
inhibitor identified by the methods provided herein for the
manufacture of a medicament. Described herein, in certain
embodiments, are methods of treatment comprising administering to a
subject in need thereof a therapeutically effective amount of the
pharmaceutical composition, wherein the composition comprises a
suitable pharmaceutical carrier. In some embodiments, the subject
has cancer. In some embodiments, the subject has an AR-mediated
cancer. In some embodiments, the cancer is a prostate cancer,
breast cancer, liver (i.e. hepatocellular) cancer or bladder
cancer. In some embodiments, the cancer is a castration resistant
prostate cancer. In some embodiments, the subject expresses a
mutant AR. In some embodiments, the mutant AR comprises a
substitution or a deletion of the amino acid at amino acid position
876 in the androgen receptor polypeptide. In some embodiments, the
substitution is F876L. In some embodiments, the third-generation AR
antagonist is administered with an additional therapeutic agent. In
some embodiments, the third-generation AR antagonist and the
additional therapeutic agent are administered sequentially,
simultaneously or intermittently. In some embodiments, the
additional therapeutic agent is selected from among hormones,
hormone receptor agonists or antagonists, corticosteroids,
anti-emetic agents, analgesics, anti-cancer agents,
anti-inflammatory agents, kinase inhibitors, HSP90 inhibitors,
histone deacetylase (HDAC) inhibitors. In some embodiments, the
additional therapeutic agent is a gonadotropin-releasing hormone
(GnRH) agonist or antagonist. In some embodiments, the GnRH agonist
is leuprolide, bruserelin or goserelin.
[0026] Described herein, in certain embodiments, is a microchip
comprising the mutant AR polypeptide provided herein or a nucleic
acid encoding modified AR polypeptide provided herein. In some
embodiments, the modified AR polypeptide comprises a modification
at amino acid 876. In some embodiments, the modification is an
amino acid substitution that is F876L.
[0027] Described herein, in certain embodiments, are kits
comprising the mutant AR polypeptide provided herein. Described
herein, in certain embodiments, is a kit comprising the isolated
nucleic acid encoding a mutant AR polypeptide provided herein.
Described herein, in certain embodiments, are kits comprising one
or more reagents for the detection of a mutant AR polypeptide
comprising a modification at amino acid position 876. Described
herein, in certain embodiments, is a kit comprising one or more
reagents for the detection of nucleic acid encoding a mutant AR
polypeptide comprising a modification at amino acid position 876.
In some embodiments, the modification is an amino acid substitution
that is F876L. In some embodiments, the kits comprises a pair
oligonucleotide primers that flank the nucleic acid region encoding
amino acid 876 of a AR polypeptide. In some embodiments, the kits
comprises an oligonucleotide primer that (a) binds to nucleic acid
encoding a modified AR that is modified at amino acid position 876;
and (b) does not bind to nucleic acid encoding the wild-type AR
having phenylalanine at amino acid position 876. In some
embodiments, the kits comprises a microchip comprising (a) a
modified AR polypeptide having a modification that is F876S, or a
portion thereof comprising a modification that is F876S; or (b) a
nucleic acid molecule encoding a mutant AR polypeptide having a
modification that is F876S, or a portion thereof comprising a
modification that is F876S.
[0028] Described herein, in certain embodiments, are systems for
detecting a modified AR that is resistant to inhibition with an a
first- or second-generation AR antagonist in a subject, comprising:
(a) a sample containing a nucleic acid molecule encoding a AR
polypeptide from the subject; and (b) a microarray comprising
nucleic acid encoding a mutant AR polypeptide or a portion thereof
that is modified at an amino acid position corresponding to amino
acid position 876 of the amino acid sequence set forth in SEQ ID
NO: 1. In some embodiments, the microarray is contained on a
microchip. Described herein, in certain embodiments, are systems
for detecting a modified AR that is resistant to inhibition with a
first- or second-generation AR antagonist in a subject, comprising:
(a) a sample containing a nucleic acid molecule encoding a AR
polypeptide from the subject; and (b) a sequence specific nucleic
acid probe, wherein the sequence specific nucleic acid probe: (i)
binds to nucleic acid encoding a modified AR that is modified at
amino acid position 876; and (ii) does not bind to nucleic acid
encoding the wild-type AR having phenylalanine at amino acid
position 876. Described herein, in certain embodiments, are systems
for detecting a modified AR that is resistant to inhibition with a
first- or second-generation AR antagonist in a subject, comprising:
(a) a sample containing a nucleic acid molecule encoding a AR
polypeptide from the subject; and (b) a pair oligonucleotide
primers that flank the nucleic acid region encoding amino acid 876
of a AR polypeptide.
[0029] Described herein, in certain embodiments, are isolated
antibodies that bind to a modified androgen receptor polypeptide
provided herein, wherein the antibody does not bind to or binds
with lower affinity to a wild-type AR polypeptide having the
sequence of amino acids set forth in SEQ ID NO: 1. In some
embodiments, the modified AR polypeptide comprises a modification
at amino acid 876. In some embodiments, the modification is an
amino acid substitution that is F876L.
[0030] Described herein, in certain embodiments, are methods for
maintenance therapy in a patient having a cancer, comprising: (a)
administering to the patient a maintenance therapy regimen
comprising administering a therapeutically effective dose of first-
or second-generation AR antagonist; and (b) monitoring the patient
at predetermined intervals of time over the course of the
maintenance therapy regimen to determine whether the subject has
mutation in an endogenous gene encoding AR that results in a
modification at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1.
In some embodiments, monitoring comprises: testing a sample
containing a nucleic acid molecule encoding a AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1.
In some embodiments, the methods further comprise discontinuing
maintenance therapy regimen if the subject has the mutation or
continuing maintenance therapy regimen if the subject does not have
the modification. In some embodiments, the methods further comprise
administering third-generation AR antagonist that inhibits the
modified AR if the subject has the modification. In some
embodiments, the modification in the AR polypeptide is F876L. In
some embodiments, the first- or second-generation AR antagonist
inhibits a wild-type AR polypeptide by competitive antagonism. In
some embodiments, the second-generation AR antagonist is selected
from among ARN-509, enzalutamide (MDV3100), and RD162. In some
embodiments, the cancer is a prostate cancer, a breast cancer, a
bladder cancer, or a hepatocellular cancer. In some embodiments,
the cancer is prostate cancer. In some embodiments, the cancer is a
castration resistant prostate cancer. In some embodiments, the
predetermined interval of time is every week, every month, every 2
months, every 3 months, every 4 months, every 5 months, every 6
months, every 7 months, every 8 months, every 9 months, every 10
months, every 11 months, or every year.
[0031] Other objects, features and advantages of the polypeptides,
nucleic acids, compounds, methods and compositions described herein
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating specific embodiments, are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the instant disclosure
will become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGS. 1A-D illustrate ARN-509 and enzalutamide resistance.
FIG. 1A shows LNCaP and LNCaP ARN-509r1 cell proliferation. LNCaP
and LNCaP ARN-509r1 cells were cultured in the presence of hormone
depleted medium for 2 days followed by ligand addition.
Proliferation is quantified by CellTiter-Glo.RTM. (Promega Corp.)
luminescence based viability assay after 7 day compound treatment.
FIG. 1B is an agonist proliferation assay of LNCaP/AR-Luc,
LNCaP/AR-LucENZr2 and LNCaP ARN-509r2 cell lines. Cells were
cultured in the presence of hormone depleted medium for 2 days
followed by ligand treatment for 7 days. Proliferation is
quantified by CellTiter-Glo.RTM. luminescence based viability
assay. FIG. 1C is an antagonist proliferation assay of parental and
2nd generation anti-androgen resistant cell lines. Cells were
cultured in the presence of hormone depleted medium for 2 days
followed by ligand treatment in the presence of R1881 (final
concentration=100 pM). Proliferation is quantified by CellTiter-Glo
luminescence based viability assay. FIG. 1D is a schematic
representation of AR domain structure showing amino acids that when
mutated display altered ligand activity in CRPC.
[0033] FIG. 2 illustrates AR levels in parental and 2nd generation
anti-androgen resistant cell lines. Protein extracts were generated
from cells cultured in hormone depleted medium for 3 days. AR
protein levels were analyzed and by western blot. AR levels were
quantified and normalized to .alpha.-tubulin and expressed relative
to LNCaP cells.
[0034] FIGS. 3A-B illustrate ARN-509 and enzalutamide are partial
agonists of AR F876L. FIG. 3A shows transcriptional agonist and
antagonist activity of ARN-509 and enzalutamide on wild-type or
F876L AR. Transcriptional activation of a 4X ARE-luciferase
reporter was measured in the presence of increasing compound
concentration in the absence or presence of 1 nM R1881 (for
wild-type AR) or 5 nM R1881 (for F876L AR). FIG. 3B shows
transcriptional agonist activity of 1st and 2.sup.nd generation
anti-androgens and prednisone on wild-type, F876L, T877A.
F876L/T877A, L701H, H874Y and W741C AR dependent activation of 4X
ARE-Luciferase reporter was measured in transiently transfected
HepG2 cells.
[0035] FIGS. 4A-B illustrate VP16-AR (FIG. 4A) and F876L VP16-AR
(FIG. 4B) agonist and antagonist activity of ARN-509 and
enzalutamide. 4X ARE-luciferase reporter activity was monitored in
the presence of increasing compound concentration in the absence or
presence of 90 pM R1881 (for wild-type VP16-AR) or 1 nM R1881 (for
F876L VP16-AR).
[0036] FIGS. 5A-B illustrate a competitive binding assay of
wild-type AR (FIG. 5A) vs. F876L AR (FIG. 5B). 3H-R1881 binding
performed in PC3 cell extracts expressing wild-type or F876L AR.
Data is representative of 3 independent experiments. Error bars,
SEM; n=2.
[0037] FIG. 6 illustrates AR levels in AR overexpressing cell
lines. Protein extracts were generated from LNCaP, LNCap/AR(cs),
LNCaP/SR.alpha.F876L and LNCaP/pCDNAF876L cells cultured in hormone
depleted medium for 3 days. AR protein levels were analyzed and by
Western blot. AR levels were quantified and normalized to actin and
expressed relative to LNCaP cells.
[0038] FIGS. 7A-B illustrate that the F876L AR mutation confers
partial agonist activity to ARN-509 and enzalutamide. FIG. 7A shows
LNCaP/AR(cs) and LNCaP/SR.alpha.F876L cell proliferation. Cells
were cultured in the presence of hormone depleted medium for 2 days
followed by ligand treatment for 7 days. Proliferation is
quantified by CellTiter-Glo luminescence based viability assay.
FIG. 7B shows LNCaP/AR(cs), LNCaP/SR.alpha.F876L and
LNCaP/pCDNAF876L cell proliferation. Cells were cultured in hormone
depleted medium for 2 days followed by ligand treatment for 7 days.
For antagonist assays, compounds were added in the presence of 200
pM R1881 (100 pM final concentration). Proliferation was quantified
using the luminescence based CellTiter-Glo.RTM. assay.
[0039] FIG. 8 illustrates AR N/C interaction assay. Ligand induced
N/C interaction was monitored via mammalian two hybrid assay in
HepG2 cells. Antagonists were assayed at 8 .mu.M, R1881 at 1
nM.
[0040] FIG. 9 illustrates AR ChIP analysis of AR target genes. ChIP
assays were performed on LNCaP/AR(cs) and LNCaP/SR.alpha.F876L
cells incubated for 3 days in hormone depleted medium followed by 4
hour ligand treatment. Cells were treated in with 10 .mu.M
antagonist in the presence or absence of 1 nM R1881. Data for AR
and non-specific IgG control is presented as percent input.
[0041] FIG. 10 illustrates AR F876L mutation confers ARN-509 and
enzalutamide resistance in vivo. LNCaP/AR(cs) and
LNCaP/SR.alpha.F876L tumor xenografts. Castrate male mice bearing
tumors were treated daily with vehicle or 30 mg/kg/day compound.
Tumor growth for each group is presented as the average tumor
volume.+-.SEM.
[0042] FIG. 11 illustrates the dosing schedule for the open-label
phase 1/2 safety, pharmacokinetic, and proof-of-concept study of
ARN-509 in patients with progressive advanced, metastatic
Castration-Resistant Prostate Cancer.
[0043] FIGS. 12A-B illustrate identification of AR-F876L in ARN-509
treated patients. FIG. 12A shows PSA response of 29 patients
analyzed for F876L mutation. Terminal end of PSA response line is
time at which the patient plasma was screened for F876L mutation
using BEAMing analysis. The plasma used in this study was initially
collected to determine pharmacokinetics of ARN-509 and as such the
samples were not prepared using methodology to maximize ratio of
ctDNA to lymphocyte DNA. FIG. 12B shows PSA response of patient
positive for F876L. PSA response of patient 7 at indicated
treatment cycle. Circulating plasma was analyzed for the F876L at
times indicated with arrows. The plasma samples with no detectable
mutant are notated as "w.t.", the presence of the F876L mutation is
represented by "m". A plasma sample was called positive for the
mutant if the percent of mutant beads was above the cut-off (0.02%)
and the number of mutant copies estimated to be .gtoreq.0.5 (number
of genome equivalents in the plasma sample x mutant bead
fraction=.gtoreq.0.5).
[0044] FIG. 13 illustrates the relative amount of prostate specific
antigen (PSA) detected over the course of the Phase 1/2 ARN-509
clinical study in each of the three patients (7, 10, and 13)
bearing detectable somatic F876L mutations in AR. Arrow indicates
sample in which the mutation(s) were detected.
DETAILED DESCRIPTION OF THE INVENTION
Certain Terminology
[0045] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs.
All patents, patent applications, published applications and
publications, GENBANK sequences, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. In the event that there is a plurality of definitions for
terms herein, those in this section prevail. Where reference is
made to a URL or other such identifier or address, it is understood
that such identifiers can change and particular information on the
internet can come and go, but equivalent information is known and
can be readily accessed, such as by searching the internet and/or
appropriate databases. Reference thereto evidences the availability
and public dissemination of such information. Generally, the
procedures for cell culture, cell infection, antibody production
and molecular biology methods are methods commonly used in the art.
Such standard techniques can be found, for example, in reference
manual, such as, for example, Sambrook et al. (2000) and Ausubel et
al. (1994).
[0046] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. In this application, the use of the singular includes
the plural unless specifically stated otherwise. As used herein,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, use of the term "including" as well as other forms
(e.g., "include", "includes", and "included") is not limiting.
[0047] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.g" means "about 5 .mu.g" and also "5
.mu.g." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0048] As used herein, an androgen receptor (AR) polypeptide refers
to any androgen receptor protein or polypeptide, including, but not
limited to, a recombinantly produced protein, a synthetically
produced protein, a native androgen receptor protein, and an
androgen receptor protein extracted from cells or tissues. An AR
polypeptide includes related polypeptides from different species
including, but not limited to animals of human and non-human
origin. AR polypeptides of non-human origin include, but are not
limited to, non-human primate (e.g. chimpanzee and ape), murine
(e.g., mouse and rat), canine (dog), feline (cat), leporine
(rabbit), avian (bird), bovine (cow), ovine (sheep), porcine (pig),
equine (horse), piscine (fish), ranine (frog) and other mammalian
or non-mammalian AR polypeptides. Exemplary AR polypeptides
include, for example, SEQ ID NOS: 1-17. An androgen receptor
polypeptide includes wild-type androgen receptor, allelic variant
isoforms, somatic mutations including those found in tumors,
synthetic molecules from nucleic acids, protein isolated from human
tissue and cells, and modified forms thereof. The androgen receptor
polypeptides provided herein can be further modified by
modification of the primary amino acid sequence, by deletion,
addition, or substitution of one or more amino acids. An androgen
receptor polypeptide includes any AR polypeptide or a portion
thereof having AR activity, including for example, fusion
polypeptides of an AR ligand binding domain to a heterologous DNA
binding domain.
[0049] As used herein, a mutant androgen receptor (AR) polypeptide,
a mutant AR protein, a modified AR polypeptide, or a modified AR
protein or are used interchangeably herein and refer to an androgen
receptor polypeptide that is modified at one or more amino acid
positions. Exemplary modifications include, but are not limited to,
substitutions, deletions or additions of amino acids.
[0050] As used herein, the term "anti-androgen" refers to a group
of hormone receptor antagonist compounds that are capable of
preventing or inhibiting the biologic effects of androgens on
normally responsive tissues in the body.
[0051] As used herein, the term "AR inhibitor" or "AR antagonist"
are used interchangeably herein and refer to an agent that inhibits
or reduces at least one activity of an AR polypeptide. Exemplary AR
activities include, but are not limited to, co-activator binding,
DNA binding, ligand binding, or nuclear translocation.
[0052] As used herein, a "full antagonist" refers to an antagonist
which, at an effective concentration, essentially completely
inhibits an activity of an AR polypeptide. As used herein, a
"partial antagonist" refers an antagonist that is capable of
partially inhibiting an activity of an AR polypeptide, but that,
even at a highest concentration is not a full antagonist. By
`essentially completely` is meant at least about 80%, at least
about 90%, at least about 95%, at least about 96%, at least about
97%, at least about 98% at least about 99%, or greater inhibition
of the activity of an AR polypeptide.
[0053] As used herein, the term "third-generation AR inhibitor" or
"third-generation AR antagonist" are used interchangeably herein
and refer to an agent that inhibits at least one activity of an AR
polypeptide containing one or more amino acid modifications that
confers resistance to inhibition by a second-generation AR
antagonist, such as, for example, ARN-509 (CAS No. 956104-40-8),
enzalutamide (also known as MDV3100; CAS No: 915087-33-1), or RD162
(CAS No. 915087-27-3). In some embodiments, a third-generation AR
inhibitor inhibits at least one activity of a wild-type AR
polypeptide, such as, but not limited to, co-activator binding, DNA
binding, ligand binding, or nuclear translocation. In some
embodiments, a third-generation AR inhibitor inhibits an activity
of a mutant AR polypeptide that is also inhibited by a first- or
second-generation AR inhibitor.
[0054] As used herein, the phrase "resistant to inhibition" refers
to a decrease in ability of an AR antagonist to inhibit (i.e.
antagonize) one or more activities of a modified AR polypeptide
compared to a wild type AR polypeptide. For example, the AR
antagonist is less effective at inhibiting a modified AR
polypeptide compared to a wild type AR polypeptide. In some
embodiments, the decreased antagonistic activity against a modified
AR polypeptide is about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 90%, 95%, 98% or 100% compared to the antagonistic activity
against a wildtype AR polypeptide. Exemplary AR activities include,
but are not limited to, co-activator binding, DNA binding, ligand
binding, or nuclear translocation. In some embodiments, the AR
antagonist is unable to bind to or binds with decreased affinity to
a modified AR polypeptide.
[0055] As used herein, the term "second-generation AR inhibitor" or
"second-generation AR antagonist" are used interchangeably herein
and refer to an agent that exhibits full antagonist activity of a
wild-type AR polypeptide, but does not exhibit full antagonist
activity of an AR polypeptide containing one or more amino acid
modifications that confers resistance to inhibition, such as a
modification at amino acid position 876 in the AR polypeptide. In
some embodiments, the second-generation AR inhibitor does not
exhibit full antagonist activity on an AR polypeptide having an
amino acid substitution that is F876L. In some embodiments, the
second-generation AR inhibitor induces activity of AR (i.e. is an
AR agonist) at concentrations that are equivalent or higher than
the concentration required to inhibit a wild-type AR.
Second-generation AR inhibitors differ from first-generation AR
inhibitors, such as bicalutamide and flutamide, in that
second-generation AR inhibitors act as full antagonists in cells
expressing elevated levels of AR, such as for example, in
castration resistant prostate cancers (CRPC). First-generation AR
inhibitors, such as bicalutamide and flutamide, act as agonists in
CRPC. Exemplary second-generation AR inhibitors include ARN-509,
enzalutamide and RD162. In some embodiments, a second-generation AR
inhibitor is an agonist of a mutant AR polypeptide provided herein.
In some embodiments, a second-generation AR inhibitor binds to an
AR polypeptide at or near the ligand binding site of the AR
polypeptide.
[0056] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides, ribonucleosides, or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogs of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless
specifically limited otherwise, the term also refers to
oligonucleotide analogs including PNA (peptidonucleic acid),
analogs of DNA used in antisense technology (e.g.,
phosphorothioates, phosphoroamidates). Unless otherwise indicated,
a particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (including but not limited
to, degenerate codon substitutions) and complementary sequences as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions are achieved by generating sequences in which
the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al. (1991) Nucleic Acid Res, 19:5081; Ohtsuka et al. (1985) J.
Biol. Chem. 260:2605-2608; and Cassol et al. (1992) Mol. Cell.
Probes 6, 327-331; and Rossolini et al. (1994) Mol. Cell. Probes
8:91-98).
[0057] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally encoded amino acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine
and selenocysteine. Amino acid analogs refers to agents that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, such as, homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (such as, norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid.
[0058] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0059] The terms "polypeptide", peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to naturally occurring amino acid
polymers as well as amino acid polymers in which one or more amino
acid residues is a non-naturally occurring amino acid, e.g., an
amino acid analog. The terms encompass amino acid chains of any
length, including full length proteins, wherein the amino acid
residues are linked by covalent peptide bonds.
[0060] As used herein, modification refers to modification of a
sequence of amino acids of a polypeptide or a sequence of
nucleotides in a nucleic acid molecule and includes deletions,
insertions, and replacements of amino acids and nucleotides,
respectively.
[0061] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences can be aligned for
optimal comparison purposes (e.g., gaps are 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 can then be 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
homology 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 some embodiments the two sequences are
the same length.
[0062] To determine percent homology between two sequences, 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 is used. 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 are
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to a nucleic acid molecules
described or disclose herein. BLAST protein searches are performed
with the XBLAST program, score=50, wordlength=3. To obtain gapped
alignments for comparison purposes, Gapped BLAST is utilized as
described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) are used. See the website of the National Center for
Biotechnology Information for further details (on the World Wide
Web at ncbi.nlm.nih.gov). Proteins suitable for use in the methods
described herein also includes proteins having between 1 to 15
amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15 amino acid substitutions, deletions, or additions,
compared to the amino acid sequence of any protein described
herein. In other embodiments, the altered amino acid sequence is at
least 75% identical, e.g., 77%, 80%, 82%, 85%, 88%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino
acid sequence of any protein described herein. Such
sequence-variant proteins are suitable for the methods described
herein as long as the altered amino acid sequence retains
sufficient biological activity to be functional in the compositions
and methods described herein. Where amino acid substitutions are
made, the substitutions should be conservative amino acid
substitutions. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. Those of skill in this art
recognize that, in general, single amino acid substitutions in
non-essential regions of a polypeptide do not substantially alter
biological activity (see, e.g., Watson et al. Molecular Biology of
the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.
224). The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff et al (1992) Proc.
Natl. Acad. Sci. USA, 89:10915-10919). Accordingly, the BLOSUM62
substitution frequencies are used to define conservative amino acid
substitutions that, in some embodiments, are introduced into the
amino acid sequences described or disclosed herein. Although it is
possible to design amino acid substitutions based solely upon
chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0063] As used herein, corresponding residues refers to residues
that occur at aligned loci. Related or variant polypeptides are
aligned by any method known to those of skill in the art. Such
methods typically maximize matches, and include methods such as
using manual alignments and by using the numerous alignment
programs available (for example, BLASTP) and others known to those
of skill in the art. By aligning the sequences of polypeptides, one
skilled in the art can identify corresponding residues, using
conserved and identical amino acid residues as guides.
Corresponding positions also can be based on structural alignments,
for example by using computer simulated alignments of protein
structure. In other instances, corresponding regions can be
identified.
[0064] As used herein, an allelic variant or allelic variation
references to a polypeptide encoded by a gene that differs from a
reference form of a gene (i.e. is encoded by an allele). Typically
the reference form of the gene encodes a wild-type form and/or
predominant form of a polypeptide from a population or single
reference member of a species. Typically, allelic variants, which
include variants between and among species, have at least 80%, 90%
or greater amino acid identity with a wild-type and/or predominant
form from the same species; the degree of identity depends upon the
gene and whether comparison is interspecies or intraspecies.
Generally, intraspecies allelic variants have at least about 80%,
85%6, 90% or 95% identity or greater with a wild-type and/or
predominant form, including 96%, 97%, 98%, 99% or greater identity
with a wild-type and/or predominant form of a polypeptide.
[0065] As used herein, species variants refer to variants of the
same polypeptide between and among species. Generally, interspecies
variants have at least about 60/%, 70%, 80%, 85%, 90%, or 95%
identity or greater with a wild-type and/or predominant form from
another species, including 96%, 97%, 98%, 99% or greater identity
with a wild-type and/or predominant form of a polypeptide.
[0066] As used herein, the terms "treat," "treating" or
"treatment," and other grammatical equivalents, include
alleviating, abating or ameliorating one or more symptoms of a
disease or condition, ameliorating, preventing or reducing the
appearance, severity or frequency of one or more additional
symptoms of a disease or condition, ameliorating or preventing the
underlying metabolic causes of one or more symptoms of a disease or
condition, inhibiting the disease or condition, such as, for
example, arresting the development of the disease or condition,
relieving the disease or condition, causing regression of the
disease or condition, relieving a condition caused by the disease
or condition, or inhibiting the symptoms of the disease or
condition either prophylactically and/or therapeutically. In a
non-limiting example, for prophylactic benefit, a third-generation
AR inhibitor compound disclosed herein is administered to an
individual at risk of developing a particular disorder, predisposed
to developing a particular disorder, or to an individual reporting
one or more of the physiological symptoms of a disorder. In some
embodiments, a third-generation AR inhibitor compound disclosed
herein is administered to a subject following treatment with one or
more therapeutic agents. In some embodiments, a third-generation AR
inhibitor compound disclosed herein is administered to a subject in
combination with treatment with one or more therapeutic agents.
[0067] As used herein, prevention or prophylaxis refers to the
reduction in the risk of developing a disease or condition.
[0068] The terms "effective amount", "therapeutically effective
amount" or "pharmaceutically effective amount" as used herein,
refer to an amount of an AR inhibitor compound that is sufficient
to treat a disorder. In some embodiments, the result is a reduction
in and/or alleviation of the signs, symptoms, or causes of a
disorder, or any other desired alteration of a biological system.
For example, an "effective amount" for therapeutic uses is the
amount of the composition comprising an AR inhibitor compound
disclosed herein required to provide a clinically significant
decrease in a disorder. An appropriate "effective" amount in any
individual case is determined using any suitable technique, (e.g.,
a dose escalation study).
[0069] The term "pharmaceutically acceptable" as used herein,
refers to a material, (e.g., a carrier or diluent), which does not
abrogate the biological activity or properties of an AR inhibitor
compound described herein, and is relatively nontoxic (i.e., the
material is administered to an individual without causing
undesirable biological effects or interacting in a deleterious
manner with any of the components of the composition in which it is
contained).
[0070] As used herein, a control refers to a sample that is
substantially identical to the test sample, except that it is not
treated with a test parameter, or, if it is a plasma sample, it can
be from a normal volunteer not affected with the condition of
interest. A control also can be an internal control.
[0071] As used herein, the terms "subject", "individual" and
"patient" are used interchangeably. None of the terms are to be
interpreted as requiring the supervision of a medical professional
(e.g., a doctor, nurse, physician's assistant, orderly, hospice
worker). As used herein, the subject can be any animal, including
mammals (e.g., a human or non-human animal) and non-mammals. In one
embodiment of the methods and compositions provided herein, the
mammal is a human.
[0072] As used herein, "contacting" refers to refers to the act of
touching, making contact, or of bringing substances into immediate
proximity. "Contacting" can be achieved by mixing the components in
a fluid or semi-fluid mixture.
Overview: AR Function and Drug Resistance in Cancer
[0073] Androgen receptor (AR) is a member of the steroid and
nuclear receptor superfamily. Among this large family of proteins,
only five vertebrate steroid receptors are known and include the
androgen receptor, estrogen receptor, progesterone receptor,
glucocorticoid receptor, and mineralocorticoid receptor. AR is a
soluble protein that functions as an intracellular transcription
factor. AR function is regulated by the binding of androgens, which
initiates sequential conformational changes of the receptor that
affect receptor-protein interactions and receptor-DNA
interactions.
[0074] AR is mainly expressed in androgen target tissues, such as
the prostate, skeletal muscle, liver, and central nervous system
(CNS), with the highest expression level observed in the prostate,
adrenal gland, and epididymis. AR in certain instance is activated
by the binding of endogenous androgens, including testosterone and
5.alpha.-dihydrotestosterone (5.alpha.-DHT).
[0075] AR is a 110 kD nuclear receptor that, upon activation by
androgens, mediates transcription of target genes that modulate
growth and differentiation of prostate epithelial cells. AR is
encoded by the AR gene located on the X chromosome at Xq11-12. The
AR gene comprises 8 exons encoding the full-length androgen
receptor. Similar to the other steroid receptors, unbound AR is
mainly located in the cytoplasm and associated with a complex of
heat shock proteins (HSPs) through interactions with the
ligand-binding domain. Upon agonist binding, AR goes through a
series of conformational changes: the heat shock proteins
dissociate from AR, and the transformed AR undergoes dimerization,
phosphorylation, and translocation to the nucleus, which is
mediated by the nuclear localization signal. The translocated
receptor then binds to the androgen response element (ARE), which
is characterized by the two hexameric six-nucleotide half-site
consensus sequence 5'-TGTTCT-3' arranged as inverted repeats spaced
by three random nucleotides and is located in the promoter or
enhancer region of AR gene targets. Recruitment of other
transcription co-regulators (including co-activators and
co-repressors) and transcriptional machinery further ensures the
appropriate modulation of AR-regulated gene expression. These
processes are initiated by the ligand-induced conformational
changes in the ligand-binding domain.
[0076] AR signaling is crucial for the development and maintenance
of male reproductive organs including the prostate gland, as
genetic males harboring loss of function AR mutations and mice
engineered with AR defects do not develop prostates. This
dependence of prostate cells on AR signaling continues even upon
neoplastic transformation. Androgen depletion (e.g. using
gonadotropin-releasing hormone (GnRH) agonists) continues to be the
mainstay of prostate cancer treatment. However, androgen depletion
is usually effective for a limited duration and prostate cancer
evolves to regain the ability to grow despite low levels of
circulating androgens.
[0077] Prostate cancer is the most prevalent cancer in men.
Prostate cancer represents approximately 29 percent of all new
cancer cases diagnosed and 10 percent of cancer deaths in males. In
addition, American men over their lifetime have a roughly 17
percent chance of developing invasive prostate cancer. At initial
diagnosis, a large percentage of prostate cancers have low to
medium risk, meaning that the 10 year mortality risk is relatively
low (up to 24%) with little intervention. However, advanced and
metastatic prostate cancers have mean survival of 2.5-3 years and
are subject to aggressive treatment including surgery and chemical
castration therapy.
[0078] Given that most prostate cancer cells depend on AR for their
proliferation and survival, treatment generally consists of
administration of agents that block production of testosterone
(e.g. GnRH agonists), alone or in combination with anti-androgens
(e.g. bicalutamide), which antagonize the effect of any residual
testosterone. Unfortunately, while often initially successful as
evidenced by a drop in prostate specific antigen (PSA) and
regression of visible tumor if present, metastatic tumors
inevitably become resistant to hormonal therapy at which stage no
curative treatment exists.
[0079] Prostate cancers resistant to hormonal therapies are
currently referred to as `castration resistant`, implying that they
have progressed beyond the point at which drugs targeting any point
on the androgen axis would have any clinical utility. Pre-clinical
and clinical evidence indicate that the AR is a viable therapeutic
target even in castration resistant cancers. AR mutations have been
reported to occur in up to 33 percent of prostate cancer cases and
are most commonly observed following treatment in the AR dependent
castration resistant state. Mutations have been found that alter
ligand specificity and potency and that result in ligand
independent receptor activity. The specific mutations vary widely
but appear to be dependent upon treatment regimen. Additionally,
upregulation of the AR itself has been associated with progression
to the castration resistant state in both patients and animal
models.
[0080] Two agents, abiraterone acetate (Zytiga; CAS No.
154229-19-3) and MDV3100 (enzalutamide; CAS No. 915087-33-7), have
recently been employed in late stage clinical testing for the
treatment of men with castration-resistant prostate cancer (CRPC).
Abiraterone acetate targets 7-.alpha.-hydroxylase/17,20-lyase
(CYP17A), thereby inhibiting residual androgen biosynthesis.
MDV3100 is an anti-androgen discovered in a screen for potent
anti-androgens lacking agonist activity in context of AR
overexpression. The clinical efficacies of MDV3100 and abiraterone
acetate support the hypothesis that AR continues to promote growth
and survival of castration resistant prostate cancer.
Unfortunately, similar to first-generation androgen ablation
therapies, prolonged treatment with abiraterone acetate or
second-generation AR antagonists ultimately results in
resistance.
[0081] Resistance to abiraterone acetate and MDV3100 has been
observed in both models of prostate cancer and in patients.
Preliminary data suggests that, similar to castration resistant
prostate cancer, second-generation anti-androgen resistance
develops through multiple mechanisms and AR is thought to remain a
therapeutic target in this setting. In both xenograft models and in
patients, CYP17A upregulation and the presence of picogram levels
of androgens have been noted in resistant populations. CYP17A
upregulation presumably promotes resistance via AR activation by
intratumoral androgen synthesis. In other cases, resistance
correlates with increased AR levels as well as nuclear
localization. Additionally, numerous cellular signaling pathways
known to activate AR in the absence of endogenous ligands may
promote second-generation therapy resistance. The observation that
tumors with high levels of activated Src respond poorly to MDV3100
and abiraterone acetate treatment supports this hypothesis. To
date, no AR mutations have been described that confer resistance to
MDV3100, ARN-509 or abiraterone.
[0082] ARN-509 is a synthetic thiohydantoin compound discovered
using structure-activity relationship (SAR)-guided medicinal
chemistry to identify nonsteroidal anti-androgens that retain full
antagonist activity in the setting of increased AR expression.
ARN-509 exhibits anti-tumor activity in castration-sensitive and
resistant xenograft models of prostate cancer and anti-androgenic
effects in dogs that phenocopy castration.
Identification of AR Inhibitor-Resistant Cell Lines and a Mutant AR
Polypeptide
[0083] Described herein are working examples demonstrating the
production of drug resistant cell lines. In some embodiments, the
methods provided are employed for the generation of a cell line
that is resistant to inhibition by a second-generation-AR
antagonist, such as, but not limited to, ARN-509, enzalutamide
(MDV3100) or RD162. In some embodiments, the methods provided are
employed for the generation of a cell line that is resistant to
inhibition by an AR antagonist that inhibits androgen production
and exhibits binding to an AR receptor. In some embodiments, the AR
antagonist that inhibits androgen production is a CYP17A inhibitor
and exhibits binding to AR. In some embodiments, the AR antagonist
that inhibits androgen production and exhibits AR binding is
galeterone (TOK001) or abiraterone acetate. In some embodiments,
the AR antagonist that inhibits androgen production and exhibits AR
binding is TAK-700.
[0084] As described herein, resistant cell lines were generated in
vivo in a castration resistant prostate cancer (CRPC) cell
xenograft and in vitro in androgen responsive prostate cancer cell
lines by exposure to increasing concentrations of the anti-androgen
drugs ARN-509 or MDV3100. In cell proliferation and transcriptional
assays, the cell lines segregated into two distinct classes.
[0085] Class 1 cell lines expressed a higher level of AR compared
to their parental cell lines and proliferated in the absence of
added androgens. Ligand-independent growth of the class 1 cells was
unaltered in the presence of ARN-509, MDV3100 or bicalutamide. The
synthetic androgen, R1881, inhibited proliferation in the class 1
cells and is antagonized by either MDV3100 or ARN-509, indicating
that AR in these cell lines still binds to MDV3100 and ARN-509.
[0086] Class 2 resistant cell lines remained androgen dependent for
growth similar to their parental cell lines. While ARN-509 and
MDV3100 inhibited proliferation of the parental cell line, both
compounds exhibited agonist activity and stimulated proliferation
of the class 2 cell lines. Analysis of the nucleic acid encoding AR
in the class 2 cell lines revealed that the cell lines expressed a
mutant AR with a mutation in the ligand binding domain. The
mutation was a thymidine (T) to cytosine (C) missense mutation that
resulted in an amino acid substitution of phenylalanine at position
876 of the wild-type AR for leucine (F876L).
[0087] As described herein, in the examples, mutations in the AR
gene have been identified in plasma samples from prostate cancer
patients undergoing ARN-509 Phase 1/2 clinical studies. The
mutations identified include a thymidine (T) to cytosine (C)
missense mutation at position 2988 (first position of the codon
encoding phenylanine, which is encoded in exon 8 of the AR gene),
and a cytosine (C) to alanine (A) missense mutation at position
2990 (third position of the codon encoding phenylalanine) in an
amino acid substitution of phenylalanine at position 876 of the
wild-type AR for leucine (F876L). The patients identified as having
these mutations also exhibited increasing levels of prostate
specific antigen (PSA) over the course of the study indicating
increasing resistance to treatment.
[0088] Described herein are mutant AR polypeptides that contain an
amino acid substitution of phenylalanine at position 876 of the
wild-type AR for leucine (F876L) and nucleic acids encoding the
polypeptides. Also described herein are methods of producing the
mutant AR nucleic acids and polypeptides described herein. Also
described herein are compositions, combinations and kits containing
the mutant AR nucleic acids and polypeptides described herein. Also
provided are methods of using the mutant AR polypeptides for
identifying mutant AR interacting molecules, including androgen
inhibitors, including third-generation AR inhibitor compounds. Also
provided are compositions containing the mutant AR interacting
molecules, including pharmaceutical compositions thereof. Also
provided are methods of treatment using the identified mutant AR
interacting molecules. Also described herein are mutant AR nucleic
acids that are synthetic nucleic acids. Also described herein are
mutant AR nucleic acids that are cDNA molecules. Also described
herein are mutant AR polypeptides produced by mutant AR nucleic
acids that are synthetic nucleic acids. Also described herein are
mutant AR polypeptides produced by mutant AR nucleic acids that are
cDNA molecules. Also described herein are mutant AR nucleic acids
that do not contain AR genomic DNA. Also described herein are
mutant AR nucleic acids that are unmethylated. Also described
herein are mutant AR nucleic acids that do not contain AR intron
sequences. Also described herein are mutant AR nucleic acids that
comprises a sequence of nucleotides from two or more exons of the
AR genomic sequence. In some embodiments, the mutant AR nucleic
acids comprise a sequence of nucleotides that encode phenylalanine
at a position corresponding to position 876 of the wild-type AR
polypeptide.
[0089] As described herein, identification of a mutation at
position 876 in the AR, such as for example F876L, allows for the
design and screening of inhibitors effective for inhibition of a
mutant AR having one or more resistance mutations. Such inhibitors
are useful in clinical and therapeutic applications. In some
embodiments, the inhibitors are useful for the treatment of a
cancer, such as for example, an AR-mediated cancer, such as, for
example, a prostate cancer, breast cancer, liver (i.e.
hepatocellular) cancer, or bladder cancer, such as for example, a
drug resistant prostate, breast, liver (i.e. hepatocellular), or
bladder cancer.
[0090] As described herein, in some embodiments, subjects are
screened for the identification of a mutation at position 876 in
AR, such as for example F876L. In some embodiments, identification
of such a mutation allows for the prescription of a cancer
treatment or modification of a cancer treatment. In some
embodiments, identification of such a mutation is used to stratify
subjects for a particular therapy, such as for example, therapy
with an inhibitor that inhibits the activity of the mutant AR (i.e.
a third-generation AR inhibitor). In some embodiments,
identification of such a mutation is used to characterize a subject
as having a high risk of relapse of a disease or condition, such
as, for example, a prostate cancer, breast cancer, liver (i.e.
hepatocellular) cancer, or bladder cancer. In some embodiments,
identification of such a mutation is used to characterize a subject
as lacking responsiveness to particular AR inhibitor, such as for
example ARN-509, MDV3100, or RD162. In some embodiments,
identification of such a mutation is used to characterize a subject
as lacking responsiveness to an AR inhibitor that is a CYP17A
inhibitor, such as, for example galeterone (TOK001), TAK-700 or
abiraterone acetate.
Mutant AR Polypeptides
[0091] Provided herein are mutant AR polypeptides. In some
embodiments, the isolated mutant AR polypeptides are isolated
mutant AR polypeptides. In some embodiments, the isolated mutant AR
polypeptides are non-native mutant AR polypeptides. In some
embodiments, the isolated mutant AR polypeptides are recombinant
mutant AR polypeptides. In some embodiments, the mutant AR
polypeptides provided herein exhibit androgen receptor activity.
For example, the mutant AR polypeptides bind to androgen response
elements and promote the expression of AR-responsive genes. In some
embodiments, the mutant AR polypeptides contain one or more amino
acid substitutions that confers resistance to full antagonism by an
anti-androgen. In some embodiments, the mutant AR polypeptides
contain one or more amino acid substitutions that confers
resistance to full antagonism by a second-generation AR inhibitor,
such as, but not limited to, ARN-509, enzalutamide (MDV3100) or
RD162. In some embodiments, the mutant AR polypeptides contain one
or more amino acid substitutions that confers resistance to
inhibition by ARN-509. In some embodiments, the mutant AR
polypeptides contain one or more amino acid substitutions that
confers resistance to inhibition by enzalutamide (MDV3100). In some
embodiments, the mutant AR polypeptides contain one or more amino
acid substitutions that confers resistance to inhibition by
RD162.
[0092] In some embodiments, treatment of a mutant AR polypeptide
provided herein with a second-generation AR inhibitor induces AR
activity. For example, the second-generation AR inhibitor acts as
an agonist of the mutant AR polypeptide. In some embodiments,
treatment of a mutant AR polypeptide provided herein with ARN-509
induces AR activity. In some embodiments, treatment of a mutant AR
polypeptide provided herein with enzalutamide (MDV3100) induces AR
activity. In some embodiments, treatment of a mutant AR polypeptide
provided herein with RD162 induces AR activity.
[0093] In some embodiments, the mutant AR polypeptide comprises a
modification at a position corresponding to amino acid position 876
of the wild-type AR polypeptide set forth in SEQ ID NO: 1
(Accession No. P10275) or a corresponding position in the wild-type
AR polypeptide set forth in SEQ ID NO: 2 (Accession No.
NP_000035.2). In some embodiments, the modification is a
substitution of the amino acid phenylalanine at the position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1. In some embodiments, the
mutant androgen receptor (AR) polypeptide does not comprise
phenylalanine at the position corresponding to amino acid position
876 of the wild-type AR polypeptide set forth in SEQ ID NO: 1.
[0094] In some embodiments, the modification is a substitution of
the amino acid phenylalanine at the position corresponding to amino
acid position 876 of the wild-type AR polypeptide set forth in SEQ
ID NO: 1, and the substituted amino acid is selected from among
leucine, isoleucine, valine, alanine, glycine, methionine, serine,
threonine, cysteine, tryptophan, lysine, arginine, histidine,
proline, tyrosine, asparagine, glutamine, aspartic acid and
glutamic acid. In some embodiments, the mutant androgen receptor
(AR) polypeptide comprises a leucine, isoleucine, valine, alanine,
glycine, methionine, serine, threonine, cysteine, tryptophan,
lysine, arginine, histidine, proline, tyrosine, asparagine,
glutamine, aspartic acid or glutamic acid at the position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1. In some embodiments, the
modification is a substitution of the amino acid phenylalanine at
the position corresponding to amino acid position 876 of the
wild-type AR polypeptide set forth in SEQ ID NO: 1, and the
substituted amino acid is selected from among leucine, isoleucine,
valine, alanine, glycine, methionine and tryptophan. In some
embodiments, the mutant androgen receptor (AR) polypeptide
comprises a leucine, isoleucine, valine, alanine, glycine,
methionine or tryptophan at the position corresponding to amino
acid position 876 of the wild-type AR polypeptide set forth in SEQ
ID NO: 1. In some embodiments, the modification is a substitution
of the amino acid phenylalanine to leucine at the position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1. In some embodiments, the
mutant AR polypeptide comprises a leucine at the position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1.
[0095] In some embodiments, the modification comprises a deletion
of the amino acid phenylalanine at the position corresponding to
amino acid position 876 of the wild-type AR polypeptide set forth
in SEQ ID NO: 1.
[0096] In some embodiments, the mutant AR polypeptide comprises a
sequence of amino acids set forth in SEQ ID NO: 5. In some
embodiments the mutant AR polypeptide comprises a polypeptide
having a leucine at the position corresponding to amino acid
position 876 and having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% amino acid sequence identity to the polypeptide
having the sequence set forth in SEQ ID NO: 5. In some embodiments
the mutant AR polypeptide comprises a polypeptide not having a
phenylalanine at the position corresponding to amino acid position
876 and having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% amino acid sequence identity to the polypeptide having the
sequence set forth in SEQ ID NO: 5.
[0097] In some embodiments, the mutant AR polypeptide comprises a
modification at amino acid position 876 and a modification at one
or more additional amino acid positions. In some embodiments, the
mutant AR polypeptide comprises a modification at amino acid
position 876 and a modification at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid
positions. In some embodiments, the mutant AR polypeptide comprises
a modification at position 876 and a modification at one additional
amino acid position. In some embodiments, the mutant AR polypeptide
comprises a leucine at amino acid position 876 and a modification
at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more additional amino acid positions. In some
embodiments, the mutant AR polypeptide comprises a leucine at
position 876 and a modification at one additional amino acid
position. In some embodiments, the modification at amino acid
position 876 is a substitution that is F876L.
[0098] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification at an additional
amino acid position that confers resistance to a first- or
second-generation AR antagonist or an AR antagonist that inhibits
androgen production. In some embodiments, the modification at the
amino acid position in addition to the modification at amino acid
876 increases the resistance of the AR polypeptide to a first- or
second-generation AR antagonist or an AR antagonist that inhibits
androgen production compared to an AR polypeptide comprising the
modification at amino acid position 876 alone. In some embodiments,
the modification at amino acid position 876 is a substitution that
is F876L.
[0099] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification selected from among
AR modifications described in, for example. Grasso et al. (2012)
Nature 487(7406):239-43; Ning et al. (2012) Urology 80(1):216-8;
Cong et al. (2012) Gene 500(2):220-3; Hay et al. (2012) PLoS One
2012; 7(3):e32514; Koochekpour (2010) Asian J Androl. 12(5):639-57;
Waltering et al. (2012) Mol Cell Endocrinol. 360:38-43; Robbins
(2012) Mol Cell Endocrinol. 352(1-2):26-33; or Gottlieb et al.
(2012) Hum Mutat. 33(5):887-94. In some embodiments, the
modification at amino acid position 876 is a substitution that is
F876L.
[0100] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification selected from among
AR modifications associated with castration resistant prostate
cancer. In some embodiments, the modification associated with
castration resistant prostate cancer is an amino acid substitution
such as, for example, T877A, W741C, W741L, W741R. L701H or H874Y.
In some embodiments, the modification at amino acid position 876 is
a substitution that is F876L.
[0101] In some embodiments, the mutant AR polypeptide comprises a
modification at amino acid position 876 and a modification at amino
acid position 877. In some embodiments, the mutant AR polypeptide
comprises a leucine at position 876 and a modification at amino
acid position 877. In some embodiments, the modification at amino
acid position 877 is a substitution of the amino acid threonine. In
some embodiments, the substituted amino acid at amino acid position
877 is selected from among leucine, isoleucine, valine, alanine,
phenylalanine, glycine, methionine, serine, cysteine, tryptophan,
lysine, arginine, histidine, proline, tyrosine, asparagine,
glutamine, aspartic acid and glutamic acid. In some embodiments,
the substituted amino acid at amino acid position 877 is alanine.
In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and an alanine at amino acid position
877. In some embodiments, the mutant AR polypeptide comprises a
leucine at position 876 and an alanine at amino acid position
877.
[0102] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification at amino acid
position 741. In some embodiments, the mutant AR polypeptide
comprises a leucine at position 876 and a modification at amino
acid position 741. In some embodiments, the modification at amino
acid position 741 is a substitution of the amino acid tryptophan.
In some embodiments, the substituted amino acid at amino acid
position 741 is selected from among leucine, isoleucine, valine,
alanine, phenylalanine, glycine, methionine, serine, threonine,
cysteine, lysine, arginine, histidine, proline, tyrosine,
asparagine, glutamine, aspartic acid and glutamic acid. In some
embodiments, the substituted amino acid at amino acid position 741
is leucine, cysteine or arginine. In some embodiments, the mutant
AR polypeptide comprises a modification at position 876 and a
leucine at amino acid position 741. In some embodiments, the mutant
AR polypeptide comprises a modification at position 876 and a
cysteine at amino acid position 741. In some embodiments, the
mutant AR polypeptide comprises a modification at position 876 and
an arginine at amino acid position 741. In some embodiments, the
mutant AR polypeptide comprises a leucine at position 876 and a
leucine at amino acid position 741. In some embodiments, the mutant
AR polypeptide comprises a leucine at position 876 and a cysteine
at amino acid position 741. In some embodiments, the mutant AR
polypeptide comprises a leucine at position 876 and an arginine at
amino acid position 741.
[0103] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification at amino acid
position 701. In some embodiments, the mutant AR polypeptide
comprises a leucine at position 876 and a modification at amino
acid position 701. In some embodiments, the modification at amino
acid position 701 is a substitution of the amino acid leucine. In
some embodiments, the substituted amino acid at amino acid position
701 is selected from among isoleucine, valine, alanine,
phenylalanine, glycine, methionine, histidine, serine, threonine,
cysteine, lysine, arginine, tryptophan, proline, tyrosine,
asparagine, glutamine, aspartic acid and glutamic acid. In some
embodiments, the substituted amino acid at amino acid position 701
is histidine. In some embodiments, the mutant AR polypeptide
comprises a modification at position 876 and a histidine at amino
acid position 701. In some embodiments, the mutant AR polypeptide
comprises a leucine at position 876 and a histidine at amino acid
position 701.
[0104] In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a modification at amino acid
position 874. In some embodiments, the mutant AR polypeptide
comprises a leucine at position 876 and a modification at amino
acid position 874. In some embodiments, the modification at amino
acid position 874 is a substitution of the amino acid histidine. In
some embodiments, the substituted amino acid at amino acid position
874 is selected from among leucine, isoleucine, valine, alanine,
phenylalanine, glycine, methionine, serine, threonine, cysteine,
lysine, arginine, tryptophan, proline, tyrosine, asparagine,
glutamine, aspartic acid and glutamic acid. In some embodiments,
the substituted amino acid at amino acid position 874 is tyrosine.
In some embodiments, the mutant AR polypeptide comprises a
modification at position 876 and a tyrosine at amino acid position
874. In some embodiments, the mutant AR polypeptide comprises a
leucine at position 876 and a tyrosine at amino acid position
874.
[0105] In some embodiments, the mutant AR polypeptide is an AR
polypeptide variant that comprises a modification at position 876
and one or more additional amino acid positions relative to the
wild-type AR polypeptide set forth in SEQ ID NO: 1. Exemplary
variants include, for example, species variants, allelic variants.
RNA splicing variants and variants that contain conservative and
non-conservative amino acid mutations. In some embodiments, the AR
polypeptide variant comprises a polyglutamine tract of about 6
consecutive glutamine residues to about 39 consecutive glutamine
residues. In some embodiments, the AR polypeptide variant comprises
a polyglutamine tract of about 16 consecutive glutamine residues to
about 29 consecutive glutamine residues. In some embodiments, the
AR polypeptide variant comprises a polyglutamine tract of about 21
consecutive glutamine residues. In some embodiments, the AR
polypeptide variant comprises a polyglutamine tract of about 22
consecutive glutamine residues. In some embodiments, the AR
polypeptide variant comprises a polyglutamine tract of about 23
consecutive glutamine residues. In some embodiments, the AR
polypeptide variant comprises a polyglycine tract of about 10
consecutive glycine residues to about 27 consecutive glycine
residues. In some embodiments, the AR polypeptide variant comprises
a polyglycine tract of about 23 consecutive glycine residues. In
some embodiments, the AR polypeptide variant comprises a
polyglycine tract of about 24 consecutive glycine residues.
[0106] In some embodiments, the mutant AR polypeptide comprises a
portion of the mutant AR polypeptide set forth in SEQ ID NO: 5. In
some embodiments, the portion exhibits an activity of an AR
polypeptide. In some embodiments, the mutant AR polypeptide
comprises the DNA binding domain of an AR polypeptide and the
ligand binding domain of the AR polypeptide comprising the
modification at amino acid position 876 of the mutant AR
polypeptide set forth in SEQ ID NO: 5. In some embodiments, the
mutant AR polypeptide consists essentially of the DNA binding
domain of an AR polypeptide and the ligand binding domain of the AR
polypeptide comprising the modification at amino acid position 876
of the mutant AR polypeptide set forth in SEQ ID NO: 5. In some
embodiments, the mutant AR polypeptide comprises the sequence of
amino acids from about amino acid position 554 to about amino acid
position 919 of the mutant AR polypeptide set forth in SEQ ID NO:
5. In some embodiments, the mutant AR polypeptide comprises the
ligand binding domain of the mutant AR polypeptide. In some
embodiments, the mutant AR polypeptide consists essentially of the
ligand binding domain of the mutant AR polypeptide. In some
embodiments, the mutant AR polypeptide comprises the sequence of
amino acids from about amino acid position 554 to about amino acid
position 919.
[0107] In some embodiments, an AR polypeptide is a fusion protein
comprising the ligand binding domain of an AR polypeptide
comprising the modification at amino acid position 876 of the
wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
heterologous polypeptide. In some embodiments, the modification is
an amino acid substitution that is F876L. Methods for the
generation of fusion proteins are known in the art and include
standard recombinant DNA techniques. For example, in some
embodiments, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In some embodiments, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. In some embodiments, PCR amplification
of gene fragments can be carried out using anchor primers which
give rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example. Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). In some embodiments, expression vectors are
commercially available that encode a fusion moiety (e.g., a GST
polypeptide). A nucleic acid encoding a modified AR polypeptide can
be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the modified AR polypeptide.
[0108] In some embodiments, an AR polypeptide is a fusion protein
comprising the ligand binding domain of an AR polypeptide
comprising a modification at amino acid position 876 of the
wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
heterologous DNA binding domain. In some embodiments, an AR
polypeptide is a fusion protein comprising the ligand binding
domain of a mutant AR polypeptide set forth in SEQ ID NO: 5 linked
to a heterologous DNA binding domain. In some embodiments the
heterologous DNA binding domain is GAL4 DNA binding domain. In some
embodiments the heterologous DNA binding domain is LexA DNA binding
domain. In some embodiments, the ligand binding domain of an AR
polypeptide comprising a modification at amino acid position 876 of
the wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
heterologous DNA binding domain via a peptide linker. In some
embodiments, an AR polypeptide is a fusion protein comprising the
ligand binding domain of a mutant AR polypeptide set forth in SEQ
ID NO: 5 linked to a heterologous DNA binding domain via a peptide
linker.
[0109] In some embodiments, an AR polypeptide is a fusion protein
comprising the ligand binding domain of an AR polypeptide
comprising a modification at amino acid position 876 of the
wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
heterologous peptide for use in an protein interaction assay, such
as, but not limited to a yeast two hybrid assay, a mammalian cell
based two hybrid (M2H) system, a Forster (Fluorescence) Resonance
Energy Transfer (FRET) assay, a Bioluminescence Resonance Energy
Transfer (BRET), or a Homogeneous Time Resolved Fluorescence
(HTRF.RTM.) assay. In some embodiments, an AR polypeptide is a
fusion protein comprising the ligand binding domain of a mutant AR
polypeptide set forth in SEQ ID NO: 5 linked to a heterologous
peptide for use in an protein interaction assay, such as, but not
limited to a yeast two hybrid assay, a mammalian cell based two
hybrid (M2H) system, a Forster (Fluorescence) Resonance Energy
Transfer (FRET) assay, a Bioluminescence Resonance Energy Transfer
(BRET), or a Homogeneous Time Resolved Fluorescence (HTRF.RTM.)
assay.
[0110] In some embodiments, the ligand binding domain of an AR
polypeptide comprising a modification at amino acid position 876 of
the wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
detectable polypeptide. In some embodiments, the ligand binding
domain of a mutant AR polypeptide set forth in SEQ ID NO: 5 is
linked to a detectable polypeptide. In some embodiments, the ligand
binding domain of an AR polypeptide comprising a modification at
amino acid position 876 of the wild-type AR polypeptide set forth
in SEQ ID NO: 1 linked to a fluorescent protein, such as, but
limited to, a green (GFP), red (RFP), cyan (CFP), yellow (YFP), or
blue (BFP) fluorescent protein. In some embodiments, the ligand
binding domain of a mutant AR polypeptide set forth in SEQ ID NO: 5
is linked to a fluorescent protein, such as, but limited to, a
green (GFP), red (RFP), cyan (CFP), yellow (YFP), or blue (BFP)
fluorescent protein. In some embodiments, the ligand binding domain
of an AR polypeptide comprising a modification at amino acid
position 876 of the wild-type AR polypeptide set forth in SEQ ID
NO: 1 linked to a bioluminescent protein. In some embodiments, the
ligand binding domain of a mutant AR polypeptide set forth in SEQ
ID NO: 5 is linked to a bioluminescent protein. In some
embodiments, the ligand binding domain of an AR polypeptide
comprising a modification at amino acid position 876 of the
wild-type AR polypeptide set forth in SEQ ID NO: 1 linked to a
peptide tag. In some embodiments, the ligand binding domain of a
mutant AR polypeptide set forth in SEQ ID NO: 5 is linked to a
peptide tag. In some embodiments, the peptide tag is an epitope tag
recognized by a tag-specific antibody. In some embodiments the tag
is an epitope tag, such as, but not limited to a c-myc, V-5,
hemagglutinin (HA), FLAG. In some embodiments the tag is an
affinity tag, such as, but not limited to, biotin, strep-tag,
chitin binding protein (CBP), maltose binding protein (MBP),
glutathione-S-transferase (GST), or a poly(His) tag.
[0111] In some embodiments, provided herein is an array comprising
a mutant AR polypeptide provided herein. In some embodiments, the
mutant AR polypeptide is bound to a microchip. In some embodiments,
the mutant AR polypeptide is bound directly to the microchip. In
some embodiments, the mutant AR polypeptide is bound indirectly to
the microchip via a linker. In some embodiments, provided herein is
a microchip array comprising a mutant AR polypeptide provided
herein.
Nucleic Acids
[0112] Provided herein are nucleic acids encoding mutant AR
polypeptides. Provided herein are nucleic acids encoding any of the
mutant AR polypeptides described herein. Methods for deducing
nucleic acids that encode particular polypeptides are known in the
art and involve standard molecular biology techniques. Exemplary
nucleic acids encoding mutant AR polypeptides provided herein are
provided. It is understood that due to the degeneracy of the
genetic code multiple variants nucleic acids exist that encode the
same polypeptide. Nucleic acids that encode the mutant AR
polypeptides provided herein encompass such variants. In some
embodiments, the mutant AR nucleic acids are synthetic nucleic
acids. In some embodiments, the mutant AR nucleic acids are cDNA
molecules. In some embodiments, the mutant AR nucleic acids do not
contain genomic DNA. In some embodiments, the mutant AR nucleic
acids are unmethylated. In some embodiments, the mutant AR nucleic
acids are do not contain AR genomic intron sequences. In some
embodiments, the mutant AR nucleic acids comprise a sequence of
nucleotides from two or more exons of the AR genomic sequence,
including exon 8 or a portion thereof comprising the nucleic acid
sequence encoding position 876 of the AR polypeptide. In some
embodiments, the mutant AR nucleic acids comprise a sequence of
nucleotides that encode phenylalanine at a position corresponding
to position 876 of the wild-type AR polypeptide.
[0113] In some embodiments, the nucleic acid encoding mutant AR
polypeptides comprises a modification relative to a nucleic acid
encoding a wild-type AR polypeptide. In some embodiments, the
nucleic acid encoding a mutant AR polypeptide comprises a
modification where the encoded polypeptide comprises a substitution
of the amino acid phenylalanine at the position corresponding to
amino acid position 876 of the wild-type AR polypeptide set forth
in SEQ ID NO: 1. In some embodiments, the nucleic acid encoding a
mutant AR polypeptide comprises a modification where encoded
polypeptide does not comprise phenylalanine at the position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1.
[0114] In some embodiments the nucleic acid modification is a
missense mutation or a deletion of one or more codons that encode
the polypeptide. In some embodiments, the modification is a
missense mutation that changes the nucleic acid codon that encodes
phenylalanine at amino position 876 of the AR polypeptide. In some
embodiments, the nucleic acid codon that encodes phenylalanine at
amino position 876 of the AR polypeptide is TTC or TTT. In some
embodiments, the nucleic acid codon that encodes phenylalanine at
amino position 876 of the AR polypeptide is TTC. In some
embodiments, the nucleic acid codon that encodes phenylalanine at
amino position 876 of the AR polypeptide is TIT. In some
embodiments, the modification changes the nucleic acid codon that
encodes phenylalanine at amino position 876 of the AR polypeptide
from TTC to a nucleic acid codon that encodes leucine. In some
embodiments, the modification changes the nucleic acid codon that
encodes phenylalanine at amino position 876 of the AR polypeptide
from TIT to a nucleic acid codon that encodes leucine. In some
embodiments, the nucleic acid codon that encodes leucine is
selected from among TTA, TTG, CTT, CTC, CTA or CTG.
[0115] In some embodiments, the modification is a missense mutation
that comprises a substitution of Thymine (T) for Cytosine (C). In
some embodiments, the modification changes the nucleic acid codon
that encodes phenylalanine at amino position 876 of the AR
polypeptide from TTC to CTC. In some embodiments, the modification
is a missense mutation that comprises a substitution of Thymine (T)
for Cytosine (C) at nucleic acid position 2626 of the AR nucleic
acid sequence set forth in SEQ ID NO: 18.
[0116] In some embodiments, the modification is a missense mutation
that comprises a substitution of Thymine (T) for Adenine (A). In
some embodiments, the modification changes the nucleic acid codon
that encodes phenylalanine at amino position 876 of the AR
polypeptide from TTT to TTA. In some embodiments, the modification
is a missense mutation that comprises a substitution of Thymine (T)
for Adenine (A) at nucleic acid position 2628 of the AR nucleic
acid sequence set forth in SEQ ID NO: 18.
[0117] In some embodiments, the modification is a missense mutation
that comprises a substitution of Thymine (T) for Guanine (G). In
some embodiments, the modification changes the nucleic acid codon
that encodes phenylalanine at amino position 876 of the AR
polypeptide from TTT to TTG. In some embodiments, the modification
is a missense mutation that comprises a substitution of Thymine (T)
for Guanine (G) at nucleic acid position 2628 of the AR nucleic
acid sequence set forth in SEQ ID NO: 18.
[0118] In some embodiments, the modification comprises a missense
mutation that comprises a substitution of Thymine (T) for Cytosine
(C) at the first position of the codon that encodes F876 of the AR
polypeptide and a second missense mutation that comprises a
substitution of Thymine (T) for Adenosine (A) at the third position
of the codon that encodes F876 of the AR polypeptide. In some
embodiments, the modification changes the nucleic acid codon that
encodes phenylalanine at amino position 876 of the AR polypeptide
from TTT to CTA. In some embodiments, the modification is a
missense mutation that comprises a substitution of Thymine (T) for
Cytosine (C) at nucleic acid position 2626 and a second missense
mutation that comprises a substitution of Thymine (T) for Adenosine
(A) at nucleic acid position 2628 of the AR nucleic acid sequence
set forth in SEQ ID NO: 18.
[0119] In some embodiments, the modification comprises a missense
mutation that comprises a substitution of Thymine (T) for Cytosine
(C) at the first position of the codon that encodes F876 of the AR
polypeptide and a second missense mutation that comprises a
substitution of Thymine (T) for Guanine (G) at the third position
of the codon that encodes F876 of the AR polypeptide. In some
embodiments, the modification changes the nucleic acid codon that
encodes phenylalanine at amino position 876 of the AR polypeptide
from TTT to CTG. In some embodiments, the modification is a
missense mutation that comprises a substitution of Thymine (T) for
Cytosine (C) at nucleic acid position 2626 and a second missense
mutation that comprises a substitution of Thymine (T) for Guanine
(G) at nucleic acid position 2628 of the AR nucleic acid sequence
set forth in SEQ ID NO: 18.
[0120] In some embodiments the nucleic acid encoding the mutant AR
polypeptide comprises a sequence of nucleotides set forth in SEQ ID
NO: 19. In some embodiments the nucleic acid encoding the mutant AR
polypeptide comprises a nucleic acid having 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide acid sequence
identity to the nucleic acid having the sequence of nucleotides set
forth in SEQ ID NO: 19, where the encoded mutant AR comprises a
modification relative to the wild-type AR polypeptide at a position
corresponding to amino acid position 876. In some embodiments the
nucleic acid encoding the mutant AR polypeptide comprises a nucleic
acid having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97/%, 98%, or
99% nucleotide acid sequence identity to the nucleic acid having
the sequence of nucleotides set forth in SEQ ID NO: 19, where the
encoded mutant AR does not comprise a phenylalanine at the position
corresponding to amino acid position 876. In some embodiments the
nucleic acid encoding the mutant AR polypeptide comprises a nucleic
acid having 650/%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% nucleotide acid sequence identity to the nucleic acid having
the sequence of nucleotides set forth in SEQ ID NO: 19, where the
encoded mutant AR comprises a leucine at the position corresponding
to amino acid position 876.
[0121] In some embodiments, the nucleic acid provided herein
encoding a mutant AR polypeptide is an isolated nucleic acid. In
some embodiments, the nucleic acid provided herein encoding a
mutant AR polypeptide is a DNA molecule. In some embodiments, the
nucleic acid provided herein encoding a mutant AR polypeptide is a
cDNA molecule. In some embodiments, the nucleic acid provided
herein encoding a mutant AR polypeptide is an RNA molecule. In some
embodiments, the nucleic acid provided herein encoding a mutant AR
polypeptide is an inhibitory RNA molecule (i.e. RNAi). In some
embodiments, the nucleic acid provided herein is a nucleic acid
molecule that is complementary, or binds to, an nucleic acid
encoding a mutant AR polypeptide.
[0122] In some embodiments, the nucleic acid provided herein
encoding a mutant AR polypeptide or a portion thereof contains
nucleic acid encoding an amino acid at position 876 that is not
phenylalanine. In some embodiments, the nucleic acid provided
herein encoding a mutant AR polypeptide or a portion thereof
contains nucleic acid encoding leucine at amino acid position
876.
[0123] In some embodiments, the nucleic acid provide herein is an
oligonucleotide that encodes a portion of the mutant AR
polypeptide. In some embodiments the nucleic acid provided herein
is an oligonucleotide that encodes a portion of the mutant AR
polypeptide that contains a nucleotide codon encoding the amino
acid corresponding to amino acid position 876. In some embodiments,
the codon encodes an amino acid that is not phenylalanine. In some
embodiments, the codon encodes an amino acid that is leucine.
[0124] In some embodiments, the nucleic acid provided herein is a
vector that comprises nucleic acid encoding any of the mutant AR
polypeptides provided herein. In some embodiments, the nucleic acid
provided herein is a vector that comprises nucleic acid encoding
any of the mutant AR polypeptides provided herein is an expression
vector. In some embodiments, the nucleic acid provided herein is a
vector that comprises nucleic acid encoding any of the mutant AR
polypeptides provided herein is operably linked to a promoter for
the expression of the mutant AR polypeptides.
[0125] In some embodiments, the vector is a plasmid vector. In some
embodiments, the vector is a viral vector. In some embodiments, the
viral vector is a DNA or RNA viral vector. Exemplary viral vectors
include, but are not limited to, a vaccinia, adenovirus,
adeno-associated virus (AAV), retrovirus, or herpesvirus
vector.
[0126] In some embodiments, provided herein is an array comprising
a nucleic acid encoding any of the mutant AR polypeptides provided
herein. In some embodiments, the mutant AR nucleic acid is bound to
a microchip. In some embodiments, the mutant AR nucleic acid is
bound directly to the microchip. In some embodiments, the mutant AR
nucleic acid is bound indirectly to the microchip via a linker. In
some embodiments, provided herein is a microchip array comprising a
nucleic acid encoding any of the mutant AR polypeptides provided
herein.
Production of Nucleic Acids and Polypeptides
[0127] In some embodiments, an isolated nucleic acid molecule
encoding a mutant AR polypeptide provided herein is generated by
standard recombinant methods. In some embodiments, an isolated
nucleic acid molecule encoding a mutant AR polypeptide provided
herein is generated by amplification of a mutant AR sequence from
genomic DNA. In some embodiments, an isolated nucleic acid molecule
encoding a mutant AR polypeptide provided herein is generated by
polymerase chain reaction using AR sequence specific primers. In
some embodiments, an isolated nucleic acid molecule encoding a
mutant AR polypeptide provided herein is generated by reverse
transcription of mRNA encoding a mutant AR polypeptide.
[0128] In some embodiments, an isolated nucleic acid molecule
encoding a mutant AR polypeptide provided herein is inserted into
an expression vector and expressed in a host cell or a non-cell
extract. In some embodiments, an isolated nucleic acid molecule
encoding a mutant AR polypeptide provided herein is operatively
linked to a promoter for expression of the encoding polypeptide in
a cell or non-cell extract. In some embodiments, the promoter is a
constitutive promoter. In some embodiments, the promoter is an
inducible promoter.
[0129] In some embodiments, the nucleic acid molecule encoding a
mutant AR polypeptide provided herein is "exogenous" to a cell,
which means that it is foreign to the cell into which the vector is
being introduced or that the sequence is homologous to a sequence
in the cell but in a position within the host cell nucleic acid in
which the sequence is ordinarily not found. Vectors include
plasmids, cosmids, viruses (bacteriophage, animal viruses, and
plant viruses), and artificial chromosomes (e.g., YACs). One of
skill in the art would be well equipped to construct a vector
through standard recombinant techniques, which are described in
Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated
herein by reference.
[0130] Methods for the expression of a protein in a cell are well
known in the art and include, for example, expression in cells,
such as animal and plant cells. Exemplary animal cells for the
expression of mutant AR polypeptides provided herein include but
are not limited to bacteria, yeast, insect cells, and mammalian
cells, such as for example, human, primate, rodent, bovine, and
ovine cells. In some embodiments, the nucleic acid encoding the
mutant AR is integrated into the genome of the host cell.
[0131] In some embodiments, a method for the expression of a mutant
AR polypeptide provided herein comprises culturing a host cell
containing an expression vector encoding a mutant AR polypeptide
such that the mutant AR polypeptide is produced by the cell. In
some methods, the nucleic acid encoding the mutant polypeptide is
connected to nucleic acid encoding a signal sequence such that the
signal sequence is expressed as a fusion peptide with the mutant AR
polypeptide. In some embodiments the signal sequence allows for the
secretion of the mutant AR polypeptide by the host cell.
[0132] In some embodiments the mutant AR polypeptide is isolated
from a host cell expressing the mutant polypeptide. In some
embodiments an extract is prepared from the host cell and the
mutant AR polypeptide is isolated by purification methods such as
but not limited to chromatography or immunoaffinity with an
antibody that is specific for AR polypeptides or specific to the
mutant AR polypeptide in particular.
Antibodies
[0133] In some embodiments the antibody binds to a mutant AR
polypeptide provided herein, and binds with less affinity or does
not bind to a wild-type AR polypeptide. In some embodiments the
antibody binds to a mutant AR polypeptide in the presence of a
second-generation inhibitor, such as, but not limited to ARN-509,
enzalutamide (MDV3100) or RD162.
[0134] In some embodiments, mutant AR polypeptide provided herein
are detected using antibodies that specifically recognize the
mutant AR polypeptides, but do not recognize wild-type AR
polypeptides. In some embodiments, mutant AR polypeptide provided
herein are detected using antibodies that specifically recognize a
mutant AR polypeptide having a leucine at amino acid position 876,
but do not recognize wild-type AR polypeptides. In some
embodiments, antibodies are raised against one or more allelic
forms of the mutant AR polypeptide provided herein. Techniques for
using a specific protein or an oligopeptide as an antigen to elicit
antibodies that specifically recognize epitopes on the peptide or
protein are well known. In one embodiment, the DNA sequence of the
desired allelic form of the target gene is cloned by insertion into
an appropriate expression vector and translated into protein in a
prokaryotic or eukaryotic host cell. The protein can be recovered
and used as an antigen to elicit the production of specific
antibodies. In another embodiment, the DNA of the desired allelic
form of the target gene is amplified by PCR technology and is
subsequently translated in vitro into protein to be used as the
antigen to elicit the production of specific antibodies. In another
embodiment, the DNA sequence of the alternative alleles is used as
a basis for the generation of synthetic peptides representing the
amino acid sequence of the alleles for use as the antigen to elicit
the production of specific antibodies.
[0135] In some embodiments, antibodies are generated either by
standard monoclonal antibody techniques or generated through
recombinant based expression systems. See generally, Abbas,
Lichtman, and Pober, Cellular and Molecular Immunology, W. B.
Saunders Co. (1991). The term "antibodies" is meant to include
intact antibody molecules as well as antibody fragments or
derivatives, such as Fab and F(ab')2, which are capable of
specifically binding to antigen. The antibodies so produced
preferentially bind only the mutant protein produced in the allelic
form which was used as an antigen to create the antibody. Methods
of generating allele-specific antibodies are also described in U.S.
Pat. No. 6,200,754 and U.S. Pat. No. 6,054,273, the entire contents
of which are incorporated herein by reference.
[0136] In some embodiments, the antibody provided herein is a
humanized antibody. A "humanized antibody" refers to a type of
engineered antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one or more human immunoglobulin(s). In
some embodiments, framework support residues are altered to
preserve binding affinity (see, e.g., Queen et al. Proc. Natl. Acad
Sci USA, 86:10029-10032 (1989), Hodgson et al. Bio/Technology.
9:421 (1991)). In some embodiments, a suitable human acceptor
antibody is one selected from a conventional database, e.g., the
KABAT.RTM. database, Los Alamos database, and Swiss Protein
database, by homology to the nucleotide and amino acid sequences of
the donor antibody. In some embodiments, a human antibody
characterized by a homology to the framework regions of the donor
antibody (on an amino acid basis) is suitable to provide a heavy
chain constant region and/or a heavy chain variable framework
region for insertion of the donor CDRs. In some embodiments, a
suitable acceptor antibody capable of donating light chain constant
or variable framework regions is selected in a similar manner. In
some embodiments, the acceptor antibody heavy and light chains
originate from the same acceptor antibody. In some embodiments, the
acceptor antibody heavy and light chains originate from the
different acceptor antibodies. The prior art describes several ways
of producing such humanized antibodies-see, for example,
EP-A-0239400 and EP-A-054951.
[0137] In some embodiments, antibodies specific for mutant AR
polypeptide provided herein can be used to detect the presence of a
mutant AR polypeptide provided herein in a sample, e.g., an assay
sample, a cell sample, a cell extract, a biological sample, or a
patient sample, using techniques known in the art. These techniques
include, for example, Western blot, immunohistochemistry, indirect
immunofluorescence, and antibody microarray. In some embodiments,
antibodies which specifically recognize mutant AR polypeptide are
third-generation AR inhibitors. In some embodiments, the ability of
an antibody which specifically recognizes a mutant AR polypeptide
to inhibit the biological activity of the mutant AR polypeptide can
be determined using the methods described herein for identifying
third-generation AR inhibitors.
Diagnostic Assays for Detecting Mutant AR Polypeptides and Nucleic
Acids Encoding Mutant AR Polypeptides
[0138] Provided herein are diagnostic methods that involve the
detection of a mutant AR polypeptide in a subject or a nucleic acid
encoding a mutant AR polypeptide in a subject. In some embodiments,
the subject has an AR-mediated disease or condition. In some
embodiments, the diagnostic methods are employed for the screening
subjects having a cancer that is resistant to therapy with an
anti-androgen, such as a first- or second-generation AR antagonist,
identifying subjects for the treatment with anti-androgen, such as
a first- or second-generation AR antagonist, monitoring the therapy
of subjects receiving an anti-androgen therapy, such as a first- or
second-generation AR antagonist, optimizing the therapy of subjects
receiving an anti-androgen therapy, such as a first- or
second-generation AR antagonist, and combinations thereof. In some
embodiments, the methods comprises selecting a subject for therapy
with a third-generation AR antagonist. In some embodiments, the
methods further comprise administering to the subject a
third-generation AR antagonist as described herein. In some
embodiments, the mutant AR polypeptide detected comprises a
modification at a position corresponding to amino acid position 876
of the wild-type AR polypeptide set forth in SEQ ID NO: 1. In some
embodiments, the mutant AR polypeptide detected comprises a
substitution of the amino acid phenylalanine to leucine at the
position corresponding to amino acid position 876 of the wild-type
AR polypeptide set forth in SEQ ID NO: 1. In some embodiments, a
subject having a mutant AR polypeptide comprising a modification at
amino acid position 876 is resistant to inhibition with a first- or
a second-generation antagonist.
[0139] In some embodiments, a subject having a mutant AR
polypeptide comprising a modification at amino acid position 876 is
resistant to inhibition with a first- or a second-generation
antagonist. In some embodiments, a subject having a mutant AR
polypeptide comprising a modification at amino acid position 876 is
resistant to inhibition with a first- and a second-generation
antagonist. In some embodiments, a subject having a mutant AR
polypeptide comprising a leucine at amino acid position 876 is
resistant to inhibition with a first- or a second-generation
antagonist. In some embodiments, a subject having a mutant AR
polypeptide comprising a leucine at amino acid position 876 is
resistant to inhibition with a first- and a second-generation
antagonist. In some embodiments, a subject having a mutant AR
polypeptide comprising a modification at amino acid position 876 is
resistant to inhibition with a CYP17A inhibitor that binds to AR,
such as for example, galeterone (TOK001). TAK-700 or abiraterone
acetate. In some embodiments, a subject having a mutant AR
polypeptide comprising a modification at amino acid position 876 is
resistant to inhibition with a CYP17A inhibitor that binds to AR,
such as for example, galeterone (TOK001), TAK-700 or abiraterone
acetate.
[0140] In some embodiments, provided are methods for characterizing
an AR polypeptide that is resistant to inhibition with a
second-generation AR antagonist in a subject, comprising: (a)
assaying a sample containing a nucleic acid molecule encoding an AR
polypeptide from the subject to determine whether the encoded AR
polypeptide is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1; and (b) characterizing the AR as resistant to inhibition
with a second-generation AR antagonist if the subject has the
modification. In some embodiments, the method further comprises
administration of a third-generation AR antagonist provided herein
for inhibition of the mutant AR. In some embodiments, the method
further comprises not administering a second generation AR
antagonist. In some embodiments, the method further comprises
administering a second generation AR antagonist if the subject does
not have the modification. In some embodiments, the subject has
cancer. In some embodiments, the subject has a prostate cancer. In
some embodiments, the subject has a castration resistant prostate
cancer. In some embodiments, the method further comprises a step of
obtaining the nucleic acid sample from the subject.
[0141] In some embodiments, provided are methods for characterizing
an AR that is resistant to inhibition with a first-generation AR
antagonist in a subject, comprising: (a) assaying a sample
containing a nucleic acid molecule encoding an AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1;
and (b) characterizing the AR as resistant to inhibition with a
first-generation AR antagonist if the subject has the modification.
In some embodiments, the method further comprises administration of
a third-generation AR antagonist provided herein for inhibition of
the mutant AR. In some embodiments, the method further comprises
not administering a second generation AR antagonist. In some
embodiments, the method further comprises administering a second
generation AR antagonist if the subject does not have the
modification. In some embodiments, the subject has cancer. In some
embodiments, the subject has a prostate cancer. In some
embodiments, the subject has a castration resistant prostate
cancer. In some embodiments, the method further comprises obtaining
the nucleic acid sample from the subject.
[0142] In some embodiments, provided are methods for charactering
an AR that is resistant to inhibition with an AR antagonist that is
a CYP17A inhibitor in a subject, comprising: (a) assaying a sample
containing a nucleic acid molecule encoding an AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1;
and (b) characterizing the AR as resistant to inhibition with an AR
antagonist that is a CYP17A inhibitor if the subject has the
modification. In some embodiments, the method further comprises
administration of a third-generation AR antagonist provided herein
for inhibition of the mutant AR. In some embodiments, the method
further comprises not administering a second generation AR
antagonist. In some embodiments, the method further comprises
administering a second generation AR antagonist if the subject does
not have the modification. In some embodiments, the subject has
cancer. In some embodiments, the subject has a prostate cancer. In
some embodiments, the subject has a castration resistant prostate
cancer. In some embodiments the CYP17A inhibitor is galeterone
(TOK001). TAK-700 or abiraterone acetate. In some embodiments, the
method further comprises obtaining the nucleic acid sample from the
subject.
[0143] In some embodiments, provided are methods for selecting a
subject for therapy with a second-generation AR antagonist,
comprising (a) assaying a sample containing a nucleic acid molecule
encoding an AR polypeptide from the subject to determine whether
the encoded AR polypeptide is modified at an amino acid position
corresponding to amino acid position 876 of the amino acid sequence
set forth in SEQ ID NO: 1; and (b) characterizing the subject as a
candidate for therapy with a second-generation AR antagonist if the
subject does not have the modification. In some embodiments, the
method further comprises characterizing the subject as not a
candidate for therapy with a second-generation AR antagonist if the
subject has the modification. In some embodiments, the method
further comprises administration of a third-generation AR
antagonist provided herein for inhibition of the mutant AR. In some
embodiments, the subject has cancer. In some embodiments, the
subject has a prostate cancer. In some embodiments, the subject has
a castration resistant prostate cancer. In some embodiments, the
method further comprises a step of obtaining the nucleic acid
sample from the subject.
[0144] In some embodiments, provided are methods for selecting a
subject for therapy with a first-generation AR antagonist,
comprising (a) assaying a sample containing a nucleic acid molecule
encoding an AR polypeptide from the subject to determine whether
the encoded AR polypeptide is modified at an amino acid position
corresponding to amino acid position 876 of the amino acid sequence
set forth in SEQ ID NO: 1; and (b) characterizing the subject as a
candidate for therapy with a first-generation AR antagonist if the
subject does not have the modification. In some embodiments, the
method further comprises characterizing the subject as not a
candidate for therapy with a first-generation AR antagonist if the
subject has the modification. In some embodiments, the method
further comprises administration of a third-generation AR
antagonist provided herein for inhibition of the mutant AR. In some
embodiments, the subject has cancer. In some embodiments, the
subject has a prostate cancer. In some embodiments, the subject has
a castration resistant prostate cancer. In some embodiments, the
method further comprises a step of obtaining the nucleic acid
sample from the subject.
[0145] In some embodiments, provided are methods for selecting a
subject for therapy with an AR antagonist that is a CYP17A
inhibitor, comprising (a) assaying a sample containing a nucleic
acid molecule encoding an AR polypeptide from the subject to
determine whether the encoded AR polypeptide is modified at an
amino acid position corresponding to amino acid position 876 of the
amino acid sequence set forth in SEQ ID NO: 1; and (b)
characterizing the subject as a candidate for therapy with an AR
antagonist that is a CYP17A inhibitor if the subject does not have
the modification. In some embodiments, the method further comprises
characterizing the subject as not a candidate for therapy with a
CYP17A inhibitor if the subject has the modification. In some
embodiments, the method further comprises administration of a
third-generation AR antagonist provided herein for inhibition of
the mutant AR. In some embodiments, the subject has cancer. In some
embodiments, the subject has a prostate cancer. In some
embodiments, the subject has a castration resistant prostate
cancer. In some embodiments the CYP17A inhibitor is galeterone
(TOK001), TAK-700 or abiraterone acetate. In some embodiments, the
method further comprises a step of obtaining the nucleic acid
sample from the subject.
[0146] In some embodiments, provided are methods for selecting a
subject for therapy with a third-generation AR antagonist,
comprising (a) assaying a sample containing a nucleic acid molecule
encoding an AR polypeptide from the subject to determine whether
the encoded AR polypeptide is modified at an amino acid position
corresponding to amino acid position 876 of the amino acid sequence
set forth in SEQ ID NO: 1; and (b) characterizing the subject as a
candidate for therapy with a third-generation AR antagonist if the
subject has the modification. In some embodiments, the method
further comprises administration of a third-generation AR
antagonist provided herein for inhibition of the mutant AR. In some
embodiments, the subject has cancer. In some embodiments, the
subject has a prostate cancer. In some embodiments, the subject has
a castration resistant prostate cancer. In some embodiments, the
method further comprises a step of obtaining the nucleic acid
sample from the subject.
[0147] In some embodiments, provided are methods for determining
whether a subject is or is likely to become resistant to therapy
with a second-generation AR antagonist, comprising: (a) assaying a
sample containing a nucleic acid molecule encoding an AR
polypeptide from the subject to determine whether the encoded AR
polypeptide is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1; and (b) characterizing the subject as resistant or is
likely to become resistant to therapy with a second-generation AR
antagonist if the subject has the modification. In some
embodiments, the method further comprises administration of a
third-generation AR inhibitor provided herein for inhibition of the
mutant AR. In some embodiments, the subject has cancer. In some
embodiments, the subject has a prostate cancer. In some
embodiments, the subject has a castration resistant prostate
cancer. In some embodiments, the method further comprises a step of
obtaining the nucleic acid sample from the subject.
[0148] In some embodiments, provided are methods for determining
whether a subject is or is likely to become resistant to therapy
with a first-generation AR antagonist, comprising: (a) assaying a
sample containing a nucleic acid molecule encoding an AR
polypeptide from the subject to determine whether the encoded AR
polypeptide is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1; and (b) characterizing the subject as resistant or is
likely to become resistant to therapy with a first-generation AR
antagonist if the subject has the modification. In some
embodiments, the method further comprises administration of a
third-generation AR inhibitor provided herein for inhibition of the
mutant AR. In some embodiments, the subject has cancer. In some
embodiments, the subject has a prostate cancer. In some
embodiments, the subject has a castration resistant prostate
cancer. In some embodiments, the method further comprises a step of
obtaining the nucleic acid sample from the subject.
[0149] In some embodiments, provided are methods for determining
whether a subject is or is likely to become resistant to therapy
with an AR antagonist that is a CYP17A inhibitor, comprising: (a)
assaying a sample containing a nucleic acid molecule encoding an AR
polypeptide from the subject to determine whether the encoded AR
polypeptide is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1 and (b) characterizing the subject as resistant or is
likely to become resistant to therapy with an AR antagonist that is
a CYP17A inhibitor if the subject has the modification. In some
embodiments, the method further comprises administration of a
third-generation AR inhibitor provided herein for inhibition of the
mutant AR. In some embodiments, the subject has cancer. In some
embodiments, the subject has a prostate cancer. In some
embodiments, the subject has a castration resistant prostate
cancer. In some embodiments the CYP17A inhibitor is galeterone
(TOK001), TAK-700 or abiraterone acetate. In some embodiments, the
method further comprises a step of obtaining the nucleic acid
sample from the subject.
[0150] In some embodiments, provided are methods for monitoring
whether a subject receiving a second-generation AR antagonist for
treatment of a cancer has developed or will develop resistance to
the therapy, comprising (a) assaying a sample containing a nucleic
acid molecule encoding an AR polypeptide from the subject to
determine whether the encoded AR polypeptide is modified at an
amino acid position corresponding to amino acid position 876 of the
amino acid sequence set forth in SEQ ID NO: 1; and (b)
characterizing the subject as resistant or will become resistant to
therapy with a second-generation AR antagonist if the subject has
the modification. In some embodiments, the method further comprises
administration of a third-generation AR antagonist provided herein
for inhibition of the mutant AR. In some embodiments, the subject
has cancer. In some embodiments, the subject has a prostate cancer.
In some embodiments, the subject has a castration resistant
prostate cancer. In some embodiments, the method further comprises
a step of obtaining the nucleic acid sample from the subject.
[0151] In some embodiments, provided are methods for monitoring
whether a subject receiving a first-generation AR antagonist for
treatment of a cancer has developed or will develop resistance to
the therapy, comprising (a) assaying a sample containing a nucleic
acid molecule encoding an AR polypeptide from the subject to
determine whether the encoded AR polypeptide is modified at an
amino acid position corresponding to amino acid position 876 of the
amino acid sequence set forth in SEQ ID NO: 1; and (b)
characterizing the subject as resistant or will become resistant to
therapy with a first-generation AR antagonist if the subject has
the modification. In some embodiments, the method further comprises
administration of a third-generation AR antagonist provided herein
for inhibition of the mutant AR. In some embodiments, the subject
has cancer. In some embodiments, the subject has a prostate cancer.
In some embodiments, the subject has a castration resistant
prostate cancer. In some embodiments, the method further comprises
a step of obtaining the nucleic acid sample from the subject.
[0152] In some embodiments, provided are methods for monitoring
whether a subject receiving an AR antagonist that is a CYP17A
inhibitor for treatment of a cancer has developed or will develop
resistance to the therapy, comprising (a) assaying a sample
containing a nucleic acid molecule encoding an AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1;
and (b) characterizing the subject as resistant or will become
resistant to therapy with an AR antagonist that is a CYP17A
inhibitor if the subject has the modification. In some embodiments,
the method further comprises administration of a third-generation
AR antagonist provided herein for inhibition of the mutant AR. In
some embodiments, the subject has cancer. In some embodiments, the
subject has a prostate cancer. In some embodiments, the subject has
a castration resistant prostate cancer. In some embodiments the
CYP17A inhibitor is galeterone (TOK001), TAK-700 or abiraterone
acetate. In some embodiments, the method further comprises a step
of obtaining the nucleic acid sample from the subject.
[0153] In some embodiments, provided are methods for optimizing the
therapy of a subject receiving a second-generation AR antagonist
for treatment of a cancer, comprising: (a) assaying a sample
containing a nucleic acid molecule encoding an AR polypeptide from
the subject to determine whether the encoded AR polypeptide is
modified at an amino acid position corresponding to amino acid
position 876 of the amino acid sequence set forth in SEQ ID NO: 1;
and (b) discontinuing treatment with the second-generation AR
antagonist if the subject has the modification or continuing
treatment with the second-generation AR antagonist if the subject
does not have the modification. In some embodiments, the method
further comprises a step of obtaining the nucleic acid sample from
the subject.
[0154] In some embodiments, provided are methods for optimizing the
therapy of a subject receiving a first-generation AR antagonist for
treatment of a cancer, comprising: (a) assaying a sample containing
a nucleic acid molecule encoding an AR polypeptide from the subject
to determine whether the encoded AR polypeptide is modified at an
amino acid position corresponding to amino acid position 876 of the
amino acid sequence set forth in SEQ ID NO: 1; and (b)
discontinuing treatment with the first-generation AR antagonist if
the subject has the modification or continuing treatment with the
first-generation AR antagonist if the subject does not have the
modification. In some embodiments, the method further comprises a
step of obtaining the nucleic acid sample from the subject.
[0155] In some embodiments, provided are methods for optimizing the
therapy of a subject receiving an AR antagonist that is a CYP17A
inhibitor for treatment of a cancer, comprising: (a) assaying a
sample containing a nucleic acid molecule encoding an AR
polypeptide from the subject to determine whether the encoded AR
polypeptide is modified at an amino acid position corresponding to
amino acid position 876 of the amino acid sequence set forth in SEQ
ID NO: 1; and (b) discontinuing treatment with the AR antagonist
that is a CYP17A inhibitor if the subject has the modification or
continuing treatment with the AR antagonist that is a CYP17A
inhibitor if the subject does not have the modification. In some
embodiments the CYP17A inhibitor is galeterone (TOK001), TAK-700 or
abiraterone acetate. In some embodiments, the method further
comprises a step of obtaining the nucleic acid sample from the
subject.
[0156] In some embodiments, the modified AR is resistant to full
antagonism by a second-generation AR antagonist, such as, for
example, ARN-509, enzalutamide (MDV3100) or RD162. In some
embodiments, a second-generation AR antagonist, such as, for
example, ARN-509, enzalutamide (MDV3100) or RD162 exhibits agonist
activity toward the modified AR. In some embodiments, the modified
AR is resistant to full antagonism by a first-generation AR
antagonist. In some embodiments, the modified AR is resistant to
full antagonism by an AR antagonist that is a CYP17A.
[0157] In some embodiments, the subject has an AR dependent or AR
mediated disease or condition. In some embodiments, the AR
dependent or AR mediated disease or condition is benign prostate
hyperplasia, hirsutism, acne, adenomas and neoplasms of the
prostate, benign or malignant tumor cells containing the AR,
hyperpilosity, seborrhea, endometriosis, polycystic ovary syndrome,
androgenic alopecia, hypogonadism, osteoporosis, suppression of
spermatogenesis, libido, cachexia, anorexia, androgen
supplementation for age related decreased testosterone levels,
prostate cancer, breast cancer, bladder cancer, liver cancer,
endometrial cancer, uterine cancer, hot flashes, Kennedy's disease,
muscle atrophy and weakness, skin atrophy, bone loss, anemia,
arteriosclerosis, cardiovascular disease, loss of energy, loss of
well-being, type 2 diabetes or abdominal fat accumulation.
[0158] In some embodiments, the subject has cancer. In some
embodiments, the subject has a solid tumor. In some embodiments,
the cancer is a prostate cancer, a breast cancer, a liver cancer,
or a bladder cancer. In some embodiments, the cancer is a prostate
cancer.
[0159] In some embodiments, the sample is from any tissue or fluid
from an organism. Samples include, but are not limited, to whole
blood, dissociated bone marrow, bone marrow aspirate, pleural
fluid, peritoneal fluid, central spinal fluid, abdominal fluid,
pancreatic fluid, cerebrospinal fluid, brain fluid, ascites,
pericardial fluid, urine, saliva, bronchial lavage, sweat, tears,
ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk,
amniotic fluid, and secretions of respiratory, intestinal or
genitourinary tract. In particular embodiments, the sample is a
tumor biopsy sample. In particular embodiments, the sample is from
a fluid or tissue that is part of, or associated with, the
lymphatic system or circulatory system. In some embodiments, the
sample is a blood sample that is a venous, arterial, peripheral,
tissue, cord blood sample. In particular embodiments, the sample is
a serum sample. In some embodiments, the sample contains one or
more circulating tumor cells (CTCs). In some embodiments, the
sample contains one or more disseminated tumor cells (DTC, e.g. in
a bone marrow aspirate sample).
[0160] Methods for the isolation of nucleic acids and proteins from
cells contained in tissue and fluid samples are well-known in the
art. In particular embodiments, the nucleic acid sample obtained
from the subject is isolated from cells contained in a tumor biopsy
from the subject. In particular embodiments, the nucleic acid
sample obtained from the subject is isolated from cells in a bone
marrow aspirate. In particular embodiments, the nucleic acid sample
obtained from the subject is isolated from cells contained serum
sample. In particular embodiments, the nucleic acid sample obtained
from the subject is isolated from cells contained lymph sample.
[0161] In some embodiments, the samples are obtained from the
subject by any suitable means of obtaining the sample using
well-known and routine clinical methods. Procedures for obtaining
fluid samples from a subject are well known. For example,
procedures for drawing and processing whole blood and lymph are
well-known and can be employed to obtain a sample for use in the
methods provided. Typically, for collection of a blood sample, an
anti-coagulation agent (e.g. EDTA, or citrate and heparin or CPD
(citrate, phosphate, dextrose) or comparable substances) is added
to the sample to prevent coagulation of the blood. In some
examples, the blood sample is collected in a collection tube that
contains an amount of EDTA to prevent coagulation of the blood
sample.
[0162] In some embodiments, the sample is a tissue biopsy and is
obtained, for example, by needle biopsy, CT-guided needle biopsy,
aspiration biopsy, endoscopic biopsy, bronchoscopic biopsy,
bronchial lavage, incisional biopsy, excisional biopsy, punch
biopsy, shave biopsy, skin biopsy, bone marrow biopsy, and the Loop
Electrosurgical Excision Procedure (LEEP). Typically, a
non-necrotic, sterile biopsy or specimen is obtained that is
greater than 100 mg, but which can be smaller, such as less than
100 mg, 50 mg or less, 10 mg or less or 5 mg or less; or larger,
such as more than 100 mg. 200 mg or more, or 500 mg or more, 1 gm
or more, 2 gm or more, 3 gm or more, 4 gm or more or 5 gm or more.
The sample size to be extracted for the assay depends on a number
of factors including, but not limited to, the number of assays to
be performed, the health of the tissue sample, the type of cancer,
and the condition of the patient. In some embodiments, the tissue
is placed in a sterile vessel, such as a sterile tube or culture
plate, and is optionally immersed in an appropriate media.
Typically, the cells are dissociated into cell suspensions by
mechanical means and/or enzymatic treatment as is well known in the
art. Typically, the cells are collected and then subjected to
standard procedures for the isolation of nucleic acid for the
assay.
[0163] In some embodiments, the samples are obtained from the
subject at regular intervals, such as, for example, one day, two
days, three days, four days, five days, six days, one week, two
weeks, weeks, four weeks, one month, two months, three months, four
months, five months, six months, one year, daily, weekly,
bimonthly, quarterly, biyearly or yearly. In some embodiments, the
collection of samples is performed at a predetermined time or at
regular intervals relative to treatment with one or more
anti-cancer agents. In some embodiments, the collection of samples
is performed at a predetermined time or at regular intervals
relative to treatment with an AR antagonist, such as a first- or
second-generation AR antagonist. For example, a sample is collected
at a predetermined time or at regular intervals prior to, during,
or following treatment or between successive treatments. In
particular examples, a sample is obtained from the subject prior to
administration of an anti-cancer therapy and then again at regular
intervals after treatment has been effected. In particular
examples, a sample is obtained from the subject prior to
administration of an AR antagonist, such as a first- or
second-generation AR antagonist, therapy and then again at regular
intervals after treatment has been effected. In some embodiments,
the AR antagonist is selected from among bicalutamide, flutamide,
hydroxyflutamide, nilutamide, ARN-509, enzalutamide (MDV3100), and
RD162. In some embodiments, the AR antagonist is a CYP17A
inhibitor. In some embodiments, the CYP17A inhibitor is selected
from among galeterone (TOK001), TAK-700 or abiraterone acetate.
[0164] The volume of a fluid sample can be any volume that is
suitable for the detection of an AR mutant in the methods provided.
In some examples, the volume for the fluid sample is dependent on
the particular assay method used. For example, particular assay
methods can require a larger or smaller fluid sample volumes
depending on factors such as, but not limited to, the capacity of
the device or method used and level of throughput of the assay
method. In some examples a fluid sample is diluted in an
appropriate medium prior to application of the assay method. In
some examples, a fluid sample is obtained from a subject and a
portion or aliquot of the sample is used in the assay method. The
portion or aliquot can be diluted in an appropriate medium prior to
application of the assay method.
[0165] In some embodiments, the sample is obtained from a subject
that is mammal. Exemplary mammalian subjects include, but are not
limited to primates, such as humans, apes and monkeys; rodents,
such as mice, rats, rabbits, and ferrets; ruminants, such as goats,
cows, deer, and sheep; horses, pigs, dogs, cats, and other animals.
In some embodiments, the sample is obtained from a patient. In some
examples, the patient is a human patient.
[0166] In some embodiments, the nucleic acid sample obtained from
the subject is a genomic nucleic acid sample. In some embodiments,
the nucleic acid sample obtained from the subject is an RNA sample.
In some embodiments, mRNA is isolated from the total RNA in an RNA
sample. In some embodiments, the RNA sample is reverse transcribed
into cDNA. In some embodiments, the genomic nucleic acid sample is
amplified by a nucleic acid amplification method. In some
embodiments, the nucleic acid amplification method is polymerase
chain reaction (PCR). In some embodiments, the genomic nucleic acid
sample is amplified using a set of nucleotide primers specific for
the AR gene. In some embodiments, the set of nucleotide primers
flank the nucleic acid sequence encoding amino acid position 876 of
the AR polypeptide. In some embodiments, the amplification product
is a nucleic acid encoding amino acid position 876 of the AR
polypeptide. In some embodiments, a sequence specific primer is
conjugated to a detectable molecule, such as a fluorescent label, a
bioluminescent label, a chemiluminescent label, a radiolabel, an
enzyme label, a detectable substrate, or a peptide or molecule that
binds to a second detectable molecule.
[0167] In some embodiments, assaying comprises sequencing the
nucleic acid sample. In some embodiments, the nucleic acid encoding
AR in a nucleic acid sample is first amplified by a method such as
polymerase chain reaction (PCR) using sequence specific primers,
and the amplified PCR fragment is then sequenced. Exemplary
sequencing methods for use in the methods provide herein are well
known in the art and include, but are not limited to, dideoxy or
chain termination methods, Maxam-Gilbert sequencing, massively
parallel signature sequencing (or MPSS), polony sequencing,
pyrosequencing, Illumina dye sequencing, SOLiD (or sequencing by
ligation) sequencing, ion semiconductor sequencing, DNA nanoball
sequencing, heliscope sequencing, and single molecule real time
(SMRT) sequencing.
[0168] In some embodiments, the samples is a plasma or serum sample
containing circulating tumor DNA (ctDNA), RNA (ctRNA) or microRNA
(see e.g. Chan et al. (2007) Br J Cancer. 96(5):681-5). In some
embodiments, the DNA encoding the mutant AR is assessed by BEAMing
(beads, amplification, emulsion, magnetic) PCR sequencing method
(see, e.g. Li et al. (2006) Nat Methods. 3(2):95-7; Li et al.
(2006) Nat Methods. 3(7):551-9; and Diehl et al. (2008) Nat Med.
14(9): 985-990). BEAMing is a technique in which individual DNA
molecules are attached to magnetic beads in water-in-oil emulsions
and then subjected to compartmentalized PCR amplification. The
mutational status of DNA bound to beads is then determined by
hybridization to fluorescent allele-specific probes for mutant or
wild-type AR. Flow cytometry is then used to quantify, the level of
mutant DNA present in the plasma or serum (see e.g. Higgins et al.
(2012) Clin Cancer Res 18: 3462-3469).
[0169] In some embodiments, assaying a sample for detecting the
presence of DNA encoding the mutant AR comprises detection of the
mutation with a sequence specific oligonucleotide probe that is
specific for nucleic acid that encodes the mutant AR but not the
wild-type AR. In some embodiments, assaying comprises (a)
contacting a sample with a mutant AR nucleic acid sequence specific
oligonucleotide probe, whereby if the mutant nucleic acid sequence
is present in the sample, a probe-DNA complex is formed, and (b)
detecting the probe-DNA complex. In some embodiments, the
oligonucleotide probe is specific for nucleic acid encoding leucine
at a position corresponding to amino acid 876 of an AR polypeptide.
In some embodiments, the sequence specific probe is conjugated to a
detectable molecule, such as a fluorescent label, a bioluminescent
label, a chemiluminescent label, a radiolabel, an enzyme label, a
detectable substrate, or a peptide or molecule that binds to a
second detectable molecule.
[0170] In some embodiments, single nucleotide changes are
detectable by PCR using PCR-based cleaved amplified polymorphic
sequences (CAPS) markers which create restriction sites in the
mutant sequences (Michaels et al (1998) Plant J. 14(3):381-5) or
sequence specific hairpin probes attached to detectable moieties,
such as, but not limited to, a fluorophore (Mhlanga and Malmberg
(2001) Methods 25:463-471). In some embodiments, the sequence
specific probe is conjugated to a detectable molecule, such as a
fluorescent label, a bioluminescent label, a chemiluminescent
label, a radiolabel, an enzyme label, a detectable substrate, or a
peptide or molecule that binds to a second detectable molecule. In
some embodiments, the oligonucleotide probe is specific for nucleic
acid encoding leucine at a position corresponding to amino acid 876
of an AR polypeptide.
[0171] In some embodiments, assaying a sample for detecting the
presence of DNA encoding the mutant AR is performed using an
oligonucleotide array (see e.g. Hastia et al. (1999) J Med Genet.
36(10):730-6). In some embodiments, the sample containing nucleic
acid from the subject is hybridized directly to the chip. In some
embodiments, the sample containing nucleic acid from the subject is
amplified using an amplification method, such as, but not limited
to polymerase chain reaction (PCR), and the amplified nucleic acid
is hybridized to the chip. In some embodiments, the oligonucleotide
array is contained on a microchip. In some embodiments, single
nucleotide changes are detectable using microchips.
[0172] In some embodiments, assaying a sample comprises detection
of the mutation with an antibody specific for the mutant AR
polypeptide. In some embodiments, the method of detecting a mutant
AR polypeptide comprises obtaining a sample from a subject, wherein
the sample comprises an AR polypeptide and testing the sample for
the presence of a mutant AR polypeptide by contacting the sample
with an antibody that is specific for binding to the mutant AR
polypeptide, and does not bind or bind with decreased affinity for
the wild-type AR polypeptide, wherein the presence of the mutant AR
polypeptide creates an antibody-mutant AR polypeptide complex. In
some embodiments, the method further comprises detecting the
antibody-mutant AR polypeptide complex. In some embodiments, the
method further comprises detecting the antibody-mutant AR
polypeptide complex with a detection reagent. In some embodiments,
the mutant AR specific antibody is conjugated to a detectable
molecule, such as a fluorescent label, a bioluminescent label, a
chemiluminescent label, a radiolabel, an enzyme label, a detectable
substrate, or a peptide or molecule that binds to a second
detectable protein (e.g. a secondary antibody). In some
embodiments, binding of the mutant AR specific antibody is detected
by assaying for the detectable molecule. In some embodiments,
binding of the mutant AR specific antibody is detected by using a
secondary (e.g. anti-IgG) antibody. In some embodiments, the sample
is a tumor biopsy sample, a bone marrow aspirate, a blood sample, a
serum sample, or a lymph sample.
Identification of Molecules that Interact with Mutant Androgen
Receptor
[0173] Provided herein are methods of using the mutant AR
polypeptides for screening of compounds that inhibit the mutant
receptor (i.e. third-generation AR inhibitor compounds). In some
embodiments, the methods are employed for the identification of
third-generation AR inhibitor compounds for the treatment of
cancer. In some embodiments, the methods are employed for the
identification of third-generation AR inhibitor compounds for the
treatment of resistant cancers, such as prostate cancer resistant
to treatment with second-generation AR antagonists, such as
ARN-509, enzalutamide (MDV3100) or RD162.
[0174] In some embodiments, a method for identifying
third-generation AR inhibitor compounds comprises (a) expressing a
mutant AR polypeptide provided herein in a cell, (b) contacting the
cell with a test compound, and (c) detecting the level of AR
activity in the cell. In some embodiments, the cell is contacted
with an AR agonist prior to or at the same time as contacting the
cell with the test compound. In some embodiments the cell is
contacted with an AR agonist about 1 hour, 2, hours, 3 hours, 4
hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16
hours, 18 hours, 20 hours, 22 hours, 24 hours or longer prior to
contacting the cell with the test compound. In some embodiments the
cell is contacted with an AR agonist at the same time as the cell
is contacted with test compound. In some embodiments, the AR
agonist is selected from among methyltrienolone (R1881), DHT,
mibolerone (Mb) and testosterone. In some embodiments, the mutant
AR polypeptide comprises an amino acid substitution at a position
corresponding to amino acid position 876 of the wild-type AR
polypeptide set forth in SEQ ID NO: 1. In some embodiments, the
mutant AR polypeptide does not contain a phenylalanine at amino
acid position 876 in the polypeptide. In some embodiments, the
mutant AR polypeptide contains a leucine at amino acid position 876
in the polypeptide.
[0175] In some embodiments, a cell line that can be transfected
with nucleic acid encoding the mutant AR polypeptide and in which
AR activity can be monitored is used. In some embodiments, the cell
does not express the wild-type AR. In some embodiments, the cell
expresses a low level of wild-type AR. In some embodiments, the
cell expresses an endogenous mutant AR polypeptide. In some
embodiments, the endogenous mutant AR polypeptide comprises a
modification at an amino acid corresponding to amino acid 877 of a
wild-type AR polypeptide. In some embodiments, the endogenous
mutant AR polypeptide comprises a modification that is a
substitution of Threonine to Alanine at amino acid position 877
(T877A). In some embodiments, the mutant AR polypeptide comprises a
modification at an amino acid corresponding to amino acid 874 of a
wild-type AR polypeptide. In some embodiments, the mutant AR
polypeptide comprises a modification that is a substitution of
Histidine to Tyrosine at amino acid position 874 (H874Y). In some
embodiments, the cell is a selected from among HeLa, CV1, COS7,
HepG2, HEK-293, DU145, PC3, and TSY-PR1. In some embodiments, the
cell line is a prostate cancer cell line. In some embodiments, the
cell line is selected from among CWR, LNCaP, VCaP and LAPC4.
[0176] In some embodiments, the cell stably expresses the mutant AR
polypeptide. In some embodiments, the nucleic acid encoding the
mutant AR is integrated into the genome of the cell. In some
embodiments, the level of AR activity is detected using a reporter
gene operably linked to an AR-responsive promoter. In some
embodiments, the AR-responsive promoter comprises one or more
androgen response elements (AREs) to which the mutant AR
polypeptide binds. In some embodiments, the promoter is selected
from among a probasin (Pb), a prostate specific antigen (PSA),
mouse mammary tumor virus long terminal repeat (MMTV LTR), fatty
acid synthase (FASN), six transmembrane epithelial antigen of the
prostate 4 (STEAP4), transmembrane protease, serine 2 (TMPRSS2),
alpha-1-acid glycoprotein 1 (ORM1), or human homeobox gene NKX3.1
promoter. In some embodiments, the promoter is a synthetic promoter
containing one or more AREs.
[0177] In some embodiments, the AR-responsive promoter is operably
linked to a suitable reporter gene that encodes a detectable
protein. Exemplary detectable proteins include, but are not limited
to, luciferase, fluorescent proteins, bioluminescent proteins,
.beta.-galactosidase, alkaline phosphatase, and chloramphenicol
acetyltransferase. In some embodiments, a decrease in the
expression of the reporter gene following exposure to the test
compound compared to a suitable control indicates that the test
compound is effective for inhibition of the mutant AR polypeptide.
In some embodiments, the control is basal expression of the
reporter gene prior to exposure of the cell to the test compound.
In some embodiments the expression of the reporter gene is assayed
using a cell extract prepared from the test cells.
[0178] In some embodiments, the level of AR activity is detected by
measuring the expression of one or more endogenous androgen
responsive genes in a cell. In some embodiments, the androgen
responsive gene is upregulated (i.e. induced) in response to
androgen treatment. In some embodiments, the androgen responsive
gene is downregulated (i.e. repressed) in response to androgen
treatment. Exemplary androgen responsive genes include, but are not
limited to, prostate specific antigen (PSA), prostate specific
membrane antigen (PSMA), prostasin, snail homolog 2 (SLUG),
transmembrane protease, serine 2 (TMPRSS2), six transmembrane
epithelial antigen of prostate family member 4 (STEAP4), FK506
binding protein 5 (FKBP5), orosomucoid 1/alpha-1-acid glycoprotein
1 (ORM1), solute carrier family 35 (SLC35F2/NOV), insulin-like
growth factor I (IGF-1) IGF binding protein-3 and -5,
CCAAT-enhancer binding protein-.delta., phosphatase and tensin
homolog deleted on chromosome 10 (PTEN), FASN, NKX3.1, AMIGO2,
BDNF, CAMK2N1, HPGD, NCAPD3, PLD1, IL-15, IL-18, and
ERBB2/HER2.
[0179] In some embodiments, the level of AR activity is detected by
measuring the expression of one or more endogenous genes that are
induced by exposure to an androgen or an AR agonist. In some
embodiments, a decrease in the expression of one or more androgen
inducible genes following exposure to the test compound compared to
a suitable control indicates that the test compound is effective
for inhibition of the mutant AR polypeptide. Exemplary androgen
inducible genes include but are not limited to, prostate specific
antigen (PSA), prostasin, snail homolog 2 (SLUG), transmembrane
protease, serine 2 (TMPRSS2), six transmembrane epithelial antigen
of prostate family member 4 (STEAP4). FK506 binding protein 5
(FKBP5), and orosomucoid 1/alpha-1-acid glycoprotein 1 (ORM1). In
some embodiments, expression of the inducible gene is assessed in
the presence of an AR agonist. In some embodiments, the cells are
contacted with an androgen agonist prior to or simultaneously with
the test compound.
[0180] In some embodiments, the level of AR activity is detected by
measuring the expression of one or more endogenous genes that are
repressed by exposure to an androgen or an AR agonist. In some
embodiments, an increase in or failure to repress the expression of
one or more androgen repressed genes following exposure to the test
compound compared to a suitable control indicates that the test
compound is effective for inhibition of the mutant AR polypeptide.
In some embodiments, the control is basal expression of the gene
prior to exposure of the cell to the test compound. Exemplary
androgen repressed genes include, but are not limited to, prostate
specific membrane antigen (PSMA), solute carrier family 35
(SLC35F2/NOV), IGF binding protein-3 and -5, CCAAT-enhancer binding
protein-6, phosphatase and tensin homolog deleted on chromosome 10
(PTEN). IL-15, IL-18, and ERBB2/HER2. In some embodiments,
expression of the repressible gene is assessed in the presence of
an AR agonist. In some embodiments, the cells are contacted with an
androgen agonist prior to or simultaneously with the test
compound.
[0181] Methods to measure the expression of endogenous genes is
well-known in the art. Exemplary methods for the measurement of
gene expression include, but are not limited to protein analytical
methods such as, for example, immunohistochemistry, immunoblotting
(e.g. Western analysis), chromatography, and nucleic acids
analytical methods, such as, for example, polymerase chain reaction
(PCR), quantitative PCR (qPCR), real time PCR (RT-PCR), Northern
analysis.
[0182] In some embodiments, the activity of a mutant AR polypeptide
is measured using an assay such as, but not limited to, a AR
coactivator binding assay (e.g. immunoprecipitation assays,
two-hybrid assays, Forster (fluorescence) resonance energy transfer
assays, for example, LanthaScreen.TM. TR-FRET androgen receptor
coactivator assay), a AR conformational profiling assay (see, e.g.,
Joseph et al. (2009) PNAS 106(29): 12178-12183), an AR DNA binding
assay (see, e.g., Roche et al. (1992) Mol. Endocrinol.
6(12):2229-35), chromatin immunoprecipitation, N/C terminal
interaction assay (see, e.g., Hsu et al. (2005) Mol. Endocrinology
19(2) 350-361 and Ghali et al. (2003) J Clin Endocrinol Metab.
88(5):2185-93).
[0183] In some embodiments, a method for identifying
third-generation AR inhibitor compounds comprises selecting a
potential third-generation AR inhibitor compound using
computer-assisted modeling with a three-dimensional crystal or
solution structure of a mutant AR polypeptide provided herein. In
some embodiments, the mutant AR polypeptide comprises an amino acid
substitution at a position corresponding to amino acid position 876
of the wild-type AR polypeptide set forth in SEQ ID NO: 1. In some
embodiments, the mutant AR polypeptide does not contain a
phenylalanine at amino acid position 876 in the polypeptide. In
some embodiments, the mutant AR polypeptide contains a leucine at
amino acid position 876 in the polypeptide. In some embodiments,
the method comprises contacting the mutant AR polypeptide with the
test compound and detecting the interaction of the test compound
with the mutant AR polypeptide. In some embodiments, a test
compound that interacts with the mutant AR polypeptide is
identified as a candidate third-generation AR inhibitor
compound.
[0184] In some embodiments, a test compound for use in the methods
provided is a member of a library of compounds. In some
embodiments, generation of a library of test compounds is by any
suitable method for the production of chemical compounds. A
"library of test compounds" refers to a panel comprising a
multiplicity of test compounds. An exemplary approach for the
synthesis of molecular libraries of small organic molecules has
been described (Carell et al. (1994). Angew. Chem. Int. Ed. Engl.
33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061).
In some embodiments the test compounds provided herein are obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
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 approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145). Other exemplary methods for the synthesis of
molecular libraries are found in the art, for example in: Erb et
al. (1994). Proc. Natl. Acad. Sci. USA 91:11422; Horwell et al.
(1996) Immunopharmacology 33:68-; and in Gallop et al. (1994): J.
Med. Chem. 37:1233-. In some embodiments, libraries of compounds
are 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 USP '409), plasmids (Cull et al. (1992)
Proc Natl 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). In some embodiments,
combinatorial polypeptides are produced from a cDNA library.
Exemplary compounds that can be screened for activity include, but
are not limited to, peptides, nucleic acids, carbohydrates, small
organic molecules, and natural product extract libraries.
AR Inhibitors Identified by the Screening Methods
[0185] The third-generation AR inhibitors identified using the
screening methods provided herein are AR modulators. In some
embodiments, the third-generation AR inhibitor inhibits or reduces
at least one activity of an AR polypeptide. Exemplary AR activities
include, but are not limited to, co-activator binding, DNA binding,
ligand binding, or nuclear translocation. In some embodiments, the
third-generation AR inhibitor inhibits an activity of an AR
polypeptide by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,
90%, 95%, 98% or 100% compared to the activity of the AR
polypeptide in the absence of the inhibitor. In some embodiments,
the third-generation AR inhibitor compounds identified using the
methods provided herein are AR inverse agonists. AR antagonists. AR
degraders, AR trafficking modulators and/or AR DNA-binding
inhibitors. In some embodiments, a third-generation AR inhibitor
compounds identified using the methods provided herein is an AR
inverse agonist. In some embodiments, the third-generation AR
inhibitor compounds identified using the methods provided herein
are AR antagonists. In some embodiments, the third-generation AR
inhibitor compounds identified using the methods provided herein
are AR degraders. In some embodiments, the third-generation AR
inhibitor compounds identified using the methods provided herein
are AR trafficking modulators. In some embodiments, the
third-generation AR inhibitor compounds identified using the
methods provided herein are AR DNA-binding inhibitors. In some
embodiments, the AR inhibitor inhibits at least one activity of a
wild-type AR polypeptide. In some embodiments, the AR inhibitor
inhibits at least one activity of a mutant AR polypeptide. In some
embodiments, the third-generation AR inhibitor compound identified
using the methods provided herein has minimal pro-convulsant
activity and/or minimal impact on seizure threshold. In some
embodiments, the third-generation AR inhibitor compound identified
using the methods provided herein displays minimal modulation of
the GABA-gated chloride channel. In some embodiments, the
third-generation AR inhibitor compound identified using the methods
provided herein displays minimal binding to the GABA-gated chloride
channel. In some embodiments, the third-generation AR inhibitor
compound identified using the methods provided herein has minimal
antagonism of the GABA-gated chloride channel. In some embodiments,
the third-generation AR inhibitor compound identified using the
methods provided herein is an AR modulator with minimal interaction
with a GABA-gated chloride channel. In some embodiments, the
third-generation AR inhibitor compound identified using the methods
provided herein is an AR modulator with minimal interaction with
the GABA.sub.A-gated chloride channel. In some embodiments, the
third-generation AR inhibitor compound identified using the methods
provided herein is an AR modulator with minimal interaction with
the GABA.sub.A-gated chloride channel and or minimal blood-brain
barrier penetration. GABA assays are known and include, but are not
limited to, those described in Ashok K. Mehta and Maharaj K. Ticku
"Characterization of the Picrotoxin Site of GABA.sub.A Receptors"
Current Protocols in Pharmacology (2000) 1.18.1-1.18.17; Copyright
.COPYRGT. 2000 by John Wiley & Sons, Inc., which is herein
incorporated by reference.
[0186] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein inhibits AR
nuclear translocation. In some embodiments, a third-generation AR
inhibitor compound identified using the methods provided herein
inhibits AR DNA binding to androgen response elements. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein inhibits coactivator recruitment
at an AR responsive promoter. In some embodiments, a
third-generation AR inhibitor compound identified using the methods
provided herein exhibits no agonist activity in AR-overexpressing
prostate cancer cells.
[0187] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein inhibits
growth of castration sensitive and castration resistant prostate
cancer xenograft models. In some embodiments, a third-generation AR
inhibitor compound identified using the methods provided herein
inhibits growth of castration sensitive and castration resistant
prostate cancer xenograft models expressing wild-type AR. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein inhibits growth of castration
sensitive and castration resistant prostate cancer xenograft models
expressing a mutant AR having a F876L mutation. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein inhibits growth of castration
sensitive and castration resistant prostate cancer xenograft models
expressing the wild-type AR and a mutant AR having a F876L
mutation. In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein inhibits
growth of castration sensitive and castration resistant prostate
cancer xenograft models expressing a mutant AR having a T877A
mutation (e.g. xenograft tumors formed from LNCaP cells). In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein inhibits growth of castration
sensitive and castration resistant prostate cancer xenograft models
expressing the wild-type AR and a mutant AR having a T877A mutation
(e.g. xenograft tumors formed from LNCaP/AR cells). In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein inhibits growth of castration
sensitive and castration resistant prostate cancer xenograft models
expressing a mutant AR having a T877A mutation and a mutant AR
having a F876L mutation.
Pharmaceutical Compositions
[0188] In some embodiments, provided is a pharmaceutical
composition comprising a therapeutically effective amount of a
third-generation AR inhibitor identified using the screening
methods provided herein. In some embodiments, provided is a use of
a third-generation AR inhibitor identified using the screening
methods provided herein for the preparation of a medicament.
[0189] In some embodiments, the pharmaceutical composition
comprising a third-generation AR inhibitor also contains at least
one pharmaceutically acceptable inactive ingredient. In some
embodiments, the pharmaceutical composition comprising a
third-generation AR inhibitor is formulated for intravenous
injection, subcutaneous injection, oral administration, or topical
administration. In some embodiments, the pharmaceutical composition
comprising a third-generation AR inhibitor is a tablet, a pill, a
capsule, a liquid, a suspension, a gel, a colloid, a dispersion, a
suspension, a solution, an emulsion, an ointment, or a lotion.
[0190] Pharmaceutical compositions are formulated in a conventional
manner using one or more pharmaceutically acceptable inactive
ingredients that facilitate processing of the active compounds into
preparations that can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen. A summary of
pharmaceutical compositions described herein can be found, for
example, in Remington: The Science and Practice of Pharmacy,
Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover,
John E., Remington's Pharmaceutical Sciences. Mack Publishing Co.,
Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,
Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;
and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh
Ed. (Lippincott Williams & Wilkins 1999), herein incorporated
by reference for such disclosure.
[0191] Provided herein are pharmaceutical compositions a
third-generation AR inhibitor, or a pharmaceutically acceptable
salt thereof, and at least one pharmaceutically acceptable inactive
ingredient. In some embodiments, the third-generation AR inhibitor
described herein is administered as pharmaceutical compositions in
which the third-generation AR inhibitor is mixed with other active
ingredients, as in combination therapy. In other embodiments, the
pharmaceutical compositions include other medicinal or
pharmaceutical agents, carriers, adjuvants, preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure, and/or buffers. In yet
other embodiments, the pharmaceutical compositions include other
therapeutically valuable substances.
[0192] A pharmaceutical composition, as used herein, refers to a
mixture of a the third-generation AR inhibitor or a
pharmaceutically acceptable salt thereof, with other chemical
components (i.e. pharmaceutically acceptable inactive ingredients),
such as carriers, excipients, binders, filling agents, suspending
agents, flavoring agents, sweetening agents, disintegrating agents,
dispersing agents, surfactants, lubricants, colorants, diluents,
solubilizers, moistening agents, plasticizers, stabilizers,
penetration enhancers, wetting agents, anti-foaming agents,
antioxidants, preservatives, or one or more combination thereof.
The pharmaceutical composition facilitates administration of the
compound to a subject, such as a mammal.
[0193] A therapeutically effective amount can vary widely depending
on the severity of the disease, the age and relative health of the
subject, the potency of the compound used and other factors. The
compounds can be used singly or in combination with one or more
therapeutic agents as components of mixtures.
[0194] The pharmaceutical formulations described herein are
administered to a subject by appropriate administration routes,
including but not limited to, oral, parenteral (e.g., intravenous,
subcutaneous, intramuscular), intranasal, buccal, topical, rectal,
or transdermal administration routes. The pharmaceutical
formulations described herein include, but are not limited to,
aqueous liquid dispersions, self-emulsifying dispersions, solid
solutions, liposomal dispersions, aerosols, solid dosage forms,
powders, immediate release formulations, controlled release
formulations, fast melt formulations, tablets, capsules, pills,
delayed release formulations, extended release formulations,
pulsatile release formulations, multiparticulate formulations, and
mixed immediate and controlled release formulations.
[0195] In some embodiments, the pharmaceutical composition further
comprises one or more additional therapeutically active agents,
including, but not limited to, corticosteroids, anti-emetic agents,
analgesics, anti-cancer agents, anti-inflammatory agents, kinase
inhibitors, HSP90 inhibitors, and histone deacetylase (HDAC)
inhibitors.
[0196] In some embodiments, provided is a pharmaceutical
composition comprising a therapeutically effective amount of a
mutant AR polypeptide provided herein. In some embodiments,
provided is a pharmaceutical composition comprising a
therapeutically effective amount of a nucleic acid encoding a
mutant AR polypeptide provided herein.
Therapeutic Methods
[0197] In some embodiments, the third-generation AR inhibitor
compounds identified using the methods provided herein are
administered for the treatment of a disease or condition. In some
embodiments, described herein are methods of treating an AR
dependent or AR mediated disease or condition in mammal comprising
administering to the mammal a therapeutically effective amount of a
third-generation AR inhibitor compound identified using the methods
provided herein, or a pharmaceutically acceptable salt thereof.
[0198] In some embodiments, provided are methods comprising
administering a third-generation AR inhibitor compound identified
using the methods provided herein to a subject (e.g. a human)
having a disease or condition that is AR meditated or AR dependent.
In some embodiments, provided is a use of a third-generation AR
inhibitor compound identified using the methods provided herein for
the preparation of medicament for the treatment of a disease or
condition that is AR meditated or AR dependent. In some
embodiments, provided is a third-generation AR inhibitor compound
identified using the methods provided herein for the treatment of a
disease or condition that is AR meditated or AR dependent. In some
embodiments, the subject (e.g. a human) is currently receiving one
or more additional therapeutically active agents other than the
third-generation AR inhibitor compound.
[0199] In some embodiments, the method further comprises
administering one or more additional therapeutically active agents
other than a third-generation AR inhibitor compound identified
using the methods provided herein. In some embodiments, the one or
more additional therapeutically active agents other than a
third-generation AR inhibitor compound identified using the methods
provided herein are selected from: hormones, hormone receptor
agonists or antagonists, corticosteroids, anti-emetic agents,
analgesics, anti-cancer agents, anti-inflammatory agents, kinase
inhibitors, HSP90 inhibitors, histone deacetylase (HDAC)
inhibitors. In some embodiments, third-generation AR inhibitor
compound is administered prior to, simultaneously, following, or
intermittently with the one or more additional therapeutically
active agents other than the third-generation AR inhibitor
compound.
[0200] In some embodiments, the subject is administered a
gonadotropin-releasing hormone (GnRH) agonist or antagonist in
combination with third-generation AR inhibitor compound provided
herein. In some embodiments, a GnRH receptor agonist such as
leuprolide, bruserelin and goserelin is administered to a subject
in combination with third-generation AR inhibitor compound provided
herein. GnRH receptor agonists cause an initial surge in hormone
production (i.e. "clinical flare") followed by the inhibition of
lutenizing hormone production, which in turn causes a suppression
of testosterone and dihydrotestosterone, on which continued growth
of prostate cancer cells depend. In some embodiments, the subject
is administered a gonadotropin-releasing hormone (GnRH) agonist or
antagonist in combination with third-generation AR inhibitor
compound provided herein for the treatment of an AR dependent or AR
mediated disease or condition such as a prostate, breast, bladder
or hepatocellular cancer. In some embodiments, the subject is
administered a gonadotropin-releasing hormone (GnRH) agonist or
antagonist in combination with third-generation AR inhibitor
compound provided herein for the treatment of a castration
resistant prostate cancer (CRPC). In some embodiments, the subject
is administered a third-generation AR inhibitor compound provided
herein to reduce or inhibit the initial surge in hormone production
caused by treatment with a GnRH receptor agonist. In some
embodiments, third-generation AR inhibitor compound is administered
prior to, simultaneously, following, or intermittently with a GnRH
receptor agonist or antagonist.
[0201] In some embodiments, the AR dependent or AR mediated disease
or condition is benign prostate hyperplasia, hirsutism, acne,
adenomas and neoplasms of the prostate, benign or malignant tumor
cells containing the androgen receptor, hyperpilosity, seborrhea,
endometriosis, polycystic ovary syndrome, androgenic alopecia,
hypogonadism, osteoporosis, suppression of spermatogenesis, libido,
cachexia, anorexia, androgen supplementation for age related
decreased testosterone levels, prostate cancer, breast cancer,
endometrial cancer, uterine cancer, bladder cancer, hepatocellular
cancer, hot flashes, and Kennedy's disease, muscle atrophy and
weakness, skin atrophy, bone loss, anemia, arteriosclerosis,
cardiovascular disease, loss of energy, loss of well-being, type 2
diabetes or abdominal fat accumulation. In some embodiments, the AR
dependent or AR mediated disease or condition is an AR dependent or
AR mediated cancer, such as, for example, a prostate, breast,
bladder or liver (i.e. hepatocellular) cancer.
[0202] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof. In some embodiments,
the cancer is a hormone dependent cancer. In some embodiments, the
hormone dependent cancer is an AR dependent cancer. In some
embodiments, the cancer is prostate cancer. In some embodiments,
the cancer is castration resistant prostate cancer. In some
embodiments, the method of treating cancer further comprises
administering to the mammal at least one additional anti-cancer
agent.
[0203] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein is used to
treat prostate cancer in a mammal, wherein the mammal has not
received treatment with an anti-cancer agent. In some embodiments,
a third-generation AR inhibitor compound identified using the
methods provided herein is used to treat prostate cancer in a
mammal, wherein the mammal has not received treatment with a
chemotherapeutic compound.
[0204] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein is used to
treat prostate cancer in a mammal, wherein the mammal has been
administered one or more anti-cancer agents. Exemplary anti-cancer
agents include, but are not limited to, hormonal therapeutic
agents, including, but not limited to first- and second-generation
AR antagonists (e.g. bicalutamide, flutamide, hydroxyflutamide,
nilutamide, ARN-509, enzalutamide (MDV3100) and RD162) and
compounds that inhibit hormone (e.g. androgen) production, such as,
for example, galeterone (TOK001). TAK-700 or abiraterone acetate,
chemotherapeutic compounds, anti-metabolites, anti-cancer
antibodies, surgery, radiation, and hyperthermal therapy. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein is used to treat prostate cancer
in a mammal, wherein the mammal has been administered one or more
chemotherapeutic compounds. In some embodiments, a third-generation
AR inhibitor compound identified using the methods provided herein
is used to treat prostate cancer in a mammal, wherein the mammal
has been treated by surgery. In some embodiments, a
third-generation AR inhibitor compound identified using the methods
provided herein is used to treat prostate cancer in a mammal,
wherein the mammal has been treated with radiation therapy. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein is used to treat prostate cancer
in a mammal, wherein the mammal has been treated with a
hyperthermal therapy.
[0205] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein is used to
treat prostate cancer in a mammal, wherein the mammal has been
administered one or more cycles of treatment with an anti-cancer
agent.
[0206] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR inverse agonist, AR antagonist, an AR degrader, an AR
trafficking modulator, an AR DNA-binding inhibitor, or combinations
thereof.
[0207] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR inverse agonist.
[0208] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR antagonist.
[0209] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR degrader.
[0210] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR trafficking modulator.
[0211] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR DNA-binding inhibitor.
[0212] In some embodiments, described herein are methods of
treating cancer in a mammal comprising administering to the mammal
a therapeutically effective amount of a third-generation AR
inhibitor compound identified using the methods provided herein, or
a pharmaceutically acceptable salt thereof, wherein the compound is
an AR protein synthesis inhibitor.
[0213] In some embodiments, the cancer is prostate cancer. In some
embodiments, the cancer is castration resistant prostate cancer. In
some embodiments, the prostate cancer is an ARN-509-resistant
prostate cancer. In some embodiments, the prostate cancer is an
enzalutamide (MDV3100)-resistant prostate cancer. In some
embodiments, the prostate cancer is an RD162-resistant prostate
cancer. In some embodiments, the prostate cancer is an abiraterone
acetate-resistant prostate cancer. In some embodiments, the
prostate cancer is a galeterone (TOK001)-resistant prostate cancer.
In some embodiments, the prostate cancer is a TAK-700-resistant
prostate cancer.
[0214] Pharmaceutical formulations described herein are
administrable to a subject in a variety of ways by multiple
administration routes, including but not limited to, oral,
parenteral (e.g., intravenous, subcutaneous, intramuscular),
buccal, topical or transdermal administration routes. The
pharmaceutical formulations described herein include, but are not
limited to, aqueous liquid dispersions, self-emulsifying
dispersions, solid solutions, liposomal dispersions, solid dosage
forms, powders, immediate release formulations, controlled release
formulations, fast melt formulations, tablets, capsules, pills,
delayed release formulations, extended release formulations,
pulsatile release formulations, multiparticulate formulations, and
mixed immediate and controlled release formulations. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein is administered orally. In some
embodiments, a third-generation AR inhibitor compound identified
using the methods provided herein is administered
intravenously.
[0215] In some embodiments, a third-generation AR inhibitor
compound identified using the methods provided herein is
administered topically. In such embodiments, a third-generation AR
inhibitor compound identified using the methods provided herein is
formulated into a variety of topically administrable compositions,
such as solutions, suspensions, lotions, gels, pastes, shampoos,
scrubs, rubs, smears, medicated sticks, medicated bandages, balms,
creams or ointments. Such pharmaceutical compounds can contain
solubilizers, stabilizers, tonicity enhancing agents, buffers and
preservatives. In one aspect, the anti-androgen compound identified
using the methods provided herein is administered topically to the
skin.
[0216] In another aspect is the use of a third-generation AR
inhibitor compound identified using the methods provided herein in
the manufacture of a medicament for treating a disease, disorder or
conditions in which the activity of AR contributes to the pathology
and/or symptoms of the disease or condition. In one aspect, the
disease or condition is any of the diseases or conditions specified
herein.
[0217] In any of the aforementioned aspects are further embodiments
in which: (a) the effective amount of the third-generation AR
inhibitor compound identified using the methods provided herein is
systemically administered to the mammal; and/or (b) the effective
amount of the third-generation AR inhibitor compound is
administered orally to the mammal; and/or (c) the effective amount
of the third-generation AR inhibitor compound is intravenously
administered to the mammal; and/or (d) the effective amount of the
third-generation AR inhibitor compound is administered by injection
to the mammal; and/or (e) the effective amount of the
third-generation AR inhibitor compound is administered topically to
the mammal; and/or (f) the effective amount of the third-generation
AR inhibitor compound is administered non-systemically or locally
to the mammal.
[0218] In any of the aforementioned aspects are further embodiments
comprising single administrations of the effective amount of the
third-generation AR inhibitor compound identified using the methods
provided herein, including further embodiments in which (i) the
third-generation AR inhibitor compound is administered once; (ii)
the third-generation AR inhibitor compound is administered to the
mammal multiple times over the span of one day; (iii) continually;
or (iv) continuously.
[0219] In any of the aforementioned aspects are further embodiments
comprising multiple administrations of the effective amount of a
third-generation AR inhibitor compound identified using the methods
provided herein, including further embodiments in which (i) the
third-generation AR inhibitor compound is administered continuously
or intermittently: as in a single dose; (ii) the time between
multiple administrations is every 6 hours; (iii) the
third-generation AR inhibitor compound is administered to the
mammal every 8 hours; (iv) the third-generation AR inhibitor
compound is administered to the mammal every 12 hours; (v) the
third-generation AR inhibitor compound is administered to the
mammal every 24 hours. In further or alternative embodiments, the
method comprises a drug holiday, % herein the administration of the
third-generation AR inhibitor compound is temporarily suspended or
the dose of the compound being administered is temporarily reduced;
at the end of the drug holiday, dosing of the compound is resumed.
In some embodiments, the length of the drug holiday varies from 2
days to 1 year.
[0220] Also provided are methods of reducing AR activation in a
mammal comprising administering to the mammal a third-generation AR
inhibitor compound identified using the methods provided herein. In
some embodiments, the method comprises reducing AR activation in
prostate cells in the mammal. In some embodiments, the method
comprises reducing AR activation in non-prostate cells. In some
embodiments, the method of reducing AR activation comprises
reducing the binding of androgens to the androgen receptor. In some
embodiments, the method of reducing AR activation comprises
reducing AR concentration in a cell.
[0221] In some cases disclosed herein is the use of a
third-generation AR inhibitor compound identified using the methods
provided herein in the manufacture of a medicament for the
treatment of diseases or conditions that are AR dependent or AR
mediated. In some embodiments, the disease or condition is prostate
cancer. In some embodiments, the AR dependent or AR mediated
disease or condition is described herein.
[0222] In some cases disclosed herein is the use of a
third-generation AR inhibitor compound identified using the methods
provided herein in the treatment or prevention of diseases or
conditions that are AR dependent or AR mediated. In some
embodiments, the AR dependent or AR mediated disease or condition
is described herein.
[0223] In any of the embodiments disclosed herein, the mammal is a
human. In some embodiments, the third-generation AR inhibitor
compound provided herein is administered to a human.
[0224] In some embodiments, the third-generation AR inhibitor
compound provided herein is used to diminish, reduce, or eliminate
the activity of AR.
[0225] In some embodiments, the third-generation AR inhibitor
compounds identified by the methods disclosed herein are selective
AR modulators. In some embodiments, the third-generation AR
inhibitor compounds identified by the methods disclosed herein have
high specificity for the AR and have desirable, tissue-selective
pharmacological activities. Desirable, tissue-selective
pharmacological activities include, but are not limited to, AR
antagonist activity in prostate cells and no AR antagonist activity
in non-prostate cells. In some embodiments, the third-generation AR
inhibitor compounds disclosed herein are anti-androgens that
display negligible or no AR agonist activity.
[0226] In some embodiments, presented herein are third-generation
AR inhibitor compounds identified by the methods disclosed herein
selected from active metabolites, tautomers, pharmaceutically
acceptable solvates, pharmaceutically acceptable salts or prodrugs
of an AR inhibitor compound identified using the methods provided
herein.
[0227] In some embodiments, the pharmaceutical composition
comprising third-generation AR inhibitor compounds is administered
in combination with one or more additional therapeutic agents,
including, but not limited to, corticosteroids, anti-emetic agents,
analgesics, anti-cancer agents, anti-inflammatory agents, kinase
inhibitors. HSP90 inhibitors, and histone deacetylase (HDAC)
inhibitors. In some embodiments, the pharmaceutical composition
comprising third-generation AR inhibitor compounds is administered
in combination with an anti-cancer agent including, but not limited
to, a hormonal therapeutic agent, including, but not limited to a
first- and second-generation AR antagonists (e.g. bicalutamide,
flutamide, hydroxyflutamide, nilutamide, ARN-509, enzalutamide
(MDV3100) and RD162), a compound that inhibits hormone (e.g.
androgen) production, such as a CYP17A inhibitor, including, for
example, galeterone (TOK001), TAK-700 or abiraterone acetate,
chemotherapeutic compounds, anti-metabolites, anti-cancer
antibodies, surgery, radiation, and hyperthermal therapy. In some
embodiments, the third-generation AR inhibitor compound and the
additional therapeutic agent is administered in the same
composition. In some embodiments, the third-generation AR inhibitor
compound and the additional therapeutic agent are administered as
separate compositions. In some embodiments, the third-generation AR
inhibitor compound and the additional therapeutic agent are
administered simultaneously, sequentially, or intermittently. In
some embodiments, the third-generation AR inhibitor compound and
the additional therapeutic agent are administered by the same route
of administration. In some embodiments, the third-generation AR
inhibitor compound and the additional therapeutic agent are
administered by different routes of administration.
Kits/Articles of Manufacture
[0228] For use in the diagnostic and therapeutic applications
described herein, kits and articles of manufacture are also
described herein. Such kits can comprise a carrier, package, or
container that is compartmentalized to receive one or more
containers such as vials, tubes, and the like, each of the
container(s) comprising one of the separate elements to be used in
a method described herein. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. The containers
are formed from any acceptable material including, e.g., glass or
plastic.
[0229] In some embodiments, the kits provided herein are for use in
detecting nucleic acid encoding a mutant AR polypeptide in a
subject or for detecting a mutant AR polypeptide in a subject (i.e.
a diagnostic kit). In some embodiments the kits are employed for
selecting patients for treatment with a third-generation AR
antagonist, for identifying subjects as resistant or likely to
become resistant to a first- or second-generation AR antagonist,
for monitoring the development of resistance to a first- or
second-generation AR antagonist therapy, or combinations thereof.
The kits provided herein contain one or more reagents for the
detection of the nucleic acid encoding a mutant AR polypeptide, for
the detection of mutant AR polypeptides, for detection of AR
activity in cells from the subject, or combinations thereof.
Exemplary reagents include but are not limited to, buffers, PCR
reagents, antibodies, substrates for enzymatic staining, chromagens
or other materials, such as slides, containers, microtiter plates,
and optionally, instructions for performing the methods. Those of
skill in the art will recognize many other possible containers and
plates and reagents that can be used for contacting the various
materials. Kits also can contain control samples, such as for
example, nucleic acids or proteins, such as for example a mutant AR
polypeptide provided herein or nucleic acids encoding a mutant AR
polypeptide provided herein. In some embodiments, kits contain one
or more set of oligonucleotide primers for detection of endogenous
androgen gene expression.
[0230] In some embodiments, the container(s) can comprise one or
more first- or second-generation AR antagonists or third-generation
AR inhibitor compounds identified by the methods described herein,
optionally in a composition or in combination with another agent as
disclosed herein. The container(s) optionally have a sterile access
port (for example the container can be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). Such kits optionally comprising a compound with an
identifying description or label or instructions relating to its
use in the methods described herein.
[0231] A kit will typically comprise one or more additional
containers, each with one or more of various materials (such as
reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of a
compound described herein. Non-limiting examples of such materials
include, but not limited to, buffers, diluents, filters, needles,
syringes; carrier, package, container, vial and/or tube labels
listing contents and/or instructions for use, and package inserts
with instructions for use. A set of instructions will also
typically be included.
[0232] A label can be on or associated with the container. A label
can be on a container when letters, numbers or other characters
forming the label are attached, molded or etched into the container
itself; a label can be associated with a container when it is
present within a receptacle or carrier that also holds the
container, e.g., as a package insert. A label can be used to
indicate that the contents are to be used for a specific
therapeutic application. The label can also indicate directions for
use of the contents, such as in the methods described herein.
[0233] Articles of manufacture, which include packaging material, a
third-generation AR inhibitor compound identified using the methods
provided herein within the packaging material, and a label that
indicates that the compound or composition, or pharmaceutically
acceptable salt, tautomers, pharmaceutically acceptable N-oxide,
pharmaceutically active metabolite, pharmaceutically acceptable
prodrug, or pharmaceutically acceptable solvate thereof, is used
for reducing, diminishing or eliminating the effects of androgen
receptors, or for the treatment, prevention or amelioration of one
or more symptoms of a disease or condition that would benefit from
a reduction or elimination of androgen receptor activity, are
provided.
Production of Anti-Androgen Resistant Cell Lines
[0234] Provided herein are methods for producing prostate cancer
cell lines resistant to treatment with an AR antagonist. In some
embodiments, the prostate cancer cell lines are resistant to
treatment with ARN-509. In some embodiments, the prostate cancer
cell lines are resistant to treatment with enzalutamide (MDV3100).
In some embodiments, the resistant cell lines generated by the
method provided express a higher level of AR compared to the
parental cell line used to generate the resistant cell line. In
some embodiments, the resistant cell lines generated by the method
express about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,
8, 8.5, 9, 9.5, 10 times or greater the amount of AR compared to
the parental cell line. In some embodiments, the resistant cell
lines express a mutant AR protein.
[0235] In some embodiments, the method comprises contacting a
prostate cancer cell line (i.e. parental cell line) with an AR
antagonist and culturing the cells for a predetermined period of
time. In some embodiments, the method comprises culturing the cells
in increasing concentrations of the AR antagonist for a
predetermined period of time. In some embodiments, the
concentration of the AR antagonist ranges from about 0.1 .mu.M to
about 100 .mu.M, such as, for example, 1 .mu.M to about 10 .mu.M.
In some embodiments, the cells are cultured at 0.8 .mu.M, 1.5
.mu.M, 3 .mu.M and 6 .mu.M of the AR antagonist. In some
embodiments, the concentration of the AR antagonist is increased 2,
3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the
concentration of the AR antagonist is increased 3 times. In some
embodiments, the cells are cultured in the presence of the AR
antagonist 1 day, 2 days, 3, days, 4 days, 5 days, 6 days, 1 week,
2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 2.5
months, 3 months or more. In some embodiments, the cells are
divided and re-plated every 2 days, every 3 days, every 4 days,
every 5 days, every 6 days, every week or longer. In some
embodiments, the culture media containing the AR antagonist is
refreshed every day, every 2 days, every 3 days, every 4 days,
every 5 days, every 6 days, every week or longer. In some
embodiments, the AR antagonist is a second-generation antagonist.
In some embodiments, the AR antagonist is ARN-509. In some
embodiments, the AR antagonist is enzalutamide (MDV3100).
[0236] In some embodiments, the prostate cancer cell line is a
human prostate adenocarcinoma cell line. In some embodiments, the
prostate cancer cell line is an LNCaP cell line. In some
embodiments, the prostate cancer cell line overexpresses the
androgen receptor. In some embodiments, the prostate cancer cell
line is LNCaP/AR(cs) or LNCaP/AR(cs)-Luc.
Examples
[0237] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1: Generation of Drug Resistant Cell Line In Vivo
[0238] Cell lines resistant to treatment with the anti-AR compound
ARN-509 were generated in vivo in mice bearing castration resistant
human prostate adenocarcinoma (LNCaP/AR(cs)) xenograft tumors. The
LNCaP/AR(cs) cell line over-expresses the androgen receptor (AR)
3-5 fold over the parental LNCaP cell line mimicking castration
resistant prostate cancer (Chen et al. (2004) Nature Medicine
10:33-39).
[0239] LNCaP-AR(cs) xenograft tumors were grown in castrated six
week old male SCID Hairless Outbred mice (SHO, Charles Rivers
Laboratories). 1.times.10.sup.6 LNCaP-ARcs cells in 50% serum-free
RPMI and 50% Matrigel.TM. were subcutaneously injected (100
.mu.l/animal) on the right flank of 10 mice 3-5 days post
castration. Tumor size was monitored daily. When tumors reached an
average volume of .about.200 mm.sup.3 (approximately 60 days
post-injection), the animals began treatment with vehicle alone
(n=1) or 30 mg/kg ARN-509 (n=9) by QD dosing regimen (i.e. orally
in 15% Vitamin E-TPGS and 65% of a 0.5% w/v carboxymethyl cellulose
(CMC) solution in 20 mM citrate buffer (pH 4.0)). Initially,
treatment with ARN-509 induced tumor regression. Following
approximately 75 days of dosing, a single tumor resumed growth and
progressed to a size greater than at treatment initiation. Once the
resistant tumor reached .about.800 mm.sup.3, the mouse was
euthanized and the tumor was harvested. Tumor cells were manually
dispersed by homogenization with a 3 mL syringe. Tumor cells were
cultured in RPMI plus 10% FBS and 10 .mu.M ARN-509. One resistant
cell line was generated by this method.
Example 2: Generation of Drug Resistant Cell Lines In Vitro
[0240] Cell lines resistant to treatment with the anti-AR compounds
ARN-509 and MDV3100 were generated in vitro using LNCaP human
prostate adenocarcinoma cell lines.
[0241] LNCaP (ATCC), LNCaP/AR(cs) (Guo et al. (2006) Cancer Cell
0:309-19) and LNCaP/AR-Luc (Tran et al. Science (2009)
324(5928):787-90; Ellwood-Yen et al. (2006) Cancer Res. 66:10513-6)
were maintained in RPMI 1640 supplemented with 10% FBS (Hyclone).
LNCaP (ATCC), LNCaP/AR(cs) or LNCaP/AR(cs)-Luc cells were cultured
in increasing concentrations of either ARN-509 or MDV3100 over a
course of 6 months. Initially, 50 mL of cells were seeded into a
225 cm.sup.2 cell culture flask at a concentration of approximately
80,000 cells/mL and grown in RPMI plus 10% FBS in the presence of
800 nM ARN-509 or MDV3100. Medium and drug were changed semiweekly
and the cells were passaged in 75 cm.sup.2 cell culture flasks as
needed. The concentration of each compound was increased several
times from about 1.5 .mu.M to about 6 .mu.M as the growth rate of
the drug-treated cells increased to that of the untreated control
cells. After approximately 6 months of drug selection, the cells
were maintained in RPMI plus 10% FBS and 6 .mu.M ARN-509 or
MDV3100. Following selection, 10 independent ARN-509 and MDV3100
resistant cell lines were obtained. Two lines were derived from
LNCaP (ATCC) cells selected in the presence of ARN-509. Four cell
lines were derived from LNCaP/AR(cs), two following treatment with
ARN-509 and two following treatment with MDV3100. LNCaP/AR(cs)-Luc
cells were used to derive 4 resistant cell lines, two from ARN-509
treatment and two from MDV3100 treatment.
Example 3: Proliferation Assays to Test Drug Resistance
[0242] Cell proliferation assays were performed to test resistance
of the cell lines to ARN-509 and MDV3100 treatment.
[0243] Proliferation assays were performed on all ARN-509 and
MDV3100 resistant cell lines by seeding 16 .mu.L/well of cells at a
density of 50,000 cells per mL in phenol-red-free RPMI 1640 (with
5% CSS) into 384-well cell culture plate (Flat Clear Bottom Black
Polystyrene TC-Treated 384 Well plates (Corning)) and incubated for
2 days at 37.degree. C. For agonist assays, 11 point semi-log
dilutions of each compound were made in culture medium and 16 .mu.L
of each dilution was added to the cells. ARN-509, MDV3100 and
bicalutamide were run at a final concentration ranging from
3.16.times.10.sup.-5 M to 3.18.times.10.sup.-10 M, while the
synthetic androgen methyltrienolone (R1881) was run at a final
concentration range of 3.16.times.10.sup.-8 M to
3.18.times.10.sup.-13 M. For the antagonist mode assay, the
compounds were diluted in culture medium also containing 200 pM
R1881 (PerkinElmer, Waltham, Mass.) (final [R1881]=100 pM) and then
added to the cells (16 .mu.L). After 7 days, 16 .mu.L of
CellTiter-Glo.RTM. Luminescent Cell Viability Assay (Promega,
Madison, Wis.) was added directly to the cell cultures and Relative
Luminescence Units (RLUs) measured according to the manufacturer's
instructions.
[0244] In the agonist mode assay, percent viability of the samples
was calculated as: % viability=100.times.([RLU sample-RLU medium
without cells]/[RLU 1881 treated cells-RLU medium without cells])
(Table 1A). In the antagonist mode assay, the percent viability of
the samples was calculated as: % viability=100.times.([RLU
sample-RLU Day 0]/[RLU R1881 treated cells-RLU Day 0]) (Table
1B).
TABLE-US-00001 TABLE 1A Agonist Proliferation Assay (% Viability)
R1881 Bicalutamide MDV3100 ARN-509 LNCaP 100.0 + + + LNCaP/AR(cs)
100.0 + + + Class 1 100.0 ++ ++ ++ Class 2 100.0 + ++ ++ `+` =
<30, `++` = >30 [R1881] = 0.1 nM, [Antagonists] = 10
.mu.M
TABLE-US-00002 TABLE 1B Antagonist Proliferation Assay (%
Viability) R1881 Bicalutamide MDV3100 ARN-509 LNCaP 100.0 + + +
LNCaP/AR(cs) 100.0 + + + Class 1 100.0 ++ ++ ++ Class 2 100.0 ++ ++
++ `+` = <30, `++` = >30 [R1881] = 0.1 nM, [Antagonists] = 10
.mu.M
[0245] In the proliferation assays, both ARN-509 and enzalutamide
were full proliferative antagonists in all three LNCaP parental
cell lines (Table 1A, FIG. 1A). The resistant cell lines segregated
into two distinct classes. Unlike their parental cell lines, the
first class of ARN-509- and MDV3100-resistant cells (Class 1)
proliferate in the absence of added androgens. The ligand
independent growth of the cells is unaltered in the presence of
ARN-509, MDV3100 or bicalutamide. The synthetic androgen, R1881,
inhibits proliferation in the class 1 cells at high concentrations.
This growth inhibitory activity of R1881 is antagonized by either
MDV3100 or ARN-509, indicating that AR is still capable of binding
MDV3100 and ARN-509 in these cell lines.
[0246] Class 1 resistant cell lines included the one cell line
derived from the LNCaP-AR(cs) xenograft tumor, the 2 cell lines
derived from LNCaP/AR(cs) cell selected in the presence of ARN-509,
the 2 cell lines derived from LNCaP/AR(cs) cells selected in the
presence of MDV3100, the 2 cell lines derived from LNCaP/AR(cs)-Luc
selected in the presence of ARN-509, and one of the cell lines
derived from LNCaP/AR(cs)-Luc selected in the presence of
MDV3100.
[0247] The second class of MDV3100- and ARN-509-resistant cell
lines (Class 2) remains androgen dependent for growth, similar to
their parental cell lines. However, unlike their activity on the
parental cell lines, ARN-509 and MDV3100 can stimulate
proliferation in the class 2 cell lines. Bicalutamide, however, did
not stimulate proliferation in the class 2 cell lines. Class 2
resistant cell lines included the two cell lines derived from LNCaP
cells selected in the presence of ARN-509 (LNCaP ARN-509r1 and
LNCaP ARN-509r2) and one of the cell lines derived from
LNCaP/AR(cs)-Luc selected in the presence of MDV3100 (LNCaP
ENZr2).
[0248] The partial agonist activity was independent of the compound
utilized for selection; ARN-509 and enzalutamide displayed partial
agonist activity in all three cell lines regardless of the compound
used to derive the resistance variants. Consistent with
proliferative activity, ARN-509 or enzalutamide only partially
antagonized androgen dependent growth of these resistant lines
(Table 1B, FIG. 1B,C).
Example 4: Transcriptional Reporter Assays to Test Drug
Resistance
[0249] Transcriptional reporter assays using an ARE response
element operatively linked to a reporter gene were performed to
test resistance of the cells to ARN-509, MDV3100, and bicalutamide
treatment.
[0250] Transcriptional reporter assays were performed on all
resistant cell lines by seeding 100 .mu.L of cells at a density of
250,000 cells/mL into 96-well cell culture plates in RPMI 1640
supplemented with 5% charcoal stripped serum and allowed to attach
overnight at 37.degree. C.
[0251] With the exception of the LNCaP/AR(cs)-Luc cells that
contain an integrated androgen responsive reporter, cells were
transiently transfected using Lipofectin.RTM. (Life Technologies)
according to the manufacturer's protocol. For LNCaP and
LNCaP/AR(cs) cells, triplicate transfections were performed using
428 ng reporter vector (pGL4 Pb-Luciferase (the rat probasin
promoter in pGL4 (Promega, Madison, Wis.))), 50 ng pRL-CMV
(normalization vector, Promega. Madison, Wis.) and 0.7 .mu.L
Lipofectin.RTM.. Following transfection, the cells were incubated
for 4 hours.
[0252] Cells were then treated with the test compounds ARN-509,
MDV3100 and bicalutamide. For agonist assays, the compounds were
serially diluted and 50 .mu.L of compound plus RPMI 1640 plus 5%
charcoal stripped FBS was added to the cells. For antagonist
assays, the compounds were serially diluted and 50 .mu.L of
compound with RPMI plus 3 nM R1881 supplemented with 5% charcoal
stripped serum was added to the cells. Following 48 hour incubation
the medium was removed and the cells were lysed in 40 .mu.L of
lysis buffer (25 mM Tris Phosphate, 2 mM CDTA, 10% Glycerol, 0.5%
Triton X-100, 2 mM DTT). Firefly luciferase activity was measured
immediately following the addition of 40 .mu.L luciferase buffer
(20 mM tricine, 0.1 mM EDTA, 1.07 mM (MgCo.sub.3).sub.4
Mg(OH).sub.2.5H.sub.2O, 2.67 mM MgSO.sub.4, 33.3 mM DTT, 270 .mu.M
Coenzyme A, 470 .mu.M luciferin, 530 .mu.M ATP). Renilla luciferase
was measured following the addition of 40 .mu.L coelenterazine
buffer (1.1 M NaCl, 2.2 mM Na.sub.2EDTA, 0.22 M K.sub.xPO.sub.4 (pH
5.1), 0.44 mg/mL BSA, 1.3 mM NaN.sub.3. 1.43 .mu.M coelenterazne,
final pH adjusted to 5.0).
[0253] The two classes of ARN-509- and MDV3100-resistant cell lines
identified in the proliferation assays also exhibited distinct
properties in the transcriptional assays. In transient transfection
assays using the pGL4 Pb-Luciferase reporters or in assays using
the integrated probasin-luciferase reporter (i.e.
LNCaP/AR(cs)-Luc-derived cells), ARN-509 and MDV3100 were effective
antagonists in the androgen-independent class 1 resistant cells as
evidenced by a decrease in luciferase activity relative to no
treatment controls (Table 2). Bicalutamide, however, exhibited an
increased agonist activity in class 1 resistant cells compared to
the parental cells. In class 2 resistant cells, MDV3100 was a weak
partial agonist in the probasin-luciferase reporter assay, while
bicalutamide and ARN-509 displayed no agonist activity.
TABLE-US-00003 TABLE 2A Agonist Transcriptional Reporter Assay (%
Max Activity) R1881 Bicalutamide MDV3100 ARN-509 LNCaP >90 + + +
LNCaP/AR(cs) >90 + + + Class 1 >90 ++ + + Class 2 >90 + ++
+ `+` = <5, `++` = >5 [R1881] = 10 nM, [Antagonists] = 50
.mu.M
TABLE-US-00004 TABLE 2B Antagonist Transcriptional Reporter Assay
(% Max Activity) R1881 Bicalutamide MDV3100 ARN-509 LNCaP >90 +
+ + LNCaP/AR(cs) >90 + + + Class 1 >90 ++ + ++ Class 2 >90
+ ++ + `+` = <5, `++` = >5 [R1881] = 10 nM, [Antagonists] =
50 .mu.M
Example 5: Endogenous Gene Transcriptional Assays
[0254] The effects of R1881, MDV3100 and bicalutamide treatment on
endogenous gene transcription was examined.
[0255] By Western blot, the AR levels in the class 1 resistant cell
lines were approximately 2 to 4-fold higher than observed in the
LNCaP/AR(cs) cell line (FIG. 2). Analogous to an approximate 3-fold
increase in AR levels being sufficient to support growth of LNCaP
cells in a castrate setting in vivo, the further 2 to 4-fold
elevation in AR levels may be sufficient to promote proliferation
in the absence of androgens in vitro. In contrast, the AR levels of
the class 2 resistant lines were similar to that observed in the
parental cell lines. Thus, the conversion of enzalutamide and
ARN-509 to partial agonists in the class 2 cell lines was not due
to AR overexpression. Minor differences in total and phosphorylated
Akt and Erk were observed with no consistent changes seen in either
class of resistant cell lines.
[0256] Total RNA was isolated using the Aurum.TM. total RNA
isolation kit (BIO-RAD, Hercules, Calif.). RNA (1 .mu.g) was
reverse transcribed using the iScript cDNA synthesis kit (BIO-RAD,
Hercules, Calif.) to produce cDNA. Real-time PCR was performed
using the Applied Biosystems 7900HT instrument and SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, Calif.). PCR reactions
were performed in 6 .mu.L according to the manufacturer's protocol
and a thermocycle protocol of 95.degree. C. for 10 minutes followed
by 40 cycles of 95.degree. C. for 15 seconds and 58.degree. C. for
1 minute. Androgen responsive gene (PSA, SLUG, TMPRSS2, STEAP4,
FKBP5, ORM1, NOV, FASN, NKX3.1, AMIGO2, BDNF, CAMK2N1, HPGD,
NCAPD3, PLD1) expression was normalized to GAPDH expression and
expressed relative to vehicle treatment of the parental cell line.
Relative expression results are provided in Table 3A. Primers
employed for PCR are listed in Table 3B.
[0257] Similar compound and class selective agonist activities were
observed on endogenous androgen-responsive genes, but in the
context of the endogenous genes the transcriptional activity of
MDV3100 is more evident (Table 3). In class 1 resistant cells,
bicalutamide displays robust transcriptional activity and MDV3100
is a weak transcriptional agonist. In contrast, in class 2
resistant cell lines, bicalutamide is a weak transcriptional
agonist while MDV3100 displays robust transcriptional agonist
activity on the genes tested.
TABLE-US-00005 TABLE 3A Endogenous Gene Transcriptional Activity
(Fold Vehicle) PSA SLUG TMPRSS2 STEAP4 FKBP5 ORM1 NOV LNCaP DMSO
1.0 1.0 1.0 1.0 1.0 1.0 1.0 R1881 ++ +++ ++ ++++ ++ +++ --
Bicalutamide + + + + + - + MDV3100 + + + - + + + LNCaP/AR(cs) DMSO
1.0 1.0 1.0 1.0 1.0 1.0 1.0 R1881 +++ +++ ++ ++++ ++ ++++ --
Bicalutamide ++ + + ++ + +++ + MDV3100 + + + + + + + Class 1 DMSO
1.0 1.0 1.0 1.0 1.0 1.0 1.0 R1881 ++ +++ + ++++ +++ ++++ --
Bicalutamide + +++ + +++ ++ ++ + MDV3100 + + + + + + + Class 2 DMSO
1.0 1.0 1.0 1.0 1.0 1.0 1.0 R1881 +++ +++ ++ ++++ +++ ++++ --
Bicalutamide + + + + + + + MDV3100 +++ +++ ++ +++ ++ +++ - [R1881]
= 10 nM, [Antagonists] = 30 .mu.M -- <0.1, - = 0.1-1, + = 1-10,
++ = 10-50, +++ 50-500, ++++ >500
TABLE-US-00006 TABLE 3B Transcriptional Real-time PCR
Oligonucleotide Sequence Gene Forward Primer Sequence Reverse
Primer Sequence AMIGO2 AGAGACTCAGAGGCGACCAT ATCAGCAAACACAGCAGCTC
(SEQ ID NO: 20) (SEQ ID NO: 21) BDNF AGAGCTGTTGGATGAGGACC
AGAAAGGCTCCAAAGGCACTTGACTACT (SEQ ID NO: 22 (SEQ ID NO: 23) CAMK2N1
GACCAAGCGGGTTGTTATTGA TGCCTTGTCGGTCATATTTTTCA (SEQ ID NO: 24) (SEQ
ID NO: 25) FKBP5 CGGAGAACCAAACGGA AAGGCTTCGCCCACAGTGAATGC (SEQ ID
NO: 26) (SEQ ID NO: 27) HPGD ACAGCAGCCGGTTTATTGTGCTTC
TGGCATTCAGTCTCACACCACTGT (SEQ ID NO: 28) (SEQ ID NO: 29) NCAPD3
ACCACTCACCATCATCTCA AGGCATGCTCTTCTTTGCCAGATCCTCGT (SEQ ID NO: 30)
(SEQ ID NO: 31) NOV GCCTTACCCTTGCAGCTTAC GAGCATGCTGTCCACTCTGT (SEQ
ID NO: 32) (SEQ ID NO: 33) ORM1 CTTGCGCATTCCCAAGTCAGATGT
TTTCCTCTCCTTCTCGTGCTGCTT (SEQ ID NO: 34) (SEQ ID NO: 35 PLD1
GAGCCTGCTACAGATGGTCA TGTCTACCAGCAGGACGAAG (SEQ ID NO: 36) (SEQ ID
NO: 37) PSA CCTCCTGAAGAATCGATTCC GAGGTCCACACACTGAAGTT (SEQ ID NO:
38) (SEQ ID NO: 39) SLUG TTTCTGGGCTGGCCAAACATAAGC
ACACAAGGTAATGTGTGGGTCCGA (SEQ ID NO: 40) (SEQ ID NO: 41) STEAP4
CGGCAGGTGTTTGTGTGTGGAAAT AGAAGACACACAGCACAGCAGACA (SEQ ID NO: 42)
(SEQ ID NO: 43) TMPRSS2 TAGTGAAACCAGTGTGTCTGCCCA
AGCGTTCAGCACTTCTGAGGTCTT (SEQ ID NO: 44) (SEQ ID NO: 45) FASN
CGCTCTGGTTCATCTGCTCTG TCATCAAAGGTGCTCTCGTCTG (SEQ ID NO: 46) (SEQ
ID NO: 47) NKX3.1 TGGAGAGGAAGTTCAGCCATCAGA AGGAGAGCTGCTTTCGCTTAGTCT
(SEQ ID NO: 48) (SEQ ID NO: 49)
[0258] In a separate experiment, gene expression of the following
genes were analyzed in LNCaP, LNCaP/AR(cs), LNCaP/AR-Luc. LNCaP
ARN-509r1, LNCaP ARN-509r2 and LNCaP/AR-Luc ENZr2 cells: PLD,
CAM2KN, NOV, BDNF, AMIGO2, FASN, TMPRSS2, NKX3.1, PSA, FKBP5, HPGD,
NCAPD3, SLUG, STEAP4, and ORM. Cells were cultured for 3 days in
hormone depleted medium followed by treatment with vehicle, 1 nM
R1881 or 30 .mu.M compound. Gene expression was normalized to GAPDH
as described above. Results are presented in Tables 4A and 4B.
TABLE-US-00007 TABLE 4A LNCaP. LNCaP/AR(cs) and resistant line
transcription LNCaP LNCaP/AR(cs) LNCaP ARN-509r1 ARN- ARN- ARN-
Gene Vehicle 509 ENZ R1881 Vehicle 509 ENZ R1881 Vehicle 509 ENZ
R1881 AMIGO2 1.00 1.12 1.20 0.68 1.00 0.72 1.13 0.54 1.00 0.62 0.99
0.21 BDNF 1.00 1.09 0.85 0.40 1.00 0.85 1.13 0.48 1.00 0.90 1.58
0.31 CAM2KN1 1.00 1.17 1.39 0.13 1.00 0.80 1.11 0.05 1.00 0.37 0.59
0.01 FASN 1.00 1.34 1.24 5.58 1.00 0.75 1.19 7.11 1.00 1.04 1.53
3.12 FKBP5 1.00 0.99 1.04 54.95 1.00 0.99 1.71 97.01 1.00 2.58 9.45
53.45 HPGD 1.00 1.48 1.78 100.43 1.00 1.18 2.01 183.55 1.00 2.36
10.20 61.39 NCAPD3 1.00 1.16 1.20 104.69 1.00 0.91 1.22 93.05 1.00
1.29 3.12 90.51 NKX3.1 1.00 0.90 1.66 30.06 1.00 1.13 2.51 8.82
1.00 6.54 11.55 8.11 NOV 1.00 1.73 2.57 0.15 1.00 1.04 1.27 0.04
1.00 1.40 1.39 0.03 ORM1 1.00 0.71 1.13 873.10 1.00 0.84 3.58
3444.31 1.00 15.89 205.07 1296.13 PLD1 1.00 1.09 0.70 0.02 1.00
1.04 0.65 0.02 1.00 0.33 0.24 0.03 PSA 1.00 0.66 1.69 47.84 1.00
1.43 2.69 19.16 1.00 32.67 57.68 85.63 SLUG 1.00 1.27 2.35 129.79
1.00 1.03 2.06 89.88 1.00 4.66 42.52 164.28 STEAP4 1.00 0.78 1.54
639.15 1.00 0.90 1.95 1314.23 1.00 3.03 32.22 1184.45 TMPRSS2 1.00
0.77 1.68 22.16 1.00 1.06 2.36 17.51 1.00 10.20 23.75 37.27 LNCaP
LNCaP ARN-509r2 Gene Vehicle ARN-509 ENZ R1881 Vehicle ARN-509 ENZ
R1881 AMIGO2 1.00 1.12 1.20 0.68 1.00 0.67 0.63 0.55 BDNF 1.00 1.09
0.85 0.40 1.00 1.04 0.77 0.47 CAM2KN1 1.00 1.17 1.39 0.13 1.00 0.46
0.38 0.02 FASN 1.00 1.34 1.24 5.58 1.00 0.98 0.93 4.82 FKBP5 1.00
0.99 1.04 54.95 1.00 8.00 3.41 48.50 HPGD 1.00 1.48 1.78 100.43
1.00 1.49 4.56 59.30 NCAPD3 1.00 1.16 1.20 104.69 1.00 1.13 1.35
54.95 NKX3.1 1.00 0.90 1.66 30.06 1.00 3.73 6.28 10.20 NOV 1.00
1.73 2.57 0.15 1.00 1.39 0.71 0.07 ORM1 1.00 0.71 1.13 873.10 1.00
3.94 23.75 625.99 PLD1 1.00 1.09 0.70 0.02 1.00 0.81 0.29 0.02 PSA
1.00 0.66 1.69 47.84 1.00 1.91 2.48 7.89 SLUG 1.00 1.27 2.35 129.79
1.00 1.31 9.58 77.17 STEAP4 1.00 0.78 1.54 639.15 1.00 1.45 4.69
377.41 TMPRSS2 1.00 0.77 1.68 22.16 1.00 3.07 4.86 15.14
TABLE-US-00008 TABLE 4B LNCaP/AR-Luc and LNCaP/AR-Luc ENZr2
transcription LNCaP/AR-Luc LNCaP/AR-Luc ENZr2 Gene Vehicle ARN-509
ENZ R1881 Vehicle ARN-509 ENZ R1881 AMIGO2 1.00 1.13 1.44 0.54 1.00
0.99 0.78 0.49 BDNF 1.00 1.17 1.08 0.18 1.00 0.56 0.44 0.12 CAM2KN1
1.00 1.13 1.51 0.11 1.00 0.65 0.57 0.11 FASN 1.00 1.08 1.40 3.48
1.00 1.72 1.20 4.44 FKBP5 1.00 1.16 1.46 20.82 1.00 2.46 3.03 23.43
HPGD 1.00 1.88 2.51 2.41 1.00 1.22 1.61 1.39 NCAPD3 1.00 1.09 1.33
17.27 1.00 1.38 1.39 31.12 NKX3.1 1.00 1.14 1.68 11.24 1.00 4.82
5.21 10.13 NOV 1.00 2.55 1.95 0.47 1.00 1.21 1.20 0.27 ORM1 1.00
1.27 1.39 7.89 1.00 15.24 17.88 347.29 PLD1 1.00 1.45 1.33 0.15
1.00 0.46 0.74 0.21 PSA 1.00 0.60 0.44 2.10 1.00 4.59 3.48 16.91
SLUG 1.00 1.13 2.73 48.84 1.00 5.98 12.30 160.90 STEAP4 1.00 0.88
1.23 20.25 1.00 1.66 4.38 79.89 TMPRSS2 1.00 0.74 1.35 3.46 1.00
4.44 4.23 7.73
Example 6: Assays for Mechanisms of Resistance
[0259] Array CGH, mRNA expression profiling and sequence analysis
of patient derived prostate cancer tumors as well as many animal
and in vitro models have implicated multiple pathways in the
progression to the castration resistant state. Three of the most
commonly activated pathways indentified in castration resistant
prostate cancer are the PI3K, Raf and AR pathways. In this example,
Akt and Erk phosphorylation was evaluated by Western blot to assess
the activation states of the PI3K and Raf pathway respectively.
[0260] To evaluate the mode of drug resistance in the class 1 and
class 2 cell lines, Western analysis was performed to assess AR
protein levels and the activation state of several cellular
signaling pathways known to modulate AR activity and commonly
activated in castration resistant prostate cancer. Relative
expression of AR, Akt, phosphorylated Akt (Ser473), p44/42 MAPK
(Erk1/2), phosphorylated p44/42 MAPK (Erk1/2) (Thr202/Tyr204),
tubulin and actin were determined.
[0261] For Western analysis, cells were grown in RPMI 1640
supplemented with 5% charcoal stripped serum for 3 days. Cells were
lysed in modified radioimmunoprecipitation buffer (mRIPA; 10 mM
Tris, 150 mM NaCl, 1% (v/v) NP-40, 0.5% deoxycholate, 0.1% SDS, 5
mM EDTA, pH 7.4) containing Halt.TM. Protease and Phosphatase
Inhibitor Cocktail (Thermo Scientific). Total protein of the
clarified lysates was quantitated by Lowry Assay (Biorad DC protein
assay). NuPAGE.RTM. LDS Sample Buffer and Sample Reducing Agent
were added to the lysates and heated to 70.degree. C. for 10
minutes. 20 .mu.g of total cell protein was separated on a NuPAGE
4-12% Bis Tris Acrylamide Gel and transferred to a nitrocellulose
membrane using an Xcell II.TM. blot module (Invitrogen). Membranes
were incubated in Blocking Buffer (LI-COR, Lincoln, Nebr.) for 30
minutes at room temperature, followed by 60 minute incubations with
primary antibodies against Androgen Receptor (Santa Cruz
Biotechnology cat. No. SC-816). Akt and Phospho-Akt (Ser473) (Cell
Signaling cat. Nos. 9272 and 4058 respectively), p44/42 MAPK
(Erk1/2) and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell
Signaling cat. Nos. 4695 and 4376s respectively) and tubulin or
actin (Sigma cat. No T6199 and A4700 respectively). Following
incubation with an IRDye.RTM. Conjugated Goat Anti Mouse or Rabbit
IgG (LI-COR), protein bands were quantified using an Odyssey.RTM.
Infrared Imaging System.
[0262] By Western blot, the AR levels in the class 1 resistant cell
lines were approximately 2 to 4-fold higher than observed in the
LNCaP/AR(cs) cell line (FIG. 2). Analogous to an approximate 3-fold
increase in AR levels being sufficient to support growth of LNCaP
cells in a castrate setting in vivo, the further 2 to 4-fold
elevation in AR levels may be sufficient to promote proliferation
in the absence of androgens in vitro. In contrast, the AR levels of
the class 2 resistant lines were similar to that observed in the
parental cell lines. Thus, the conversion of enzalutamide and
ARN-509 to partial agonists in the class 2 cell lines was not due
to AR overexpression. Minor differences in total and phosphorylated
Akt and Erk were observed with no consistent changes seen in either
class of resistant cell lines.
Example 7: Determination of Mutant AR Sequence
[0263] MDV3100 and ARN-509 are transcriptional and proliferative
agonists in the class 2 resistant cell lines, while bicalutamide
remains an antagonist. AR expression in class 2 resistant cells was
similar to the parental cell line (Example 6). Thus, the sequence
of AR in class 2 resistant cells was determined to ascertain
whether a mutation of the AR ligand binding domain promotes the
gain of function activity.
[0264] cDNA generated by reverse transcription of RNA isolated from
class 1 and class 2 cells (see Example 5) was used as a template to
sequence AR. Using a series of oligonucleotides, overlapping
segments encompassing the AR ligand binding domain (c. 2013-2757)
were generated by PCR using Phusion.RTM. polymerase (New England
Biolabs) using the manufacturer's protocol. The PCR products were
gel purified to remove non-specific bands as well as unincorporated
oligonucleotides. The purified PCR products were sequenced using
internal oligonucleotides.
[0265] A single nucleic acid mutation was identified within the AR
ligand binding domain at nucleotide position 2626 in three
independently derived cell lines. The missense mutation identified
in all lines was Thymine (T) to Cytosine (C) that converts
Phenylalanine (F) at amino acid position 876 of the encoded
polypeptide to Leucine (L) (SEQ ID NO: 19). Additionally,
sequencing of individual subcloned PCR products from the
LNCaP/AR-Luc ENZr2 cell line indicated that in all cell lines the
F876L mutation arose in the endogenous AR allele.
[0266] F876 lies in helix 11 in a region of AR ligand binding
pocket that is a hotspot for CRPC AR mutations (FIG. 1D). However,
unlike T877 and L701, which coordinate hydrogen bonding to the
17.alpha.-OH group of dihydrotestosterone, F876 contributes to a
small hydrophobic core formed by residues in helix 11 (F786, L880),
the loop between helices 11 and 12 (F891) and helix 3 (F697. L701).
While similar residues and hydrophobic ligand interactions are
conserved in the estrogen and progesterone receptor, F876 has not
been implicated in high affinity binding or steroid selectivity.
Consistent with the relatively minor role F876 is predicted to play
in steroid binding, the AR-F876L mutation has not been reported in
prostate cancer or androgen insensitive populations.
Example 8: Expression of the Mutant AR in AR-Deficient Cells
[0267] To confirm that the F876L mutation confers agonist
activities to ARN-509 and MDV3100, the point mutation was generated
in the context of the full-length wild-type AR and the LNCaP T877A
mutant receptor. The F876L and F876L/T877A mutants were generated
in the plasmid pcDNA3-AR using the QuickChange II Site-Directed
Mutagenesis Kit (Agilent Technologies, Santa Clara, Calif.)
according to the manufacturer's protocol.
[0268] Transcriptional reporter assays using an ARE response
element operatively linked to a reporter gene were performed to
test the transcriptional activity of the mutant AR in response to
ARN-509, MDV3100 and bicalutamide treatment.
[0269] Transcriptional reporter assays were performed by seeding
100 .mu.L of HEPG2 cells at a density of 250,000 cells/mL into
96-well cell culture plates in MEM supplemented with 10% charcoal
stripped serum and allowed to attach overnight at 37.degree. C.
[0270] Cells were transiently transfected using Lipofectin.RTM.
(Life Technologies) according to the manufacturer's protocol.
Triplicate transfections were performed using 378 ng reporter
vector (4X ARE-Luciferase or pGL4 Pb-Luciferase (the rat probasin
promoter in pGL4 (Promega, Madison, Wis.)), 50 ng pcDNA-AR
(wild-type or mutant) 50 ng pRL-CMV (normalization vector) and 0.7
.mu.L Lipofectin.RTM.. Following transfection, the cells were
incubated for 4 hours.
[0271] Following the incubation, the cells were treated with the
test compounds ARN-509, MDV3100 and bicalutamide. For agonist
assays, the compounds were diluted 1:6 and 50 .mu.L of compound in
MEM plus 5% charcoal stripped FBS was added to the cells for a
final concentration range of 30 .mu.M to 0.64 nM. For antagonist
assays, the compounds were serially diluted with 1 nM R1881 (for
wild-type AR) or 5 nM R1881 (for F876L AR).
[0272] Following 48 hour incubation the medium was removed and the
cells were lysed in 40 .mu.L of lysis buffer (25 mM Tris Phosphate,
2 mM CDTA, 10% Glycerol, 0.5% Triton X-100, 2 mM DTT). Firefly
luciferase activity was measured immediately following the addition
of 40 .mu.L luciferase buffer (20 mM tricine, 0.1 mM EDTA, 1.07 mM
(MgCo.sub.3).sub.4 Mg(OH).sub.2.5H.sub.2O, 2.67 mM MgSO.sub.4, 33.3
mM DTT, 270 .mu.M Coenzyme A, 470 .mu.M luciferin, 530 .mu.M ATP).
Renilla luciferase was measured following the addition of 40 .mu.L
coelenterazine buffer (1.1 M NaCl, 2.2 mM Na.sub.2EDTA, 0.22 M
K.sub.xPO.sub.4 (pH 5.1), 0.44 mg/mL BSA, 1.3 mM NaN.sub.3, 1.43
.mu.M coelenterazine, final pH adjusted to 5.0).
[0273] In transient transfection assays, second-generation AR
antagonists ARN-509 and MDV3100 induced AR dependent
transcriptional activity in the context of the F876L or F876L/T877A
mutant AR, while induction from bicalutamide was minimal (Table 5).
For example, in HepG2 cells using either a 4XARE-luciferase or
Pb-luciferase reporter, ARN-509 and MDV3100, but not bicalutamide,
induced AR dependent transcriptional activity in the context of the
F876L or F876L/T877A mutant AR. This indicates that mechanism of
resistance in the class 2 cell lines is the AR F876L mutation.
TABLE-US-00009 TABLE 5 AR Transcriptional Activity (Fold DMSO)
MDV3100 R1881 Bicalutamide (enzalutamide) ARN509 AR WT +++ + + + AR
F876L +++ + ++ ++ + = <20, ++ = 10-200, +++ > 200 [R1881] =
100 nM, [Antagonists] = 6.3 .mu.M
[0274] A second experiment was performed 4X-ARE-Luciferease report
to confirm the above results. In this experiment, first generation
anti-androgens nilutamide and hydroxyflutamide were also compared
along with bicalutamide, ARN-509 and enzalutamide. The AR
transcriptional reporter assays were performed essentially as
described above with minor modifications. Triplicate transfections
were performed using 50 ng pCDNA3-AR or pCDNA3-AR mutant, 65 ng 4X
ARE-Luciferase, 20 ng pRL (Promega), and 25 ng pCMX. For agonist
assays, compounds were serially diluted, and 50 .mu.L of compound
plus RPMI 1640 supplemented with charcoal stripped serum was added
to the cells. For antagonist assays, the compounds were serially
diluted, and 50 .mu.L of compound with RPMI supplemented with
charcoal stripped serum plus methyltrienolone (R1881) were added to
the cells. The final R1881 concentration used in the antagonist
assays was 1 nM with the exception of F876L for which 5 nM R1881
was used.
[0275] As shown in FIGS. 4A-B, in the context of wild-type AR.
ARN-509 and enzalutamide were full antagonists and with minimal
agonist activity in 4X-ARE androgen responsive transcriptional
reporter assays. However, in cells expressing AR-F876L or AR
F876L/T877A, enzalutamide and ARN-509 were partial transcriptional
agonists (FIG. 4A). Conversely, the first generation anti-androgens
bicalutamide, nilutamide and hydroxyflutamide displayed minimal
agonist activity on the F876L mutant (Table 6, FIG. 4B)
(Emax=percent maximal R1881 response). Enzalutamide and ARN-509
were full antagonists on AR mutants T877A, L701H, H874Y and W741C
that either confer resistance to first generation AR antagonists or
broaden steroidal ligand specificities in CRPC patients.
TABLE-US-00010 TABLE 6 4X ARE Reporter Assay WT IC.sub.50 WT F876L
IC.sub.50 F876L Compound (.quadrature.M) Emax (.quadrature.M) Emax
ARN-509 0.79 .+-. 0.15 1.3 .+-. 0.3 0.09 .+-. 0.06 49.7 .+-. 11.1
Enzalut- 1.12 .+-. 0.17 0.4 .+-. 0.1 0.13 .+-. 0.04 20.2 .+-. 5.2
amide Bicalutamide 1.65 .+-. 0.93 10.0 .+-. 2.9 3.63 .+-. 0.04 0.7
.+-. 0.3 Hydroxy- 0.36 .+-. 0.04 28.9 .+-. 5.0 2.60 .+-. 6.61 0.8
.+-. 0.2 flutamide Nilutamide 1.11 .+-. 0.17 5.1 .+-. 2.1 7.59 .+-.
1.18 0.6 .+-. 0.1
[0276] To test DNA binding competency of the F876L mutants VP16-AR
fusion constructs were generated. pVP16-AR was generated by
subloning full-length human AR into pVP16 (Clontech). AR point
mutations were generated in VP16-AR using the QuickChange II
Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara,
Calif.) according to the manufacturer's protocol.
[0277] Transcription assays were performed essentially as described
above. Triplicate transfections were performed using 35 ng pVP16-AR
or pVP16-F876L, 70 ng 4X ARE-Luciferase, 20 ng pRL (Promega), and
35 ng pCMX. 4X ARE-luciferase reporter activity was monitored in
the presence of increasing compound concentrations in the absence
or presence of 90 pM R1881 (for wild-type VP16-AR) or 1 nM R1881
(for F876L VP16-AR). Luciferase activity was measured as described
above.
[0278] Reflective of the transcriptional reporter assay, in the
wild-type VP16-AR assay, which monitors the DNA binding competency
of the receptor, enzalutamide and ARN-509 were full antagonists
(Table 7, FIG. 4A) (Emax=percent maximal R1881 response). However,
in the context of the VP16-AR-F876L, ARN-509 and enzalutamide
stimulated AR DNA binding. Thus, the mutation of AR F876 to L was
sufficient to convert the 2nd generation anti-androgens,
enzalutamide and ARN-509, to partial agonists.
TABLE-US-00011 TABLE 7 AR-VP16 WT IC.sub.50 WT F876L IC.sub.50
F876L Compound (.quadrature.M) Emax (.quadrature.M) Emax ARN-509
0.16 .+-. 0.06 3.98 .+-. 0.27 0.03 .+-. 0.02 53.98 .+-. 1.45
Enzalut- 0.21 .+-. 0.07 2.65 .+-. 0.73 0.05 .+-. 0.01 34.32 .+-.
5.38 amide Bicalutamide 0.18 .+-. 0.10 32.77 .+-. 5.76 2.51 .+-.
1.11 2.20 .+-. 1.35 Hydroxy- 0.03 .+-. 0.01 42.28 .+-. 4.44 0.97
.+-. 0.27 5.36 .+-. 5.35 flutamide Nilutamide 0.13 .+-. 0.08 33.53
.+-. 9.75 2.12 .+-. 0.68 2.90 .+-. 1.80
Example 9: Stable Cell Line Generation
[0279] In this example, cell lines were generated with stable
expression of AR F876L mutant. pSR.alpha.F876L, pQCXIN-AR and
pQCXIN-F876L retroviruses were first generated by co-transfecting
GP2-293 cells with pVSV-G (Clontech) according to the
manufacturer's protocol.
[0280] PC3 cells stably expressing wild-type or AR-F876L were
generated by transducing PC3 cells with either pQXIN-AR or
pQXIN-F876L retrovirus and selection in RPMI 1640 medium containing
500 .mu.g/mL gentamycin.
[0281] LNCaP cells stably expressing AR-F876L were generated by
either transfecting LNCaP cells with pCDNA-F876L or transducing
LNCaP cells with SR.alpha.F876L retrovirus. Individual clones of
LNCaP/pCDNA-F876L were isolated following selection in 400 .mu.g/mL
gentamycin. LNCaP/SR.alpha.F876L cell pools were selected 400 mg/mL
gentamycin.
[0282] AR protein expression of all cell lines was validated by
western analysis using AR N-20 antibody (Santa Cruz
Biotechnology).
Example 10: Equilibrium AR Binding Assays
[0283] Competitive binding assays of wild-type AR vs. F876L AR were
performed as described in Clegg et al. (2012) Cancer Research
72:1494-503 using PC3 cells stably expressing wild-type human AR or
AR-F876L as described above. Ki was calculated as
Ki=IC50/(1+([.sup.3H-R1881]/Kd)), [.sup.3H-R1881]=0.6 nM.
[0284] In equilibrium AR binding assays, ARN-509 and enzalutamide
bound the mutant with 30 and 48-fold higher affinity, respectively
(Table 8, FIGS. 5A-B) (Kd for R1881: AR=0.5 nM; AR-F877L=0.64 nM).
Thus, increased agonist activity on AR is associated with increased
binding affinity to both wild-type and F876L receptor, suggesting
that the agonist confirmation enables higher affinity through
decreased dissociation constant.
TABLE-US-00012 TABLE 8 AR binding, WT K.sub.i AR binding, F876L
K.sub.i Compound (nM) (nM) ARN-509 18.07 .+-. 7.46 0.68 .+-. 0.15
Enzalut- 26.30 .+-. 12.77 0.60 .+-. 0.17 amide Bicalutamide 26.56
.+-. 12.51 360.36 .+-. 283.85 Hydroxy- 14.56 .+-. 8.25 150.57 .+-.
55.10 flutamide Nilutamide 17.74 .+-. 5.65 197.42 .+-. 9.26
Example 11: Expression of AR Target Genes in and Proliferation of
F876L Stable Prostate Cancer Cell Lines
[0285] As shown above, expression of AR-F876L was sufficient to
confer enzalutamide and ARN-509 resistance in transient reporter
based assays. In this example, the effects of F876L on endogenous
AR target genes and proliferation in prostate cancer cells stably
expressing the mutant was examined. Two LNCaP cell lines
(LNCaP/SR.alpha.F876L and LNCaP/pCDNAF876L) were engineered as
described in Example 9 to overexpress AR-F876L at levels comparable
to the LNCaP/AR(cs) model.
[0286] To measure AR levels in the cells, protein extracts were
generated from LNCaP, LNCap/AR(cs), LNCaP/SR.alpha.F876L and
LNCaP/pCDNAF876L cells cultured in hormone depleted medium for 3
days. AR protein levels were analyzed by western blot. AR levels
were quantified and normalized to actin and expressed relative to
AR expression in LNCaP cells (FIG. 6)
[0287] For endogenous target gene analysis, LNCaP/AR(cs). LNCaP
SR.alpha.F876L, and LNCaP/pCDNAF876L cells were cultured for 3 days
in hormone depleted medium followed by treatment with vehicle, 1 nM
R1881 or 1, 3, 10 and 30 .mu.M ARN-509 or enzalutamide in the
presence or absence on 1 nM R1881.
[0288] For the proliferation assays, LNCaP/AR(cs), LNCaP
SR.alpha.F876L, and LNCaP/pCDNAF876L cells were cultured in the
presence of hormone depleted medium for 2 days followed by ligand
treatment for 7 days as described above. For antagonist assays,
ARN-509 or enzalutamide were added in the presence of 200 pM R1881
(100 pM final concentration). Proliferation was quantified by
CellTiter-Glo luminescence based viability assay as described
above.
[0289] In LNCaP/AR(cs) cells, ARN-509 and enzalutamide had little
effect on the induction of AR target genes or proliferative
activity (FIG. 7A, Table 9A). Both antagonists blocked R1881
induced transcription and proliferation to levels consistent with
their agonist activity at the highest concentration (FIG. 7B, Table
9B). In contrast, in F876L-AR expressing cells, both enzalutamide
and ARN-509 demonstrated robust transcriptional and proliferative
agonist activity (FIGS. 7A and 7B, Tables 9C-F).
TABLE-US-00013 TABLE 9A LNCaP/AR(cs) Agonist Transcription -R1881 1
nM Enzalutamide ARN-509 Gene Vehicle R1881 0.3 .mu.M 1 .mu.M 3
.mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M 30 .mu.M
AMIGO2 1.00 0.98 0.68 0.61 0.62 0.66 1.22 1.14 0.69 0.89 0.65 2.41
BDNF 1.00 0.86 1.04 0.92 0.88 0.95 0.98 1.15 0.93 1.03 1.03 1.80
CAM2KN1 1.00 0.04 0.80 0.70 0.71 0.64 0.64 0.90 0.76 0.99 0.78 1.56
FASN 1.00 4.12 0.47 0.32 0.29 0.31 0.58 0.46 0.40 0.42 0.41 1.30
FKBP5 1.00 100.51 0.91 0.62 0.75 0.61 0.85 0.99 0.71 0.91 0.75 1.87
HPGD 1.00 324.60 0.74 0.57 0.61 0.63 1.29 0.81 0.56 0.78 0.61 1.86
NCAPD3 1.00 95.54 0.66 0.57 0.47 0.56 0.79 0.72 0.74 0.73 0.74 1.34
NKX3.1 1.00 12.72 0.71 0.52 0.51 0.86 1.63 1.11 0.68 0.73 0.85 2.24
NOV 1.00 0.05 1.12 0.91 0.80 1.02 1.01 1.12 1.00 1.11 0.98 2.07
ORM1 1.00 6987.01 0.92 0.90 1.06 0.71 2.02 1.37 1.79 1.20 0.97 1.35
PLD1 1.00 0.03 0.77 0.62 0.63 0.56 0.56 1.28 0.66 0.88 0.65 1.33
PSA 1.00 22.14 0.42 0.32 0.38 0.74 1.60 0.44 0.49 0.58 0.61 1.13
SLUG 1.00 91.84 0.53 0.55 0.31 0.51 0.91 0.38 0.42 0.52 0.56 1.36
STEAP4 1.00 1498.46 0.75 0.73 0.52 1.16 1.28 0.94 0.77 1.05 0.56
1.01 TMPRSS2 1.00 35.42 0.75 0.53 0.63 0.89 1.45 0.99 0.64 1.04
0.62 2.78
TABLE-US-00014 TABLE 9B LNCaP/AR(cs) Antagonist Transcription +1 nM
R1881 1 nM Enzalutamide ARN-509 Gene Vehicle R1881 0.3 .mu.M 1
.mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M
30 .mu.M AMIGO2 1.00 0.98 0.49 0.19 0.48 0.78 0.92 0.30 0.31 0.52
0.60 0.76 BDNF 1.00 0.86 0.61 0.28 0.63 0.99 1.12 0.43 0.35 0.67
0.74 0.88 CAM2KN1 1.00 0.04 0.04 0.06 0.27 0.50 0.56 0.04 0.06 0.19
0.43 0.42 FASN 1.00 4.12 1.30 0.42 0.71 0.46 0.59 1.92 1.52 1.08
0.60 0.64 FKBP5 1.00 100.51 23.51 5.64 3.32 1.41 1.50 33.14 22.49
12.56 2.74 1.08 HPGD 1.00 324.60 99.16 16.30 13.19 3.85 4.25 105.97
76.25 32.19 8.36 2.78 NCAPD3 1.00 95.54 23.74 4.35 3.14 1.29 1.27
43.93 32.00 15.57 2.39 0.96 NKX3.1 1.00 12.72 7.70 3.77 6.72 4.70
5.03 8.32 9.92 12.01 7.22 5.49 NOV 1.00 0.05 0.11 0.14 0.61 1.00
0.92 0.03 0.09 0.31 0.86 0.82 ORM1 1.00 6987.01 3521.95 503.02
257.85 43.91 22.70 5100.90 2679.73 1031.40 156.90 16.42 PLD1 1.00
0.03 0.02 0.02 0.13 0.42 0.54 0.02 0.02 0.04 0.11 0.32 PSA 1.00
22.14 22.76 8.82 11.75 6.59 5.65 19.09 21.75 23.57 12.90 7.20 SLUG
1.00 91.84 50.08 10.20 10.56 5.26 5.22 71.33 62.00 50.70 11.15 5.49
STEAP4 1.00 1498.46 585.81 109.37 74.44 13.61 7.79 1019.98 742.61
256.89 32.98 6.86 TMPRSS2 1.00 35.42 19.88 7.72 10.51 3.58 4.62
19.07 24.11 23.38 11.45 5.94
TABLE-US-00015 TABLE 9C LNCaP/SR.alpha.F876L Agonist Transcription
-R1881 1 nM Enzalutamide ARN-509 Gene Vehicle R1881 0.3 .mu.M 1
.mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M
30 .mu.M AMIGO2 1.00 0.29 0.58 0.32 0.47 0.40 0.31 0.27 0.47 0.23
0.34 0.27 BDNF 1.00 1.65 1.04 1.30 1.01 0.99 1.55 0.79 1.07 0.75
0.84 0.94 CAM2KN1 1.00 0.03 0.52 0.48 0.38 0.23 0.37 0.30 0.24 0.17
0.21 0.18 FASN 1.00 3.76 0.50 0.64 0.58 1.09 1.34 0.64 1.01 0.93
1.50 2.77 FKBP5 1.00 66.89 1.38 1.14 2.54 9.61 23.67 2.66 5.95 5.56
13.02 27.89 HPGD 1.00 182.19 0.68 0.96 2.79 18.98 55.76 4.18 7.90
10.12 25.79 69.22 NCAPD3 1.00 31.69 0.77 1.01 1.09 3.56 8.83 1.39
1.58 1.69 3.53 9.35 NKX3.1 1.00 10.80 4.26 5.54 7.05 11.94 14.20
7.12 9.47 7.96 9.85 12.67 NOV 1.00 0.06 0.55 0.28 0.55 0.42 0.21
0.27 0.51 0.31 0.29 0.17 ORM1 1.00 6535.38 2.17 4.85 18.77 242.08
2114.41 44.44 58.96 101.45 459.30 1357.12 PLD1 1.00 0.02 0.67 0.76
0.60 0.42 0.41 0.49 0.44 0.30 0.45 0.36 PSA 1.00 3.43 1.95 2.25
3.02 4.05 5.02 3.71 3.26 3.00 5.07 5.16 SLUG 1.00 99.20 1.21 1.55
3.28 16.87 107.64 5.56 5.91 8.15 26.35 56.48 STEAP4 1.00 1706.02
1.13 2.15 9.98 96.53 343.81 20.36 27.49 46.06 187.24 479.64 TMPRSS2
1.00 25.55 3.11 3.85 5.39 9.20 18.61 6.36 6.29 5.84 10.90 14.20
TABLE-US-00016 TABLE 9D LNCaP/SR.alpha.F876L Antagonist
Transcription +1 nM R1881 1 nM Enzalutamide ARN-509 Gene Vehicle
R1881 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M
3 .mu.M 10 .mu.M 30 .mu.M AMIGO2 1.00 0.29 0.23 0.29 0.33 0.39 0.40
0.17 0.22 0.14 0.18 0.18 BDNF 1.00 1.65 0.53 0.64 0.62 0.51 0.71
0.74 0.76 0.57 0.50 0.63 CAM2KN1 1.00 0.03 0.08 0.12 0.19 0.24 0.32
0.03 0.03 0.06 0.11 0.17 FASN 1.00 3.76 1.29 1.22 0.85 0.94 1.58
2.23 2.03 1.39 1.30 2.66 FKBP5 1.00 66.89 17.90 14.89 10.21 17.33
40.52 36.57 39.48 26.46 29.94 22.71 HPGD 1.00 182.19 49.66 30.03
21.62 34.77 93.52 149.34 134.70 79.93 70.80 131.79 NCAPD3 1.00
31.69 6.34 4.28 2.21 3.44 13.64 19.83 15.47 8.71 6.50 11.82 NKX3.1
1.00 10.80 21.66 20.78 16.19 11.84 15.26 12.63 14.12 11.34 10.15
13.64 NOV 1.00 0.06 0.12 0.28 0.25 0.33 0.26 0.05 0.06 0.09 0.11
0.09 ORM1 1.00 6535.38 386.40 216.67 137.73 661.51 3496.87 3335.14
2343.29 1469.18 1608.16 3440.86 PLD1 1.00 0.02 0.04 0.08 0.14 0.37
0.48 0.01 0.01 0.02 0.08 0.20 PSA 1.00 3.43 5.84 5.63 4.47 4.54
4.64 4.93 4.38 4.59 5.28 4.17 SLUG 1.00 99.20 85.97 64.63 35.50
48.80 161.58 85.97 64.63 35.50 48.80 161.58 STEAP4 1.00 1706.02
259.94 147.25 83.20 275.88 645.04 259.94 147.25 83.20 275.88 645.04
TMPRSS2 1.00 25.55 14.05 13.81 11.78 12.92 20.19 14.05 13.81 11.78
12.92 20.19
TABLE-US-00017 TABLE 9E LNCaP/pCDNAF876L Agonist Transcription
-R1881 1 nM Enzalutamide ARN-509 Gene Vehicle R1881 0.3 .mu.M 1
.mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M 3 .mu.M 10 .mu.M
30 .mu.M AMIGO2 1.00 0.92 1.28 0.82 0.87 0.72 0.91 1.07 0.86 1.25
0.60 1.55 BDNF 1.00 1.01 1.54 1.04 1.11 0.82 0.64 1.01 0.76 0.87
0.85 1.16 CAM2KN1 1.00 0.02 0.55 0.96 0.51 0.37 0.23 0.47 0.55 0.34
0.41 0.27 FASN 1.00 22.20 0.91 0.72 0.48 1.27 3.66 1.16 1.42 1.59
1.79 7.86 FKBP5 1.00 145.63 2.18 2.91 4.28 10.54 42.25 7.24 7.82
9.52 17.39 55.55 HPGD 1.00 854.42 4.70 6.32 13.26 26.78 105.81
15.36 19.27 29.25 47.33 173.82 NCAPD3 1.00 169.67 1.95 1.94 3.68
9.41 35.36 5.83 6.52 8.28 20.42 51.1.3 NKX3.1 1.00 9.67 3.76 3.71
3.57 4.69 8.12 6.07 5.72 5.93 7.05 11.69 NOV 1.00 0.08 1.73 1.62
2.19 0.88 0.49 1.24 1.01 0.82 0.86 0.72 ORM1 1.00 12816.69 116.91
195.36 659.73 2530.13 4760.77 932.41 1371.33 2307.29 3322.63
5541.08 PLD1 1.00 0.75 1.37 0.80 0.69 0.29 0.45 0.78 0.69 1.01 0.68
0.48 PSA 1.00 12.17 3.56 4.04 5.29 9.53 11.70 6.74 7.19 8.25 10.06
12.99 SLUG 1.00 268.77 1.99 3.53 4.74 14.91 55.93 7.84 8.70 10.91
21.21 71.08 STEAP4 1.00 3084.81 10.38 15.58 46.50 240.68 520.15
113.39 113.29 173.77 317.80 597.09 TMPRSS2 1.00 26.85 4.98 6.46
6.09 8.51 21.78 9.49 8.51 9.70 11.09 23.01
TABLE-US-00018 TABLE 9F LNCaP/pCDNAF876L Antagonist Transcription
+1 nM R1881 1 nM Enzalutamide ARN-509 Gene Vehicle R1881 0.3 .mu.M
1 .mu.M 3 .mu.M 10 .mu.M 30 .mu.M 0.3 .mu.M 1 .mu.M 3 .mu.M 10
.mu.M 30 .mu.M AMIGO2 1.00 0.92 0.51 0.43 0.39 0.49 0.52 0.24 0.31
0.43 0.41 0.40 BDNF 1.00 1.01 0.59 0.71 0.53 0.50 0.41 0.28 0.41
0.42 0.45 0.34 CAM2KN1 1.00 0.02 0.05 0.16 0.17 0.21 0.14 0.01 0.02
0.06 0.11 0.06 FASN 1.00 22.20 5.29 2.44 1.55 2.08 4.22 3.89 5.52
3.22 2.10 3.98 FKBP5 1.00 145.63 53.18 26.29 10.03 14.22 32.97
45.69 48.95 38.89 24.82 30.26 HPGD 1.00 854.42 149.44 59.67 21.66
35.90 96.55 192.05 170.69 107.79 73.28 87.03 NCAPD3 1.00 169.67
101.65 31.49 14.34 16.69 42.06 68.41 90.52 62.27 22.96 44.10 NKX3.1
1.00 9.67 8.53 7.05 5.69 5.54 7.61 3.08 5.95 5.42 4.81 5.24 NOV
1.00 0.08 0.08 0.17 0.32 0.49 0.39 0.02 0.10 0.12 0.26 0.18 ORM1
1.00 12816.69 4375.20 1581.76 587.00 1377.92 3765.54 4802.10
4943.10 3366.19 2576.38 2470.99 PLD1 1.00 0.75 0.08 0.12 0.09 0.11
0.12 0.04 0.12 0.05 0.06 0.04 PSA 1.00 12.17 16.08 12.73 7.92 8.12
14.27 10.36 14.01 12.21 10.27 13.14 SLUG 1.00 268.77 166.04 86.01
30.23 36.56 98.34 112.06 134.56 113.34 69.55 85.38 STEAP4 1.00
3084.81 524.97 156.24 47.11 92.80 214.69 633.57 587.91 376.66
195.78 232.63 TMPRSS2 1.00 26.85 19.03 16.05 8.14 8.92 15.32 12.13
15.75 14.19 12.99 12.67
Example 12: Interaction of N and C Expression of AR Target Genes in
and Proliferation of F876L Stable Prostate Cancer Cell Lines
[0290] Interaction of the AR amino-terminus with the AR
carboxy-terminus is important for the full AR transactivation
capacity. Many AR full and partial agonists stimulate the AF2
dependent N-C interaction. A N-C two hybrid interaction assay was
performed to assess the interaction of the AR N- and C-termini in
the F87L mutant in the presence of ARN-509 and enzalutamide.
[0291] pM-AR1-660, pVP16-AR507-919 and pVP16-F876L507-919 were
generated by subcloning appropriate PCR products from pCDNA3-AR and
pCDNA3-F876L into pM and pVP16 (Clontech). For N-C terminal
interaction assays, triplicate transfections were performed using
50 ng pM-AR1-660, 75 ng pVP16-AR507-919 or pVP16-F876L507-919, 25
ng pMH100-Luc and 10 ng pRL (Promega). Transfected cells were
incubated 4 hours then treated with ligand. ARN-509 and
enzalutamide were assayed at 8 .mu.M and R1881 at 1 nM.
[0292] Consistent with their transcriptional activities,
enzalutamide and ARN-509 promoted the N-C interaction of AR-F876L
but not wild-type AR (FIG. 8). Thus, the agonist activity of
ARN-509 and enzalutamide on AF-F876L is associated with an
agonist-like AF-2 conformation.
Example 13: Chromatin Immunoprecipitation Assay of AR
[0293] Transcriptional activation of androgen regulated AR target
genes requires agonist-induced DNA binding and subsequent
recruitment of transcriptional coregulators. To confirm the VP16-AR
reporter results indicating ARN-509 and enzalutamide stimulate
AR-F876L DNA binding, we performed chromatin immunoprecipitation
(ChIP) analysis of 6 AR target genes from cells treated with R1881
and/or each antagonist was performed.
[0294] ChIP assays were performed as described in Joseph et al.
(2009) PNAS USA 106:12178-83]. Briefly, LNCaP/AR(cs) and LNCaP
SR.alpha.F876L cells were plated in 150 mm dishes (7.times.10.sup.6
cells in 20 mL) in RPMI 1640 supplemented with 10% CSS for 3 days.
Cells were treated with 10 .mu.M antagonist in the presence or
absence of 1 nM R1881 for 4 hours. Following ligand treatment,
formaldehyde was added to the media to a final concentration of 1%,
incubated for 10 min and quenched with glycine (125 mM final
concentration) for 5 minutes. Cells were washed 3.times. with PBS
containing 1.times. Halt.TM. Protease & Phosphatase Single-Use
Inhibitor Cocktail (1.times. PI, Thermo Scientific), pelleted,
lysed in 1 mL RIPA buffer (50 mM Tris pH7.5, 0.15 M NaCl, 1% NP-40,
0.5% Na-deoxycholate, 0.05% SDS, 1 mM EDTA, 1.times. PI) and
sonicated until the average DNA size fragment was .apprxeq.500 bp.
The sonicated cross-linked chromatin was diluted into 3.3 mL RIPA
and precleared with 100 mL 50% protein A/G agarose slurry (SC-2003,
Santa Cruz Biotechnology) containing 200 mg per mL sonicated salmon
sperm DNA and 500 mg per mL of BSA. One mL of precleared chromatin
was then immunoprecipitated with 15 .mu.g anti-AR (SC-816, Santa
Cruz Biotechnology) or normal rabbit IgG (SC-2027, Santa Cruz
Biotechnology), for 2 hours at 4.degree. C. and 100 mL of a 50%
slurry of protein A/G agarose beads were added and incubated
overnight at 4.degree. C. Beads were washed 2 times sequentially in
low-salt buffer (50 mM HEPES pH 7.8, 140 mM NaCl. 1 mM EDTA, 1%
Triton X-100. 0.1% Na-deoxycholate, 0.1% SDS), high-salt buffer
(same as low-salt with 500 mM NaCl), LiCl buffer (20 mM Tris pH
8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), and
TE buffer (50 mM Tris pH 8.0, 1 mM EDTA). All washing steps were
performed in the presence of 1.times. PI. Protein-DNA complexes
were eluted in 225 mL Elution buffer (50 mM Tris pH 8.0, 1 mM EDTA,
1% SDS) at 65.degree. C. twice for 15 minutes. Eluted protein-DNA
complexes were reverse cross-linked in the presence of NaCl
overnight at 65.degree. C. and further treated with EDTA and
proteinase K at 42.degree. C. for 1 hour. The DNA fragments were
purified in 10 mM Tris pH 8.5 using the QIAquick PCR purification
kit (Qiagen), diluted and analyzed by real-time PCR using iTaq SYBR
Green Supermix with ROX (Bio-Rad). The samples were amplified on
the ABI 7900HT instrument. Oligonucleotide primer sequences are
listed in Table 8.
TABLE-US-00019 TABLE 8 ChIP Real-time PCR Oligonucleotide Sequence
Gene Forward Primer Sequence Reverse Primer Sequence PSA E2
ACCTGCTCAGCCTTTGTCTCTGAT AGATCCAGGCTTGCTTACTGTCCT (SEQ ID NO: 50)
(SEQ ID NO: 51) PSA D1 ATTCTGGGTTGGGAGTGCAAGGA
AGGAGACATGCCCAGGATGAAAC A A (SEQ ID NO: 52) (SEQ ID NO: 53) STEAP4
ACTAGGCAGGACATTGACATCCC ACAGTAAACCTCTCCACACATGG A C (SEQ ID NO: 54)
(SEQ ID NO: 55) FASN TATGACACCCAGGGCTTTCGTTC
TAACGTTCCCTGCGCGTTTACAGA A (SEQ ID NO: 57) (SEQ ID NO: 56) TMPRSS2
TCCCAAATCCTGACCCCA ACCACACAGCCCCTAGGAGA (SEQ ID NO: 58) (SEQ ID NO:
59) NKX3. 1 ACAGGGTGGCCCAAATAGAAC CCTGTCTTGGACAAGCGGAGA (SEQ ID NO:
60) (SEQ ID NO: 61) ORM1 GGGTCATTTCCACCACCTCAAAC
GGAGAAAGGCCTTACAGTAGTCT A C (SEQ ID NO: 62) (SEQ ID NO: 63)
[0295] In the LNCaP/AR(cs) cells, R1881 promoted AR DNA (FIG. 9).
Consistent with the VP16-AR reporter data, both ARN-509 and
enzalutamide promoted AR DNA binding LNCaP/SR.alpha.F876L cells. In
the presence of R1881, all antagonists inhibited R1881-stimulated
AR DNA binding to levels consistent with their partial agonist or
antagonist activity in both cell lines (FIG. 9).
Example 14: In Vivo Effects of AR F876L
[0296] To determine whether the F876L alteration conveys resistance
to enzalutamide and ARN-509 in vivo, LNCaP cell lines stably
expressing F876L AR were injected (s.c.) into castrated
immune-deficient mice and tumors established.
[0297] All animal studies were carried out under protocols approved
by the Institutional Animal Care and Use Committees and
institutional guidelines for the proper, humane use of animals in
research were followed. In vivo xenograft experiments were carried
out in SCID Hairless Outbred (SHO) male mice (Charles River
Laboratories). Mice were orchiectomized under isoflorane anesthesia
and were given 7-10 days to recover. LNCaP/AR(cs) or
LNCaP/SR.alpha.F876L cells (as described above) were suspended in
50% RPMI, 50% Matrigel (BD Biosciences), and 3.times.10.sup.6
cells/xenograft were injected in a volume of 100 .mu.L. Animals
were observed weekly until tumor growth was apparent. After 40-60
days post-injection, animals were randomized into cohorts of
equivalent tumor burden mean (150-250 mm.sup.3) and range. All
compounds were administered daily by oral gavage at a dose of 30
mg/kg/day compound. For all LNCaP/AR(cs) xenograft studies ARN-509
and enzalutamide drug stocks were prepared in 18% PEG-400, 1%
Tween-80 and 1% povidone, and were formulated for dosing in 15%
Vitamin E-TPGS and 65% of a 0.5% w/v CMC solution in 20 mM citrate
buffer (pH 4.0). ARN-509 and enzalutamide pharmacokinetics were
assessed at the end of study as described previously (Clegg et al.
(2012) Cancer Research 72:1494-503).
[0298] Consistent with the in vitro data, neither ARN-509 nor
enzalutamide 30 mg/kg/day impacted the growth of
LNCaP/AR/SR.alpha.F876L tumors (FIG. 10). This lack of activity was
not a function of unexpectedly low compound exposure as day 28
plasma drug levels were quantified and were consistent with
previous LNCaP/AR xenograft studies (Table 9). In addition, in a
parallel experiment, ARN-509 30 mg/kg/day exhibited robust
anti-tumor activity in the LNCaP/AR(cs) model, consistent with
previous results (FIG. 10).
TABLE-US-00020 TABLE 9 LNCaP/SR.alpha.F876L Xenograft
Pharmacokinetics AUC.sub.0-24 C.sub.24 T.sub.1/2 (.mu.g hr/
C.sub.max T.sub.max Compound Dose (.mu.g/mL) (hr) mL) (.mu.g/mL)
(hr) Enzalutamide 30 mg/kg 9.14 9.9 527.3 33.5 1.0 ARN-509 30 mg/kg
1.02 7.1 98.9 9.11 1.0
Example 15: An Open-Label. Phase 1/2 Safety. Pharmacokinetic, and
Proof-of-Concept Study of ARN-509 in Patients with Progressive
Advanced Castration-Resistant Prostate Cancer (CRPC)
[0299] In this study, DNA was isolated from 29 patient plasma
samples patients participating in a Phase 1/2 clinical study
ARN-509 treatment for prostate cancer. These were analyzed using
the emulsion PCR-based BEAMing Technology method (Dressman et al.
(2003) PNAS USA 100:8817-22). BEAMing has been successfully used to
detect a variety of tumor derived mutations in driver oncogenes
such as PIKC3a and K-ras in ctDNA derived from human plasma (Diehl
et al. (2008) Nature Medicine 14:985-90). The AR F876L mutation was
identified in 3 of the 29 patient samples tested.
[0300] The clinical study performed was multi-institution, first in
man. Phase 1/2, dose-escalation and proof-of-concept study across 9
dose levels in which eligible patients with progressive advanced
CRPC received oral doses of ARN-509 on an outpatient basis to
determine the safety, pharmacokinetics (PK) and preliminary
evidence of the anti-tumor effects of ARN-509.
[0301] Patients with mCRPC were assigned sequentially to dose
levels in cohorts of 3 to 6 patients per dose level using standard
3.times.3 dose-escalation criteria.
[0302] The objective was to determine the maximum tolerated dose
(MTD) and/or recommended Phase 2 dose (RP2D) of ARN-509 that leads
to a dose-limiting toxicity (DLT) in a maximum of 30% of patients.
A DLT is generally defined as any Grade 1 or higher seizure, any
Grade 3-4 non-hematologic toxicity (GI toxicities must persist at
Grade 3-4 despite maximal medical therapy) and/or grade 4
hematologic toxicity of more than 5 days duration, defined by CTCAE
V4.0, and that is assessed as related to ARN-509 treatment. A
schematic of the study design is provided in FIG. 11.
[0303] Eligible patients who signed informed consent documents were
initially enrolled into a dose escalation cohort where they will
receive a single oral dose of ARN-509 followed by a one-week
observation period (PK Week). Continuous dosing began on Cycle 1
Day 1 assuming no unacceptable toxicities were observed.
[0304] A minimum of 3 subjects at each dose level were monitored
for a DLT through day 28 of Cycle 1. If no DLTs were observed in
the first 3 patients at each dose level, subsequent enrollment
proceeded at the next dose level. If 2 or more patients experienced
a DLT at a given dose level or a single episode of seizure of any
grade was observed at a given dose level, dose escalation was
stopped and the MTD was defined as the previous dose level. If no
DLTs were observed, RP2D was based on the overall pharmacokinetic
and safety profile of ARN-509 and the optimal biological dose
determined from preclinical data, and not necessarily the MTD.
[0305] The starting dose was 30 mg/day, with escalations to 60 mg,
90 mg, 120 mg, 180 mg, 240 mg, 300 mg, 390 mg, and 480 mg daily. It
was anticipated that these dose levels span the anticipated
pharmacologically active dose range and be within the safety margin
indicated by the preclinical toxicology results.
[0306] Following the selection of 240 mg as the RP2D, additional
eligible patients were enrolled in the Phase 2 portion of the
study, consisting of 3 concurrent expansion cohorts at the MTD
and/or RP2D to gather additional safety information and provide an
initial signal of anti-tumor activity. The three expansion cohorts
included:
[0307] 1. Non-metastatic CRPC treatment naive (50 patients with
non-metastatic CRPC who are chemotherapy and abiraterone treatment
naive);
[0308] 2. Metastatic CRPC treatment-naive (20 patients with mCRPC
who are chemotherapy and abiraterone naive); and
[0309] 3. Metastatic CRPC abiraterone treated (10-20 patients).
[0310] It was expected that each patient with mCRPC would receive
at least 3 cycles (12 weeks) of continuous treatment and each
patient with non-metastatic CRPC will receive at least 4 cycles (16
weeks) of continuous treatment. Treatment was discontinued at any
time for protocol-defined disease progression or unacceptable
toxicity. Tumor evaluations were performed every 3 cycles (12
weeks) for patients with mCRPC and every 4 cycles (16 weeks) for
patients with non-metastatic CRPC. Safety was assessed from the
first dose through at least four weeks after the last dose or until
resolution of drug-related toxicity, or when toxicity is deemed
irreversible, whichever was longer. The effect of food on the
absorption of ARN-509 and the effect of ARN-509 on ventricular
repolarization was evaluated in the Phase 2 expansion phase at
selected clinical sites.
[0311] Analysis of the samples using BEAMing technology in the
current ARN-509 clinical study was carried out by Inostics GMBH.
Blood was collected in K2-EDTA evacuated tubes and mix thoroughly
by slowly inverting several times. Within 30 minutes of collection
tubes were spun at 2000.times.g for 15 minutes. Plasma was
decanted, transferred to cryo storage tubes. Within 90 minutes of
decantation, plasma was stored at -70.degree. C. or lower until
analysis. DNA was purified from 300-500 .mu.l plasma aliquots and
extracted as described in Diehl et al. (2008) Nature medicine
14:985-90. Mutation detection was performed according to BEAMing
technology as described in Diehl et al. Briefly, in the initial PCR
step, the target region (.about.100 bp) was amplified using
gene-specific primers with tag sequences and subjected to an
emulsion PCR containing primer coated magnetic beads. After
emulsion PCR discrimination of wildtype and mutant beads was
performed by allele-specific hybridization followed flow cytometry.
Flow cytometry results were analyzed using FCS Express (De Novo
Software, Los Angeles, Calif.) resulting in the quantification of
the ratio of the mutant allele over the wild type alleles.
[0312] Samples were analyzed for the presence of wild type AR or 3
single point mutant alleles that could result in F876L (t2988c,
c2990a, and c2990g (or t2626c, c2628a, and c2928g, respectively,
relative to the AR nucleic acid coding sequence set forth in SEQ ID
NO: 18). Analyzed mutations and the technical sensitivity of the
BEAMing Method are shown in Table 10. For detection, the frequency
has to be above the total amount of genome equivalent used per
assay. For example, if in a sample, 1,000 genomic equivalent are
present, yet the calculated fraction of mutant DNA molecules is
0.02% (1 mutant allele in 5,000 wild-type alleles), the samples is
scored as wild type.
TABLE-US-00021 TABLE 10 AR nucleotide changes monitored by BEAMing
assay Nucleotide Nucleotide Amino Acid Amino Acid Position Change
Position Change c.2626 t > c 876 F > L c.2628 c > a 876 F
> L c.2628 c > g 876 F > L
[0313] Results
[0314] A subset of patients across all dose groups exhibited PSA
response with 14/30 patients exhibiting a 12 week.gtoreq.50%
decline in PSA compared to baseline. The PSA response for the 29
patients screened is depicted in FIG. 12A. Pre-treatment and during
treatment plasma samples were analyzed. Time of BEAMing analysis is
indicated by the terminal end of the PSA response line. Eighteen
out of the 29 patients had PSA above of baseline at time of
analysis indicating either intrinsic or acquired resistance to
ARN-509.
[0315] Three probes were designed to monitor the 3 nucleotide
changes that can encode for the F876L amino acid substitution.
Dilution mixing experiments with the mutant sequence and wild-type
DNA indicated a technical sensitivity of 0.02% (potential to detect
1 mutant sequence among 5000 wild-type). In an initial screen of
plasma samples of 29 ARN-509 treated patients, evidence of the
mutation was detected in 3 patients (Tables 11 and 12). At time of
BEAMing analysis, Patient 7 and 10 had PSA levels above baseline,
whereas Patient 13 has evidence of rising PSA above the treatment
nadir (FIG. 13). In all 3 patients, the nucleotide change c2628a
was detected. In one of these 3 patients (Patient 10) the t2626c
mutant was also detected, indicative of polyclonal disease. F876L
encoding mutations were not detected in any of pre-treatment
samples (0/29) suggesting that if present prior to ARN-509
treatment they were below the limit of detection or that the
mutations arose de novo during ARN-509 treatment. In either
scenario, the data support the hypothesis that the selective
outgrowth of lesions bearing the mutant allele to levels sufficient
to detect in ctDNA is dependent on chronic exposure to ARN-509 and
is associated with rising PSA. To further establish the association
of F876L with progressive disease, we analyzed plasma samples taken
at additional timepoints from the 3 patients scored positive during
the initial screen (Table 12) In patient 10, the mutation was not
detected at the one other timepoint analyzed (Cycle 4; PSA 102% of
TO). In Patient 13, the mutation was not detected at Cycle 4 (PSA
16.2% of baseline) or at Cycle 12 (patient was scored positive at
Cycle 11). The mutant sequence at Cycle 11 was at the limit of
detection and is estimated to arise via amplification of a single
mutant molecule. Although PSA of Patient 13 was slowly rising from
the treatment nadir at Cycle 11 and 12, at both time points PSA was
still>60% below study start, and thus frank resistance had not
yet emerged. Identification of the mutant sequence at the limit of
detection likely reflects presence of a relatively rare, mutant
clone that has potential to expand under continued selective
pressure and eventually drive progressive disease.
[0316] Given the relatively long duration of treatment of Patient
7, plasma from additional time points during evident PSA reduction;
(>90% decline from baseline; Cycle 4, 8 and 10) and at initial
PSA rise from its nadir (Cycle 15 and 19) (FIG. 13) was analyzed.
Interestingly, mutations were not detected in the 3 samples from
treatment cycle 4, 8 and 10 whereas the c2628a mutation was
detected in the 2 samples analyzed from initial PSA rise (Cycle 15
and 19). These clinical data are consistent with the preclinical
data indicating that the F876L amino acid change is sufficient to
convey resistance to ARN-509.
TABLE-US-00022 TABLE 11 BEAMing Results from F876L Positive
Patients PSA Mutant Frequency Treatment [Percent Genotypic
[mutant/w.t. beads .times. 100] Patient Cycle* of Day 0] Call
c2990a c2990g t2988c 7 0 100 Wild-type -- -- -- 7 4 1.4 Wild-type
-- -- -- 7 8 3.3 Wild-type -- -- -- 7 10 5.6 Wild-type -- -- -- 7
15 41.2 Mutant 0.162 -- -- 7 19 97.2 Mutant 5.005 -- -- 7 22 281.0
Mutant 1.002 -- -- 10 0 100 Wild-type -- -- -- 10 4 102 Wild-type
-- -- -- 10 7 245 Mutant 0.051 -- 0.12 13 0 100 Wild-type -- -- --
13 4 16.2 Wild-type -- -- -- 13 11 31.7 Mutant 0.065 -- -- 13 12
39.5 Wild-type -- -- -- *Treatment cycle was 4 weeks; Cycle 0 is
pre-treatment timepoint.
TABLE-US-00023 TABLE 12 Primary F876L BEAMing of ARN-509-001
Patients PSA at Time BEAMing of BEAMing During 12 week Analysis
Analysis Baseline Treatment PSA Treatment [Percent of Genotypic
Genotypic Patient Response Cycle* Baseline] Call.sup.# Call 1 30.49
19 195.4 w.t. w.t. 2 -61.8 10 337.08 w.t. w.t. 3 22.7 4 122 w.t.
w.t. 4 12.4 5 134 w.t. w.t. 5 -70.65 8 16 w.t. w.t. 6 -90.02 10
84.6 w.t. w.t. 7 -98.6 22 281 w.t. Mutant 8 -62.2 16 10.2 w.t. w.t.
9 26.38 7 185 w.t. w.t. 10 2.33 7 245 w.t. Mutant 11 -49.76 8
143.88 w.t. w.t. 12 -56.65 7 99.47 w.t. w.t. 13 -83.79 11 31.7 w.t.
Mutant 14 -43.21 11 150.5 w.t. w.t. 15 164.14 3 220 w.t. w.t. 16
89.09 4 189 w.t. w.t. 17 -45.86 4 54.14 w.t. w.t. 18 -95.65 6 7.34
w.t. w.t. 19 -71.19 11 196.86 w.t. w.t. 20 -30.88 21 89.3 w.t. w.t.
21 -74.43 10 46.91 w.t. w.t. 22 -82.7 25 0.19 w.t. w.t. 23 97.78 8
222.46 w.t. w.t. 24 -74.86 25 4.89 w.t. w.t. 25 -30.07 12 161 w.t.
w.t. 26 96.51 5 214.85 w.t. w.t. 27 -0.89 13 20.69 w.t. w.t. 28
-33.59 10 126 w.t. w.t. 29 -58.44 13 105 w.t. w.t.
Example 16: Method for Generation of Cell Lines for Drug
Screening
[0317] To identify compounds that retain AR antagonist activity in
the context of the F876L mutation of the androgen receptor, a
number of in vitro and in vivo assays as described above are
adapted.
[0318] In an in vitro setting, a transient transfection
transcription assays similar to that described in Example 8 is used
to identify compounds that are devoid of agonist activity and fully
antagonize both the wild-type as well as F876L mutant AR
transcriptional activity. HEPG2 cells or any eukaryotic cell in
which AR transcriptional activity can be monitored is employed for
this screen.
[0319] As an alternative to transient transfection, the
transcriptional reporter and F876L AR is stably integrated in a
number of cell lines and used for screening compounds. The stable
integration of the mutant F876L AR into an androgen sensitive
prostate cancer cell line such as LNCaP, LaPC4 or VCaP through the
use of plasmid (i.e. pCDNA3.1) or viral based integration allows
the screening and evaluation of compounds in transcriptional,
proliferation and xenograft setting. Briefly, the F876L AR is
cloned into a retroviral expression vector such as pQCXIP
(Clontech, Mountain View, Calif.). The resultant plasmid is then
used to generate high titer viral stocks for use in the generation
of stably transduced cell lines according to the manufacturer's
protocol. The resulting cell lines are used in transient
transfection transcriptional assays as described in Example 4,
endogenous gene transcriptional assays as described in Example 5,
proliferation assays as described in Example 3 or xenograft studies
as described in Example 1.
[0320] Alternatively the cells are further modified by the stable
integration of an AR responsive reporter such as Cignal Lenti AR
Reporter (Qiagen, Valencia. Calif.) allowing reporter based
compound screening without the need for transient transfection.
[0321] The examples and embodiments described herein are for
illustrative purposes only and various modifications or changes
suggested to persons skilled in the art are to be included within
the spirit and purview of this application and scope of the
appended claims.
Sequence CWU 1
1
631919PRTHomo sapiens 1Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr
Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn
Leu Phe Gln Ser Val Arg Glu 20 25 30 Val Ile Gln Asn Pro Gly Pro
Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45 Pro Pro Gly Ala Ser
Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr 65 70 75 80 Ser
Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro Gln 85 90
95 Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln
100 105 110 Gln Pro Ser Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu
Arg Gly 115 120 125 Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser
Lys Gly Leu Pro 130 135 140 Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp
Asp Ser Ala Ala Pro Ser 145 150 155 160 Thr Leu Ser Leu Leu Gly Pro
Thr Phe Pro Gly Leu Ser Ser Cys Ser 165 170 175 Ala Asp Leu Lys Asp
Ile Leu Ser Glu Ala Ser Thr Met Gln Leu Leu 180 185 190 Gln Gln Gln
Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly Arg 195 200 205 Ala
Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr Leu 210 215
220 Gly Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys Ala
225 230 235 240 Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu
His Leu Ser 245 250 255 Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr
Ala Pro Leu Leu Gly 260 265 270 Val Pro Pro Ala Val Arg Pro Thr Pro
Cys Ala Pro Leu Ala Glu Cys 275 280 285 Lys Gly Ser Leu Leu Asp Asp
Ser Ala Gly Lys Ser Thr Glu Asp Thr 290 295 300 Ala Glu Tyr Ser Pro
Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu Gly 305 310 315 320 Glu Ser
Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly Thr 325 330 335
Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp 340
345 350 Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu
Ala 355 360 365 Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro
His Ala Arg 370 375 380 Ile Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser
Ala Trp Ala Ala Ala 385 390 395 400 Ala Ala Gln Cys Arg Tyr Gly Asp
Leu Ala Ser Leu His Gly Ala Gly 405 410 415 Ala Ala Gly Pro Gly Ser
Gly Ser Pro Ser Ala Ala Ala Ser Ser Ser 420 425 430 Trp His Thr Leu
Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Cys 435 440 445 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460
Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala Pro Tyr 465
470 475 480 Gly Tyr Thr Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Ser
Asp Phe 485 490 495 Thr Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val
Ser Arg Val Pro 500 505 510 Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu
Met Gly Pro Trp Met Asp 515 520 525 Ser Tyr Ser Gly Pro Tyr Gly Asp
Met Arg Leu Glu Thr Ala Arg Asp 530 535 540 His Val Leu Pro Ile Asp
Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu 545 550 555 560 Ile Cys Gly
Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys 565 570 575 Gly
Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys 580 585
590 Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg
595 600 605 Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala
Gly Met 610 615 620 Thr Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn
Leu Lys Leu Gln 625 630 635 640 Glu Glu Gly Glu Ala Ser Ser Thr Thr
Ser Pro Thr Glu Glu Thr Thr 645 650 655 Gln Lys Leu Thr Val Ser His
Ile Glu Gly Tyr Glu Cys Gln Pro Ile 660 665 670 Phe Leu Asn Val Leu
Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly 675 680 685 His Asp Asn
Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu 690 695 700 Asn
Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys 705 710
715 720 Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala
Val 725 730 735 Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met
Gly Trp Arg 740 745 750 Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr
Phe Ala Pro Asp Leu 755 760 765 Val Phe Asn Glu Tyr Arg Met His Lys
Ser Arg Met Tyr Ser Gln Cys 770 775 780 Val Arg Met Arg His Leu Ser
Gln Glu Phe Gly Trp Leu Gln Ile Thr 785 790 795 800 Pro Gln Glu Phe
Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile 805 810 815 Pro Val
Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg Met 820 825 830
Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn 835
840 845 Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu
Asp 850 855 860 Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr
Phe Asp Leu 865 870 875 880 Leu Ile Lys Ser His Met Val Ser Val Asp
Phe Pro Glu Met Met Ala 885 890 895 Glu Ile Ile Ser Val Gln Val Pro
Lys Ile Leu Ser Gly Lys Val Lys 900 905 910 Pro Ile Tyr Phe His Thr
Gln 915 2 920 PRTHomo sapiens 2Met Glu Val Gln Leu Gly Leu Gly Arg
Val Tyr Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe
Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30 Val Ile Gln Asn Pro
Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45 Pro Pro Gly
Ala Ser Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70
75 80 Glu Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly
Ser 85 90 95 Pro Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val
Leu Asp Glu 100 105 110 Glu Gln Gln Pro Ser Gln Pro Gln Ser Ala Leu
Glu Cys His Pro Glu 115 120 125 Arg Gly Cys Val Pro Glu Pro Gly Ala
Ala Val Ala Ala Ser Lys Gly 130 135 140 Leu Pro Gln Gln Leu Pro Ala
Pro Pro Asp Glu Asp Asp Ser Ala Ala 145 150 155 160 Pro Ser Thr Leu
Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser 165 170 175 Cys Ser
Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln 180 185 190
Leu Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser 195
200 205 Gly Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp
Asn 210 215 220 Tyr Leu Gly Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys
Glu Leu Cys 225 230 235 240 Lys Ala Val Ser Val Ser Met Gly Leu Gly
Val Glu Ala Leu Glu His 245 250 255 Leu Ser Pro Gly Glu Gln Leu Arg
Gly Asp Cys Met Tyr Ala Pro Leu 260 265 270 Leu Gly Val Pro Pro Ala
Val Arg Pro Thr Pro Cys Ala Pro Leu Ala 275 280 285 Glu Cys Lys Gly
Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu 290 295 300 Asp Thr
Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu 305 310 315
320 Glu Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser
325 330 335 Gly Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser
Gly Ala 340 345 350 Leu Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr
Tyr Asn Phe Pro 355 360 365 Leu Ala Leu Ala Gly Pro Pro Pro Pro Pro
Pro Pro Pro His Pro His 370 375 380 Ala Arg Ile Lys Leu Glu Asn Pro
Leu Asp Tyr Gly Ser Ala Trp Ala 385 390 395 400 Ala Ala Ala Ala Gln
Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly 405 410 415 Ala Gly Ala
Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser 420 425 430 Ser
Ser Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly 435 440
445 Pro Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val
Ala Pro 465 470 475 480 Tyr Gly Tyr Thr Arg Pro Pro Gln Gly Leu Ala
Gly Gln Glu Ser Asp 485 490 495 Phe Thr Ala Pro Asp Val Trp Tyr Pro
Gly Gly Met Val Ser Arg Val 500 505 510 Pro Tyr Pro Ser Pro Thr Cys
Val Lys Ser Glu Met Gly Pro Trp Met 515 520 525 Asp Ser Tyr Ser Gly
Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg 530 535 540 Asp His Val
Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys 545 550 555 560
Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr 565
570 575 Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys
Gln 580 585 590 Lys Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp
Lys Phe Arg 595 600 605 Arg Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys
Cys Tyr Glu Ala Gly 610 615 620 Met Thr Leu Gly Ala Arg Lys Leu Lys
Lys Leu Gly Asn Leu Lys Leu 625 630 635 640 Gln Glu Glu Gly Glu Ala
Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr 645 650 655 Thr Gln Lys Leu
Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro 660 665 670 Ile Phe
Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala 675 680 685
Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser 690
695 700 Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp
Ala 705 710 715 720 Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp
Asp Gln Met Ala 725 730 735 Val Ile Gln Tyr Ser Trp Met Gly Leu Met
Val Phe Ala Met Gly Trp 740 745 750 Arg Ser Phe Thr Asn Val Asn Ser
Arg Met Leu Tyr Phe Ala Pro Asp 755 760 765 Leu Val Phe Asn Glu Tyr
Arg Met His Lys Ser Arg Met Tyr Ser Gln 770 775 780 Cys Val Arg Met
Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile 785 790 795 800 Thr
Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile 805 810
815 Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg
820 825 830 Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys
Arg Lys 835 840 845 Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu
Thr Lys Leu Leu 850 855 860 Asp Ser Val Gln Pro Ile Ala Arg Glu Leu
His Gln Phe Thr Phe Asp 865 870 875 880 Leu Leu Ile Lys Ser His Met
Val Ser Val Asp Phe Pro Glu Met Met 885 890 895 Ala Glu Ile Ile Ser
Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val 900 905 910 Lys Pro Ile
Tyr Phe His Thr Gln 915 920 3 369 PRTHomo sapiens 3Tyr Tyr Phe Pro
Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala 1 5 10 15 Ser Gly
Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys Val Phe 20 25 30
Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg 35
40 45 Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro Ser
Cys 50 55 60 Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly
Ala Arg Lys 65 70 75 80 Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln Glu
Glu Gly Glu Ala Ser 85 90 95 Ser Thr Thr Ser Pro Thr Glu Glu Thr
Thr Gln Lys Leu Thr Val Ser 100 105 110 His Ile Glu Gly Tyr Glu Cys
Gln Pro Ile Phe Leu Asn Val Leu Glu 115 120 125 Ala Ile Glu Pro Gly
Val Val Cys Ala Gly His Asp Asn Asn Gln Pro 130 135 140 Asp Ser Phe
Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg 145 150 155 160
Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg 165
170 175 Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp
Met 180 185 190 Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr
Asn Val Asn 195 200 205 Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val
Phe Asn Glu Tyr Arg 210 215 220 Met His Lys Ser Arg Met Tyr Ser Gln
Cys Val Arg Met Arg His Leu 225 230 235 240 Ser Gln Glu Phe Gly Trp
Leu Gln Ile Thr Pro Gln Glu Phe Leu Cys 245 250 255 Met Lys Ala Leu
Leu Leu Phe Ser Ile Ile Pro Val Asp Gly Leu Lys 260 265 270 Asn Gln
Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu 275 280 285
Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg 290
295 300 Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro Ile
Ala 305 310 315 320 Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile
Lys Ser His Met 325 330 335 Val Ser Val Asp Phe Pro Glu Met Met Ala
Glu Ile Ile Ser Val Gln 340 345 350 Val Pro Lys Ile Leu Ser Gly Lys
Val Lys Pro Ile Tyr Phe His Thr 355 360 365 Gln 4230PRTHomo sapiens
4Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn 1
5 10 15 Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys
Ala 20 25 30 Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met
Ala Val Ile 35 40 45 Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala
Met Gly Trp Arg Ser 50 55 60 Phe Thr Asn Val Asn Ser Arg Met Leu
Tyr Phe Ala Pro Asp Leu Val 65 70
75 80 Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys
Val 85 90 95 Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln
Ile Thr Pro 100 105 110 Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu
Phe Ser Ile Ile Pro 115 120 125 Val Asp Gly Leu Lys Asn Gln Lys Phe
Phe Asp Glu Leu Arg Met Asn 130 135 140 Tyr Ile Lys Glu Leu Asp Arg
Ile Ile Ala Cys Lys Arg Lys Asn Pro 145 150 155 160 Thr Ser Cys Ser
Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser 165 170 175 Val Gln
Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu 180 185 190
Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala Glu 195
200 205 Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys
Pro 210 215 220 Ile Tyr Phe His Thr Gln 225 230 5919PRTHomo sapiens
5Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1
5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg
Glu 20 25 30 Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala
Ser Ala Ala 35 40 45 Pro Pro Gly Ala Ser Leu Leu Leu Leu Gln Gln
Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Glu Thr 65 70 75 80 Ser Pro Arg Gln Gln Gln Gln
Gln Gln Gly Glu Asp Gly Ser Pro Gln 85 90 95 Ala His Arg Arg Gly
Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln 100 105 110 Gln Pro Ser
Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu Arg Gly 115 120 125 Cys
Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu Pro 130 135
140 Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro Ser
145 150 155 160 Thr Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser
Ser Cys Ser 165 170 175 Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser
Thr Met Gln Leu Leu 180 185 190 Gln Gln Gln Gln Gln Glu Ala Val Ser
Glu Gly Ser Ser Ser Gly Arg 195 200 205 Ala Arg Glu Ala Ser Gly Ala
Pro Thr Ser Ser Lys Asp Asn Tyr Leu 210 215 220 Gly Gly Thr Ser Thr
Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys Ala 225 230 235 240 Val Ser
Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser 245 250 255
Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu Gly 260
265 270 Val Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu
Cys 275 280 285 Lys Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr
Glu Asp Thr 290 295 300 Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr
Lys Gly Leu Glu Gly 305 310 315 320 Glu Ser Leu Gly Cys Ser Gly Ser
Ala Ala Ala Gly Ser Ser Gly Thr 325 330 335 Leu Glu Leu Pro Ser Thr
Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp 340 345 350 Glu Ala Ala Ala
Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala 355 360 365 Leu Ala
Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala Arg 370 375 380
Ile Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala 385
390 395 400 Ala Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly
Ala Gly 405 410 415 Ala Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala
Ala Ser Ser Ser 420 425 430 Trp His Thr Leu Phe Thr Ala Glu Glu Gly
Gln Leu Tyr Gly Pro Cys 435 440 445 Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly
Gly Gly Glu Ala Gly Ala Val Ala Pro Tyr 465 470 475 480 Gly Tyr Thr
Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Ser Asp Phe 485 490 495 Thr
Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg Val Pro 500 505
510 Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp Met Asp
515 520 525 Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala
Arg Asp 530 535 540 His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln
Lys Thr Cys Leu 545 550 555 560 Ile Cys Gly Asp Glu Ala Ser Gly Cys
His Tyr Gly Ala Leu Thr Cys 565 570 575 Gly Ser Cys Lys Val Phe Phe
Lys Arg Ala Ala Glu Gly Lys Gln Lys 580 585 590 Tyr Leu Cys Ala Ser
Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg 595 600 605 Lys Asn Cys
Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met 610 615 620 Thr
Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln 625 630
635 640 Glu Glu Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr
Thr 645 650 655 Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys
Gln Pro Ile 660 665 670 Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly
Val Val Cys Ala Gly 675 680 685 His Asp Asn Asn Gln Pro Asp Ser Phe
Ala Ala Leu Leu Ser Ser Leu 690 695 700 Asn Glu Leu Gly Glu Arg Gln
Leu Val His Val Val Lys Trp Ala Lys 705 710 715 720 Ala Leu Pro Gly
Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val 725 730 735 Ile Gln
Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg 740 745 750
Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu 755
760 765 Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln
Cys 770 775 780 Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu
Gln Ile Thr 785 790 795 800 Pro Gln Glu Phe Leu Cys Met Lys Ala Leu
Leu Leu Phe Ser Ile Ile 805 810 815 Pro Val Asp Gly Leu Lys Asn Gln
Lys Phe Phe Asp Glu Leu Arg Met 820 825 830 Asn Tyr Ile Lys Glu Leu
Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn 835 840 845 Pro Thr Ser Cys
Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp 850 855 860 Ser Val
Gln Pro Ile Ala Arg Glu Leu His Gln Leu Thr Phe Asp Leu 865 870 875
880 Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala
885 890 895 Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys
Val Lys 900 905 910 Pro Ile Tyr Phe His Thr Gln 915 6 902 PRTRattus
norvegicus 6Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro
Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln
Ser Val Arg Glu 20 25 30 Ala Ile Gln Asn Pro Gly Pro Arg His Pro
Glu Ala Ala Ser Ile Ala 35 40 45 Pro Pro Gly Ala Cys Leu Gln Gln
Arg Gln Glu Thr Ser Pro Arg Arg 50 55 60 Arg Arg Arg Gln Gln His
Pro Glu Asp Gly Ser Pro Gln Ala His Ile 65 70 75 80 Arg Gly Thr Thr
Gly Tyr Leu Ala Leu Glu Glu Glu Gln Gln Pro Ser 85 90 95 Gln Gln
Gln Ser Ala Ser Glu Gly His Pro Glu Ser Gly Cys Leu Pro 100 105 110
Glu Pro Gly Ala Ala Thr Ala Pro Gly Lys Gly Leu Pro Gln Gln Pro 115
120 125 Pro Ala Pro Pro Asp Gln Asp Asp Ser Ala Ala Pro Ser Thr Leu
Ser 130 135 140 Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser
Ala Asp Ile 145 150 155 160 Lys Asp Ile Leu Ser Glu Ala Gly Thr Met
Gln Leu Leu Gln Gln Gln 165 170 175 Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln 180 185 190 Gln Gln Gln Glu Val Ile
Ser Glu Gly Ser Ser Ser Val Arg Ala Arg 195 200 205 Glu Ala Thr Gly
Ala Pro Ser Ser Ser Lys Asp Ser Tyr Leu Gly Gly 210 215 220 Asn Ser
Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys Ala Val Ser 225 230 235
240 Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly
245 250 255 Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Ser Leu Leu Gly
Gly Pro 260 265 270 Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala
Glu Cys Lys Gly 275 280 285 Leu Ser Leu Asp Glu Gly Pro Gly Lys Gly
Thr Glu Glu Thr Ala Glu 290 295 300 Tyr Ser Ser Phe Lys Gly Gly Tyr
Ala Lys Gly Leu Glu Gly Glu Ser 305 310 315 320 Leu Gly Cys Ser Gly
Ser Ser Glu Ala Gly Ser Ser Gly Thr Leu Glu 325 330 335 Ile Pro Ser
Ser Leu Ser Leu Tyr Lys Ser Gly Ala Val Asp Glu Ala 340 345 350 Ala
Ala Tyr Gln Asn Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu Ser 355 360
365 Gly Pro Pro His Pro Pro Pro Pro Thr His Pro His Ala Arg Ile Lys
370 375 380 Leu Glu Asn Pro Ser Asp Tyr Gly Ser Ala Trp Ala Ala Ala
Ala Ala 385 390 395 400 Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His
Gly Gly Ser Val Ala 405 410 415 Gly Pro Ser Thr Gly Ser Pro Pro Ala
Thr Ala Ser Ser Ser Trp His 420 425 430 Thr Leu Phe Thr Ala Glu Glu
Gly Gln Leu Tyr Gly Pro Gly Gly Gly 435 440 445 Gly Gly Ser Ser Ser
Pro Ser Asp Ala Gly Pro Val Ala Pro Tyr Gly 450 455 460 Tyr Thr Arg
Pro Pro Gln Gly Leu Ala Ser Gln Glu Gly Asp Phe Ser 465 470 475 480
Ala Ser Glu Val Trp Tyr Pro Gly Gly Val Val Asn Arg Val Pro Tyr 485
490 495 Pro Ser Pro Ser Cys Val Lys Ser Glu Met Gly Pro Trp Met Glu
Asn 500 505 510 Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Asp Ser Thr
Arg Asp His 515 520 525 Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln
Lys Thr Cys Leu Ile 530 535 540 Cys Gly Asp Glu Ala Ser Gly Cys His
Tyr Gly Ala Leu Thr Cys Gly 545 550 555 560 Ser Cys Lys Val Phe Phe
Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr 565 570 575 Leu Cys Ala Ser
Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys 580 585 590 Asn Cys
Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr 595 600 605
Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln Glu 610
615 620 Glu Gly Glu Asn Ser Ser Ala Gly Ser Pro Thr Glu Asp Pro Ser
Gln 625 630 635 640 Lys Met Thr Val Ser His Ile Glu Gly Tyr Glu Cys
Gln Pro Ile Phe 645 650 655 Leu Asn Val Leu Glu Ala Ile Glu Pro Gly
Val Val Cys Ala Gly His 660 665 670 Asp Asn Asn Gln Pro Asp Ser Phe
Ala Ala Leu Leu Ser Ser Leu Asn 675 680 685 Glu Leu Gly Glu Arg Gln
Leu Val His Val Val Lys Trp Ala Lys Ala 690 695 700 Leu Pro Gly Phe
Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile 705 710 715 720 Gln
Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser 725 730
735 Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val
740 745 750 Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln
Cys Val 755 760 765 Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu
Gln Ile Thr Pro 770 775 780 Gln Glu Phe Leu Cys Met Lys Ala Leu Leu
Leu Phe Ser Ile Ile Pro 785 790 795 800 Val Asp Gly Leu Lys Asn Gln
Lys Phe Phe Asp Glu Leu Arg Met Asn 805 810 815 Tyr Ile Lys Glu Leu
Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro 820 825 830 Thr Ser Cys
Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser 835 840 845 Val
Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu 850 855
860 Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala Glu
865 870 875 880 Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys
Val Lys Pro 885 890 895 Ile Tyr Phe His Thr Gln 900 7899PRTMus
musculus 7Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro
Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln
Ser Val Arg Glu 20 25 30 Ala Ile Gln Asn Pro Gly Pro Arg His Pro
Glu Ala Ala Asn Ile Ala 35 40 45 Pro Pro Gly Ala Cys Leu Gln Gln
Arg Gln Glu Thr Ser Pro Arg Arg 50 55 60 Arg Arg Arg Gln Gln His
Thr Glu Asp Gly Ser Pro Gln Ala His Ile 65 70 75 80 Arg Gly Pro Thr
Gly Tyr Leu Ala Leu Glu Glu Glu Gln Gln Pro Ser 85 90 95 Gln Gln
Gln Ala Ala Ser Glu Gly His Pro Glu Ser Ser Cys Leu Pro 100 105 110
Glu Pro Gly Ala Ala Thr Ala Pro Gly Lys Gly Leu Pro Gln Gln Pro 115
120 125 Pro Ala Pro Pro Asp Gln Asp Asp Ser Ala Ala Pro Ser Thr Leu
Ser 130 135 140 Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser
Ala Asp Ile 145 150 155 160 Lys Asp Ile Leu Asn Glu Ala Gly Thr Met
Gln Leu Leu Gln Gln Gln 165 170 175 Gln Gln Gln Gln Gln His Gln Gln
Gln His Gln Gln His Gln Gln Gln 180 185 190 Gln Glu Val Ile Ser Glu
Gly Ser Ser Ala Arg Ala Arg Glu Ala Thr 195 200 205 Gly Ala Pro Ser
Ser Ser Lys Asp Ser Tyr Leu Gly Gly Asn Ser Thr 210 215 220 Ile Ser
Asp Ser Ala Lys Glu Leu Cys Lys Ala Val Ser Val Ser Met 225 230 235
240 Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly Glu Gln Leu
245 250 255 Arg Gly Asp Cys Met Tyr Ala Ser Leu Leu Gly Gly Pro Pro
Ala Val 260 265 270 Arg Pro Thr Pro Cys Ala Pro Leu Pro Glu Cys Lys
Gly Leu Pro Leu 275 280 285 Asp Glu Gly Pro Gly Lys Ser Thr Glu Glu
Thr Ala Glu Tyr Ser Ser 290 295 300 Phe Lys Gly Gly Tyr Ala Lys Gly
Leu Glu Gly Glu
Ser Leu Gly Cys 305 310 315 320 Ser Gly Ser Ser Glu Ala Gly Ser Ser
Gly Thr Leu Glu Ile Pro Ser 325 330 335 Ser Leu Ser Leu Tyr Lys Ser
Gly Ala Leu Asp Glu Ala Ala Ala Tyr 340 345 350 Gln Asn Arg Asp Tyr
Tyr Asn Phe Pro Leu Ala Leu Ser Gly Pro Pro 355 360 365 His Pro Pro
Pro Pro Thr His Pro His Ala Arg Ile Lys Leu Glu Asn 370 375 380 Pro
Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg 385 390
395 400 Tyr Gly Asp Leu Gly Ser Leu His Gly Gly Ser Val Ala Gly Pro
Ser 405 410 415 Thr Gly Ser Pro Pro Ala Thr Thr Ser Ser Ser Trp His
Thr Leu Phe 420 425 430 Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Gly
Gly Gly Gly Gly Ser 435 440 445 Ser Ser Pro Ser Asp Ala Gly Pro Val
Ala Pro Tyr Gly Tyr Thr Arg 450 455 460 Pro Pro Gln Gly Leu Thr Ser
Gln Glu Ser Asp Tyr Ser Ala Ser Glu 465 470 475 480 Val Trp Tyr Pro
Gly Gly Val Val Asn Arg Val Pro Tyr Pro Ser Pro 485 490 495 Asn Cys
Val Lys Ser Glu Met Gly Pro Trp Met Glu Asn Tyr Ser Gly 500 505 510
Pro Tyr Gly Asp Met Arg Leu Asp Ser Thr Arg Asp His Val Leu Pro 515
520 525 Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly
Asp 530 535 540 Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys Gly
Ser Cys Lys 545 550 555 560 Val Phe Phe Lys Arg Ala Ala Glu Gly Lys
Gln Lys Tyr Leu Cys Ala 565 570 575 Ser Arg Asn Asp Cys Thr Ile Asp
Lys Phe Arg Arg Lys Asn Cys Pro 580 585 590 Ser Cys Arg Leu Arg Lys
Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala 595 600 605 Arg Lys Leu Lys
Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu 610 615 620 Asn Ser
Asn Ala Gly Ser Pro Thr Glu Asp Pro Ser Gln Lys Met Thr 625 630 635
640 Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val
645 650 655 Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly His Asp
Asn Asn 660 665 670 Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu
Asn Glu Leu Gly 675 680 685 Glu Arg Gln Leu Val His Val Val Lys Trp
Ala Lys Ala Leu Pro Gly 690 695 700 Phe Arg Asn Leu His Val Asp Asp
Gln Met Ala Val Ile Gln Tyr Ser 705 710 715 720 Trp Met Gly Leu Met
Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn 725 730 735 Val Asn Ser
Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu 740 745 750 Tyr
Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg 755 760
765 His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln Glu Phe
770 775 780 Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro Val
Asp Gly 785 790 795 800 Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg
Met Asn Tyr Ile Lys 805 810 815 Glu Leu Asp Arg Ile Ile Ala Cys Lys
Arg Lys Asn Pro Thr Ser Cys 820 825 830 Ser Arg Arg Phe Tyr Gln Leu
Thr Lys Leu Leu Asp Ser Val Gln Pro 835 840 845 Ile Ala Arg Glu Leu
His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser 850 855 860 His Met Val
Ser Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser 865 870 875 880
Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe 885
890 895 His Thr Gln 8907PRTCanis familiaris 8Met Glu Val Gln Leu
Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr
Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30 Val
Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Val Ser Ala Ala 35 40
45 Pro Pro Gly Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
50 55 60 Glu Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gln Gly Asp
Asp Gly 65 70 75 80 Ser Pro Gln Ala Gln Ser Arg Gly Pro Thr Gly Tyr
Leu Ala Leu Asp 85 90 95 Glu Glu Gln Gln Pro Ser Gln Gln Arg Ser
Ala Ser Lys Gly His Pro 100 105 110 Glu Ser Ala Cys Val Pro Glu Pro
Gly Val Thr Ser Ala Thr Gly Lys 115 120 125 Gly Leu Gln Gln Gln Gln
Pro Ala Pro Pro Asp Glu Asn Asp Ser Ala 130 135 140 Ala Pro Ser Thr
Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser 145 150 155 160 Ser
Cys Ser Thr Asp Leu Lys Asp Ile Leu Ser Glu Ala Gly Thr Met 165 170
175 Gln Leu Leu Gln Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln
180 185 190 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Val Val Ser
Glu Gly 195 200 205 Ser Ser Ser Gly Arg Ala Arg Glu Ala Ala Gly Ala
Ser Thr Ser Ser 210 215 220 Lys Asp Ser Tyr Leu Gly Gly Ser Ser Thr
Ile Ser Asp Ser Ala Lys 225 230 235 240 Glu Leu Cys Lys Ala Val Ser
Val Ser Met Gly Leu Gly Val Glu Ala 245 250 255 Leu Glu His Leu Ser
Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr 260 265 270 Ala Pro Leu
Leu Gly Gly Pro Pro Ala Val Arg Pro Cys Ala Pro Leu 275 280 285 Ala
Glu Cys Lys Gly Ser Leu Leu Asp Asp Gly Pro Gly Lys Gly Thr 290 295
300 Glu Glu Thr Ala Glu Tyr Ser Pro Phe Lys Ala Gly Tyr Ala Lys Gly
305 310 315 320 Leu Asp Gly Asp Ser Leu Gly Cys Ser Ser Ser Ser Glu
Ala Gly Gly 325 330 335 Ser Gly Thr Leu Glu Met Pro Ser Thr Leu Ser
Leu Tyr Lys Ser Gly 340 345 350 Ala Leu Asp Glu Ala Ala Ala Tyr Gln
Ser Arg Asp Tyr Tyr Asn Phe 355 360 365 Pro Leu Ser Leu Gly Gly Pro
Pro Pro His Pro Pro Pro Pro His Pro 370 375 380 His Thr Arg Ile Lys
Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp 385 390 395 400 Ala Ala
Ala Ala Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His 405 410 415
Gly Ala Gly Ala Ala Gly Pro Ser Ser Gly Ser Pro Ser Ala Thr Thr 420
425 430 Ser Ser Ser Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu
Tyr 435 440 445 Gly Pro Cys Gly Gly Ser Gly Gly Gly Ser Ala Gly Asp
Gly Gly Ser 450 455 460 Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro Gln
Gly Leu Ala Gly Gln 465 470 475 480 Glu Gly Asp Phe Pro Pro Pro Asp
Val Trp Tyr Pro Gly Gly Val Val 485 490 495 Ser Arg Val Pro Phe Pro
Ser Pro Ser Cys Val Lys Ser Glu Met Gly 500 505 510 Ser Trp Met Glu
Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu 515 520 525 Thr Ala
Arg Asp His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln 530 535 540
Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly 545
550 555 560 Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala
Ala Glu 565 570 575 Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp
Cys Thr Ile Asp 580 585 590 Lys Phe Arg Arg Lys Asn Cys Pro Ser Cys
Arg Leu Arg Lys Cys Tyr 595 600 605 Glu Ala Gly Met Thr Leu Gly Ala
Arg Lys Leu Lys Lys Leu Gly Asn 610 615 620 Leu Lys Leu Gln Glu Glu
Gly Glu Ala Ser Asn Val Thr Ser Pro Thr 625 630 635 640 Glu Glu Pro
Thr Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu 645 650 655 Cys
Gln Pro Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val 660 665
670 Val Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu
675 680 685 Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His
Val Val 690 695 700 Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu
His Val Asp Asp 705 710 715 720 Gln Met Ala Val Ile Gln Tyr Ser Trp
Met Gly Leu Met Val Phe Ala 725 730 735 Met Gly Trp Arg Ser Phe Thr
Asn Val Asn Ser Arg Met Leu Tyr Phe 740 745 750 Ala Pro Asp Leu Val
Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met 755 760 765 Tyr Ser Gln
Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp 770 775 780 Leu
Gln Ile Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu 785 790
795 800 Phe Ser Ile Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe
Asp 805 810 815 Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile
Ile Ala Cys 820 825 830 Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg
Phe Tyr Gln Leu Thr 835 840 845 Lys Leu Leu Asp Ser Val Gln Pro Ile
Ala Arg Glu Leu His Gln Phe 850 855 860 Thr Phe Asp Leu Leu Ile Lys
Ser His Met Val Ser Val Asp Phe Pro 865 870 875 880 Glu Met Met Ala
Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser 885 890 895 Gly Lys
Val Lys Pro Ile Tyr Phe His Thr Gln 900 905 9912PRTCrocuta crocuta
9Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1
5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg
Glu 20 25 30 Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Thr
Ser Ala Ala 35 40 45 Pro Pro Gly Ala Arg Leu Gln Gln Gln His Gln
His Gln Gln Gln His 50 55 60 Gln His Glu Thr Ser Pro Arg Arg Gln
Gln Gln Gln Gln Pro Glu Asp 65 70 75 80 Gly Ser Pro Gln Arg Pro Ser
Arg Gly Pro Thr Ser Tyr Leu Ala Leu 85 90 95 Asp Glu Glu Gln Gln
Pro Ser Gln His Gln Ser Ala Lys Gly His Pro 100 105 110 Glu Ser Gly
Cys Val Pro Glu Pro Val Ala Met Ser Arg Thr Gly Lys 115 120 125 Gly
Leu Glu Gln Gln Gln Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala 130 135
140 Ala Pro Ser Thr Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser
145 150 155 160 Ser Cys Ser Thr Asp Leu Lys Asp Ile Leu Ser Glu Ala
Gly Thr Met 165 170 175 Gln Leu Leu Gln Arg Gln Arg Gln Arg Gln Gln
Gln Arg Gln Gln Gln 180 185 190 Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Glu 195 200 205 Val Val Ser Glu Gly Gly Ser
Ser Gly Arg Ala Arg Glu Ala Ala Gly 210 215 220 Ala Pro Thr Ser Ser
Lys Asp Ser Tyr Leu Gly Gly Ser Ser Thr Ile 225 230 235 240 Ser Asp
Ser Ala Lys Glu Leu Cys Lys Ala Val Ser Val Ser Met Gly 245 250 255
Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly Glu Gln Leu Arg 260
265 270 Gly Asp Cys Met Tyr Ala Pro Leu Leu Gly Gly Pro Pro Pro Val
Cys 275 280 285 Pro Cys Ala Pro Leu Thr Glu Cys Lys Gly Ser Val Leu
Asp Asp Gly 290 295 300 Pro Ser Lys Gly Thr Glu Glu Thr Ala Glu Tyr
Ser Pro Phe Lys Thr 305 310 315 320 Gly Tyr Ala Lys Gly Leu Asp Gly
Asp Ser Leu Gly Cys Ser Gly Ser 325 330 335 Ser Gln Ala Gly Gly Ser
Gly Thr Leu Glu Ile Pro Ser Thr Leu Ser 340 345 350 Leu Tyr Lys Ser
Gly Thr Leu Asp Glu Ala Ala Ala Tyr Gln Ser Arg 355 360 365 Asp Tyr
Tyr Asn Phe Gln Leu Ser Leu Ala Gly Pro Pro Pro Pro Pro 370 375 380
Pro Ser Pro His Pro His Ala Arg Ile Lys Leu Glu Asn Pro Leu Asp 385
390 395 400 Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg Tyr
Gly Asp 405 410 415 Leu Ala Ser Leu His Gly Gly Gly Ala Ala Gly Pro
Gly Ser Gly Ser 420 425 430 Pro Ser Ala Thr Ala Ser Ser Ser Trp His
Thr Leu Phe Thr Ala Glu 435 440 445 Glu Gly Gln Leu Tyr Gly Pro Cys
Gly Gly Ser Gly Gly Gly Gly Thr 450 455 460 Gly Glu Ser Val Ser Val
Thr Pro Tyr Gly Tyr Thr Arg Pro Gln Gln 465 470 475 480 Gly Leu Thr
Gly Gln Glu Gly Asp Phe Pro Pro Pro Asp Val Trp Tyr 485 490 495 Pro
Gly Gly Val Val Ser Arg Met Pro Tyr Pro Ser Ala Ser Cys Val 500 505
510 Lys Ser Glu Met Gly Pro Trp Met Glu Ser Tyr Ser Gly Pro Tyr Gly
515 520 525 Asp Met Arg Leu Glu Thr Thr Arg Asp His Val Leu Pro Ile
Asp Tyr 530 535 540 Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly
Asp Glu Ala Ser 545 550 555 560 Gly Cys His Tyr Gly Ala Leu Thr Cys
Gly Ser Cys Lys Val Phe Phe 565 570 575 Lys Arg Ala Ala Glu Gly Lys
Gln Lys Tyr Leu Cys Ala Ser Arg Asn 580 585 590 Asp Cys Thr Ile Asp
Lys Phe Arg Arg Lys Asn Cys Pro Pro Cys Arg 595 600 605 Leu Arg Lys
Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Arg Leu 610 615 620 Lys
Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu Ala Ser Ser 625 630
635 640 Thr Thr Ser Pro Thr Glu Glu Thr Thr Gln Lys Leu Thr Val Ser
His 645 650 655 Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val
Leu Glu Ala 660 665 670 Ile Glu Pro Gly Val Val Cys Ala Gly His Asp
Asn Asn Gln Pro Asp 675 680 685 Ser Phe Ala Ala Leu Leu Ser Ser Leu
Asn Glu Leu Gly Glu Arg Gln 690 695 700 Leu Val His Val Val Lys Trp
Ala Lys Ala Leu Pro Gly Phe Arg Asn 705 710 715 720 Leu His Val Asp
Asp Gln Met Ala Val Ile Gln Tyr Ser Trp Met Gly 725 730 735 Leu Met
Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser 740 745 750
Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr Arg Met 755
760 765 His Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg His Leu
Ser 770 775 780 Gln Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln Glu Phe
Leu Cys Met 785
790 795 800 Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro Val Asp Gly Leu
Lys Asn 805 810 815 Gln Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile
Lys Asp Leu Asp 820 825 830 Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro
Thr Ser Cys Ser Arg Arg 835 840 845 Phe Tyr Gln Leu Thr Lys Leu Leu
Asp Ser Val Gln Pro Ile Ala Arg 850 855 860 Glu Leu His Gln Phe Thr
Phe Asp Leu Leu Ile Lys Ser His Met Val 865 870 875 880 Ser Val Asp
Phe Pro Glu Met Met Ala Glu Ile Ile Ser Val Gln Val 885 890 895 Pro
Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe His Thr Gln 900 905
910 10884PRTEulemur fulvus 10Met Glu Val Gln Leu Gly Leu Gly Arg
Val Tyr Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe
Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30 Val Ile Gln Asn Pro
Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45 Pro Pro Gly
Ala Arg Leu Gln Gln Gln Gln Glu Thr Ser Pro Pro Gln 50 55 60 Gln
Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro Gln Ala Gln Ser 65 70
75 80 Arg Gly Pro Thr Gly Tyr Leu Ala Leu Asp Glu Glu Gln Gln Pro
Ser 85 90 95 Gln Gln Gln Ser Ala Leu Glu Cys His Pro Glu Ser Gly
Cys Val Pro 100 105 110 Glu Pro Gly Ala Ala Ala Ala Ala Ser Lys Gly
Leu Gln Gln Gln Pro 115 120 125 Pro Ala Pro Ser Asp Glu Asp Asp Ser
Ala Val Pro Ser Thr Leu Ser 130 135 140 Leu Leu Gly Pro Thr Phe Pro
Gly Leu Ser Ser Cys Ser Ala Asp Leu 145 150 155 160 Lys Asp Ile Leu
Ser Glu Ala Gly Thr Met Gln Leu Leu Gln Gln Gln 165 170 175 Gln Gln
Glu Ala Val Ser Glu Gly Ser Ser Ser Gly Arg Ala Arg Glu 180 185 190
Ala Ala Gly Ala Pro Thr Ser Ser Lys Asp Ser Tyr Leu Gly Gly Thr 195
200 205 Ser Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys Ala Val Ser
Val 210 215 220 Ser Met Gly Leu Gly Val Glu Thr Leu Glu His Leu Ser
Pro Gly Glu 225 230 235 240 Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro
Leu Leu Gly Gly Pro Pro 245 250 255 Ala Val Arg Pro Thr Pro Cys Ala
Pro Leu Ala Glu Cys Lys Gly Ser 260 265 270 Leu Leu Asp Asp Ser Ala
Asp Lys Gly Thr Glu Glu Pro Ala Glu Tyr 275 280 285 Thr Pro Phe Lys
Gly Ser Tyr Thr Gln Gly Leu Glu Gly Glu Ser Leu 290 295 300 Gly Cys
Ser Gly Ser Ser Glu Ala Gly Ser Ser Gly Thr Leu Glu Leu 305 310 315
320 Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu Glu Glu Ala Ala
325 330 335 Ser Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu
Ala Gly 340 345 350 Pro Pro Pro Pro Pro Leu Pro Pro His Pro His Ala
Arg Ile Lys Leu 355 360 365 Glu Asn Pro Leu Asp Tyr Gly Ser Ser Trp
Ala Ala Ala Ala Ala Gln 370 375 380 Cys Arg Phe Gly Asp Leu Ala Ser
Leu His Gly Gly Gly Ala Thr Gly 385 390 395 400 Pro Gly Ser Gly Ser
Pro Ser Ala Ala Ala Ala Ser Ser Trp His Thr 405 410 415 Leu Phe Thr
Ala Glu Glu Gly Gln Leu Tyr Gly Pro Cys Gly Gly Gly 420 425 430 Gly
Gly Gly Thr Ser Glu Ala Gly Ala Val Thr Pro Tyr Gly Tyr Ser 435 440
445 Arg Pro Pro Gln Gly Leu Ala Gly Gln Glu Gly Asp Phe Pro Ala Pro
450 455 460 Asp Val Trp Tyr Pro Ser Gly Val Val Ser Arg Val Pro Tyr
Pro Ser 465 470 475 480 Pro Ser Cys Val Lys Ser Glu Met Gly Pro Trp
Met Glu Ser Tyr Ser 485 490 495 Gly Pro Tyr Gly Asp Val Arg Leu Glu
Thr Ala Arg Asp His Val Leu 500 505 510 Pro Ile Asp Tyr Tyr Phe Pro
Pro Gln Lys Thr Cys Leu Ile Cys Gly 515 520 525 Asp Glu Ala Ser Gly
Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys 530 535 540 Lys Val Phe
Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys 545 550 555 560
Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys 565
570 575 Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu
Gly 580 585 590 Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln
Glu Glu Gly 595 600 605 Glu Ala Ser Ser Ala Thr Ser Pro Thr Glu Glu
Ser Ser Gln Lys Leu 610 615 620 Thr Val Ser His Ile Glu Gly Tyr Glu
Cys Gln Pro Ile Phe Leu Asn 625 630 635 640 Val Leu Glu Ala Ile Glu
Pro Gly Val Val Cys Ala Gly His Asp Asn 645 650 655 Asn Gln Pro Asp
Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu 660 665 670 Gly Glu
Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu Pro 675 680 685
Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln Tyr 690
695 700 Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe
Thr 705 710 715 720 Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp
Leu Val Phe Asn 725 730 735 Glu Tyr Arg Met His Lys Ser Arg Met Tyr
Ser Gln Cys Val Arg Met 740 745 750 Arg His Leu Ser Gln Glu Phe Gly
Trp Leu Gln Ile Thr Pro Gln Glu 755 760 765 Phe Leu Cys Met Lys Ala
Leu Leu Leu Phe Ser Ile Ile Pro Val Asp 770 775 780 Gly Leu Lys Asn
Gln Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile 785 790 795 800 Lys
Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser 805 810
815 Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln
820 825 830 Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu
Ile Lys 835 840 845 Ser His Met Val Ser Val Asp Phe Pro Glu Met Met
Ala Glu Ile Ile 850 855 860 Ser Val Gln Val Pro Lys Ile Leu Ser Gly
Lys Val Lys Pro Ile Tyr 865 870 875 880 Phe His Thr Gln
11777PRTRana catesbeiana 11Met Glu Val His Ile Gly Leu Gly Gly Val
Tyr Lys Gln Pro Pro Gly 1 5 10 15 Lys Met Ile Arg Gly Ala Phe Glu
Asn Leu Phe Leu Ser Val Arg Glu 20 25 30 Ala Leu Gln Gly Glu Arg
Arg Ser Ala Ala Ser Leu Asp Thr Ser Ser 35 40 45 Pro Ile Ser Ala
Cys Val His Pro His Pro Thr Trp Asn Glu Pro Ser 50 55 60 Thr Trp
Thr Glu Val Arg Gly Thr Pro Trp Arg Glu Pro Gln Gly Ala 65 70 75 80
Gln Pro Asp Pro Pro Pro Cys Ser Pro Arg Ser Gln Ala Pro Gln Phe 85
90 95 Thr Leu Ser Ser Cys Thr Thr Glu Leu Lys Glu Ile Leu Gly Glu
Gln 100 105 110 Gly Gly Met Pro Glu Glu Gly Asn Ser Glu Ser Ala Ser
Lys Glu Gly 115 120 125 Tyr Pro Glu Ser Ile Ser Asp Ser Ala Lys Glu
Ile Cys Lys Ala Val 130 135 140 Ser Val Ser Leu Gly Leu Ser Met Glu
Ala Leu Glu His Leu Ser Ala 145 150 155 160 Ala Gly Glu Trp Gln Arg
Gly Asp Cys Met Phe Ala Gly Pro Pro His 165 170 175 His Thr Met Gly
Ala Gln Thr Cys Gln Val Ala Glu Glu Asp Lys Ser 180 185 190 Asp Thr
Ser Phe Ser Gln Tyr Arg Glu Gly Ala Phe Arg Arg Ala Gly 195 200 205
Gln Ser Thr Tyr Ser Ala Gly Lys Ala Pro Glu Asp Gly Ser Ser Leu 210
215 220 Pro Thr Glu Asp Lys Glu Gln Pro Cys Thr Asp Met Ala Leu Ser
Glu 225 230 235 240 Pro Gly Ser Leu Arg Ser Arg Gly Met Glu Val Met
Pro Ser Leu Thr 245 250 255 Leu Tyr Lys Pro Thr Ala Phe Met Glu Asp
Ala Ser Ala Tyr Pro Gly 260 265 270 Arg Asp Tyr Tyr Ser Phe Gln Met
Ala Leu Ala Pro His Gly Arg Ile 275 280 285 Lys Val Glu Ser Pro Ile
Glu Phe Ala Gly Ser Ala Trp Gly Gly Pro 290 295 300 Ser Arg Tyr Ser
Glu Phe Pro Gly Phe Ser His Cys Gly Pro Ser Ala 305 310 315 320 Asn
Trp His Ser Leu Phe Glu Glu Gly Gln Ala Thr Ala Ser Tyr Thr 325 330
335 Asp Ser Ser Leu Tyr Ser Tyr Pro Arg Ser His Val Pro Ala Gly Pro
340 345 350 Asp Gly Glu Phe Ser Ala Glu Ala Trp Tyr Pro Ala Thr Ala
Met Leu 355 360 365 Gly Arg Val His Met Ala Val Pro Met Arg Pro Arg
Met Thr His Gly 370 375 380 Trp Thr Ala Thr Leu Gly Ile Arg Arg Arg
Leu Gly Trp Thr Gly Val 385 390 395 400 Glu Ser Thr Phe Tyr Pro Ile
Asp Tyr Tyr Phe Pro Pro Gln Lys Pro 405 410 415 Cys Leu Ser Cys Glu
Asp Glu Ala Ser Gly Cys His Tyr Glu Ala Leu 420 425 430 Thr Cys Gly
Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Asn 435 440 445 Gln
Lys Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe 450 455
460 Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala
465 470 475 480 Gly Met Thr Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly
Asn Leu Lys 485 490 495 Ala Gln Glu Glu Leu Glu Gly Ser Pro Gly Gln
Ser Glu Gly Arg Glu 500 505 510 Met Pro Pro Asn Met Ser Ile Pro Gln
Leu Glu Gly Tyr Ser Cys Gln 515 520 525 Pro Ile Phe Leu Asn Val Leu
Glu Ala Ile Glu Pro Met Val Val Cys 530 535 540 Ser Gly His Asp Asn
Asn Gln Pro Asp Ser Phe Ala Leu Leu Leu Ser 545 550 555 560 Ser Leu
Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp 565 570 575
Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asn Asp Gln Met 580
585 590 Thr Val Ile Gln Tyr Ser Trp Met Gly Leu Met Ile Phe Ala Met
Gly 595 600 605 Trp Arg Ser Phe Lys Asn Val Asn Ser Arg Met Leu Tyr
Phe Ala Pro 610 615 620 Asp Leu Val Phe Asn Glu Tyr Arg Met His Lys
Ser Arg Met Tyr Ser 625 630 635 640 Gln Cys Val Arg Met Arg His Leu
Ser Gln Glu Phe Gly Trp Leu Gln 645 650 655 Val Thr Pro Glu Glu Phe
Leu Cys Asp Glu Gly Pro Ser Ala Leu Ser 660 665 670 Ile Ile Pro Val
Glu Gly Leu Lys Asp Gln Lys Cys Phe Asp Glu Leu 675 680 685 Arg Met
Asn Tyr Ile Lys Glu Leu Asp Arg Val Ile Ser Cys Lys Arg 690 695 700
Asn Asn Pro Ala Ser Ser Ser Pro Arg Phe Phe Asn Leu Pro Lys Leu 705
710 715 720 Leu Gly Ser Val Gln Pro Ile Asp Val Asn Leu Val Gln Phe
Thr Phe 725 730 735 Gly Leu Phe Gly Lys Ala Gln Met Val Ser Val Asp
Phe Pro Glu Met 740 745 750 Met Ser Glu Ile Ile Ser Val Gln Val Pro
Lys Ile Leu Ser Gly Arg 755 760 765 Val Lys Pro Leu Tyr Phe His Ser
Ser 770 775 12895PRTMacaca fascicularis 12Met Glu Val Gln Leu Gly
Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg
Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30 Val Ile
Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45
Pro Pro Gly Ala Ser Leu Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr 50
55 60 Ser Pro Arg Gln Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser
Pro 65 70 75 80 Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu
Asp Glu Glu 85 90 95 Gln Gln Pro Ser Gln Pro Gln Ser Ala Pro Glu
Cys His Pro Glu Arg 100 105 110 Gly Cys Val Pro Glu Pro Gly Ala Ala
Val Ala Ala Gly Lys Gly Leu 115 120 125 Pro Gln Gln Leu Pro Ala Pro
Pro Asp Glu Asp Asp Ser Ala Ala Pro 130 135 140 Ser Thr Leu Ser Leu
Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys 145 150 155 160 Ser Thr
Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu 165 170 175
Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly 180
185 190 Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn
Tyr 195 200 205 Leu Gly Gly Thr Ser Thr Ile Ser Asp Ser Ala Lys Glu
Leu Cys Lys 210 215 220 Ala Val Ser Val Ser Met Gly Leu Gly Val Glu
Ala Leu Glu His Leu 225 230 235 240 Ser Pro Gly Glu Gln Leu Arg Gly
Asp Cys Met Tyr Ala Pro Val Leu 245 250 255 Gly Val Pro Pro Ala Val
Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu 260 265 270 Cys Lys Gly Ser
Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp 275 280 285 Thr Ala
Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu 290 295 300
Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly 305
310 315 320 Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly
Ala Leu 325 330 335 Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr
Asn Phe Pro Leu 340 345 350 Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro
Pro Pro His Pro His Ala 355 360 365 Arg Ile Lys Leu Glu Asn Pro Leu
Asp Tyr Gly Ser Ala Trp Ala Ala 370 375 380 Ala Ala Ala Gln Cys Arg
Tyr Gly Asp Leu Ala Ser Leu His Gly Ala 385 390 395 400 Gly Ala Ala
Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser 405 410 415 Ser
Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro 420 425
430 Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly
435 440 445 Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro
Gln Gly 450 455 460 Leu Ala Gly Gln Glu Gly Asp Phe Thr Ala Pro Asp
Val Trp Tyr Pro 465 470 475 480 Gly Gly Met Val Ser Arg Val Pro Tyr
Pro Ser Pro Thr Cys Val Lys 485 490 495 Ser Glu Met Gly Pro Trp Met
Asp Ser Tyr Ser Gly Pro Tyr Gly Asp
500 505 510 Met Arg Leu Glu Thr Ala Arg Asp His Val Leu Pro Ile Asp
Tyr Tyr 515 520 525 Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp
Glu Ala Ser Gly 530 535 540 Cys His Tyr Gly Ala Leu Thr Cys Gly Ser
Cys Lys Val Phe Phe Lys 545 550 555 560 Arg Ala Ala Glu Gly Lys Gln
Lys Tyr Leu Cys Ala Ser Arg Asn Asp 565 570 575 Cys Thr Ile Asp Lys
Phe Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu 580 585 590 Arg Lys Cys
Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Lys Leu Lys 595 600 605 Lys
Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu Ala Ser Ser Thr 610 615
620 Thr Ser Pro Thr Glu Glu Thr Ala Gln Lys Leu Thr Val Ser His Ile
625 630 635 640 Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val Leu
Glu Ala Ile 645 650 655 Glu Pro Gly Val Val Cys Ala Gly His Asp Asn
Asn Gln Pro Asp Ser 660 665 670 Phe Ala Ala Leu Leu Ser Ser Leu Asn
Glu Leu Gly Glu Arg Gln Leu 675 680 685 Val His Val Val Lys Trp Ala
Lys Ala Leu Pro Gly Phe Arg Asn Leu 690 695 700 His Val Asp Asp Gln
Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu 705 710 715 720 Met Val
Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser Arg 725 730 735
Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr Arg Met His 740
745 750 Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg His Leu Ser
Gln 755 760 765 Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln Glu Phe Leu
Cys Met Lys 770 775 780 Ala Leu Leu Leu Phe Ser Ile Ile Pro Val Asp
Gly Leu Lys Asn Gln 785 790 795 800 Lys Phe Phe Asp Glu Leu Arg Met
Asn Tyr Ile Lys Glu Leu Asp Arg 805 810 815 Ile Ile Ala Cys Lys Arg
Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe 820 825 830 Tyr Gln Leu Thr
Lys Leu Leu Asp Ser Val Gln Pro Ile Ala Arg Glu 835 840 845 Leu His
Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser His Met Val Ser 850 855 860
Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser Val Gln Val Pro 865
870 875 880 Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe His Thr
Gln 885 890 895 13895PRTMacaca mulatta 13Met Glu Val Gln Leu Gly
Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg
Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20 25 30 Val Ile
Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala 35 40 45
Pro Pro Gly Ala Ser Leu Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr 50
55 60 Ser Pro Arg Gln Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser
Pro 65 70 75 80 Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu
Asp Glu Glu 85 90 95 Gln Gln Pro Ser Gln Pro Gln Ser Ala Pro Glu
Cys His Pro Glu Arg 100 105 110 Gly Cys Val Pro Glu Pro Gly Ala Ala
Val Ala Ala Gly Lys Gly Leu 115 120 125 Pro Gln Gln Leu Pro Ala Pro
Pro Asp Glu Asp Asp Ser Ala Ala Pro 130 135 140 Ser Thr Leu Ser Leu
Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys 145 150 155 160 Ser Ala
Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu 165 170 175
Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly 180
185 190 Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn
Tyr 195 200 205 Leu Glu Gly Thr Ser Thr Ile Ser Asp Ser Ala Lys Glu
Leu Cys Lys 210 215 220 Ala Val Ser Val Ser Met Gly Leu Gly Val Glu
Ala Leu Glu His Leu 225 230 235 240 Ser Pro Gly Glu Gln Leu Arg Gly
Asp Cys Met Tyr Ala Pro Val Leu 245 250 255 Gly Val Pro Pro Ala Val
Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu 260 265 270 Cys Lys Gly Ser
Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp 275 280 285 Thr Ala
Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu 290 295 300
Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly 305
310 315 320 Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly
Ala Leu 325 330 335 Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr
Asn Phe Pro Leu 340 345 350 Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro
Pro Pro His Pro His Ala 355 360 365 Arg Ile Lys Leu Glu Asn Pro Leu
Asp Tyr Gly Ser Ala Trp Ala Ala 370 375 380 Ala Ala Ala Gln Cys Arg
Tyr Gly Asp Leu Ala Ser Leu His Gly Ala 385 390 395 400 Gly Ala Ala
Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser 405 410 415 Ser
Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro 420 425
430 Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly
435 440 445 Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro
Gln Gly 450 455 460 Leu Ala Gly Gln Glu Gly Asp Phe Thr Ala Pro Asp
Val Trp Tyr Pro 465 470 475 480 Gly Gly Met Val Ser Arg Val Pro Tyr
Pro Ser Pro Thr Cys Val Lys 485 490 495 Ser Glu Met Gly Pro Trp Met
Asp Ser Tyr Ser Gly Pro Tyr Gly Asp 500 505 510 Met Arg Leu Glu Thr
Ala Arg Asp His Val Leu Pro Ile Asp Tyr Tyr 515 520 525 Phe Pro Pro
Gln Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly 530 535 540 Cys
His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys 545 550
555 560 Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn
Asp 565 570 575 Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro Ser
Cys Arg Leu 580 585 590 Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly
Ala Arg Lys Leu Lys 595 600 605 Lys Leu Gly Asn Leu Lys Leu Gln Glu
Glu Gly Glu Ala Ser Ser Thr 610 615 620 Thr Ser Pro Thr Glu Glu Thr
Ala Gln Lys Leu Thr Val Ser His Ile 625 630 635 640 Glu Gly Tyr Glu
Cys Gln Pro Ile Phe Leu Asn Val Leu Glu Ala Ile 645 650 655 Glu Pro
Gly Val Val Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser 660 665 670
Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu 675
680 685 Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn
Leu 690 695 700 His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp
Met Gly Leu 705 710 715 720 Met Val Phe Ala Met Gly Trp Arg Ser Phe
Thr Asn Val Asn Ser Arg 725 730 735 Met Leu Tyr Phe Ala Pro Asp Leu
Val Phe Asn Glu Tyr Arg Met His 740 745 750 Lys Ser Arg Met Tyr Ser
Gln Cys Val Arg Met Arg His Leu Ser Gln 755 760 765 Glu Phe Gly Trp
Leu Gln Ile Thr Pro Gln Glu Phe Leu Cys Met Lys 770 775 780 Ala Leu
Leu Leu Phe Ser Ile Ile Pro Val Asp Gly Leu Lys Asn Gln 785 790 795
800 Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg
805 810 815 Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg
Arg Phe 820 825 830 Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro
Ile Ala Arg Glu 835 840 845 Leu His Gln Phe Thr Phe Asp Leu Leu Ile
Lys Ser His Met Val Ser 850 855 860 Val Asp Phe Pro Glu Met Met Ala
Glu Ile Ile Ser Val Gln Val Pro 865 870 875 880 Lys Ile Leu Ser Gly
Lys Val Lys Pro Ile Tyr Phe His Thr Gln 885 890 895 14911PRTPan
troglodytes 14Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg
Pro Pro Ser 1 5 10 15 Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe
Gln Ser Val Arg Glu 20 25 30 Val Ile Gln Asn Pro Gly Pro Arg His
Pro Glu Ala Ala Ser Ala Ala 35 40 45 Pro Pro Gly Ala Ser Leu Leu
Leu Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Thr 65 70 75 80 Ser Pro Arg
Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro Gln Ala 85 90 95 His
Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu Gln Gln 100 105
110 Pro Ser Gln Pro Gln Ser Ala Pro Glu Cys His Pro Glu Arg Gly Cys
115 120 125 Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu
Pro Gln 130 135 140 Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala
Ala Pro Ser Thr 145 150 155 160 Leu Ser Leu Leu Gly Pro Thr Phe Pro
Gly Leu Ser Ser Cys Ser Ala 165 170 175 Asp Leu Lys Asp Ile Leu Ser
Glu Ala Ser Thr Met Gln Leu Leu Gln 180 185 190 Gln Gln Gln Gln Glu
Ala Val Ser Glu Gly Ser Ser Ser Gly Arg Ala 195 200 205 Arg Glu Ala
Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr Leu Gly 210 215 220 Gly
Thr Ser Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys Ala Val 225 230
235 240 Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser
Pro 245 250 255 Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu
Leu Gly Val 260 265 270 Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro
Leu Ala Glu Cys Lys 275 280 285 Gly Ser Leu Leu Asp Asp Ser Ala Gly
Lys Ser Thr Glu Asp Thr Ala 290 295 300 Glu Tyr Ser Pro Phe Lys Gly
Gly Tyr Thr Lys Gly Leu Glu Gly Glu 305 310 315 320 Ser Leu Gly Cys
Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly Thr Leu 325 330 335 Glu Leu
Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp Glu 340 345 350
Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu 355
360 365 Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala Arg
Ile 370 375 380 Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala
Ala Ala Ala 385 390 395 400 Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser
Leu His Gly Ala Gly Ala 405 410 415 Ala Gly Pro Gly Ser Gly Ser Pro
Ser Ala Ala Ala Ser Ser Ser Trp 420 425 430 His Thr Leu Phe Thr Ala
Glu Glu Gly Gln Leu Tyr Gly Pro Cys Gly 435 440 445 Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Glu Ala
Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro Gln Gly 465 470 475
480 Leu Ala Gly Gln Glu Gly Asp Phe Thr Ala Pro Asp Val Trp Tyr Pro
485 490 495 Gly Gly Met Val Ser Arg Val Pro Tyr Pro Ser Pro Thr Cys
Val Lys 500 505 510 Ser Glu Met Gly Pro Trp Met Asp Ser Tyr Ser Gly
Pro Tyr Gly Asp 515 520 525 Met Arg Leu Glu Thr Ala Arg Asp His Val
Leu Pro Ile Asp Tyr Tyr 530 535 540 Phe Pro Pro Gln Lys Thr Cys Leu
Ile Cys Gly Asp Glu Ala Ser Gly 545 550 555 560 Cys His Tyr Gly Ala
Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys 565 570 575 Arg Ala Ala
Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp 580 585 590 Cys
Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu 595 600
605 Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Lys Leu Lys
610 615 620 Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu Ala Ser
Ser Thr 625 630 635 640 Thr Ser Pro Thr Glu Glu Thr Thr Gln Lys Leu
Thr Val Ser His Ile 645 650 655 Glu Gly Tyr Glu Cys Gln Pro Ile Phe
Leu Asn Val Leu Glu Ala Ile 660 665 670 Glu Pro Gly Val Val Cys Ala
Gly His Asp Asn Asn Gln Pro Asp Ser 675 680 685 Phe Ala Ala Leu Leu
Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu 690 695 700 Val His Val
Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu 705 710 715 720
His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu 725
730 735 Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser
Arg 740 745 750 Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr
Arg Met His 755 760 765 Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met
Arg His Leu Ser Gln 770 775 780 Glu Phe Gly Trp Leu Gln Ile Thr Pro
Gln Glu Phe Leu Cys Met Lys 785 790 795 800 Ala Leu Leu Leu Phe Ser
Ile Ile Pro Val Asp Gly Leu Lys Asn Gln 805 810 815 Lys Phe Phe Asp
Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg 820 825 830 Ile Ile
Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe 835 840 845
Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro Ile Ala Arg Glu 850
855 860 Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser His Met Val
Ser 865 870 875 880 Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser
Val Gln Val Pro 885 890 895 Lys Ile Leu Ser Gly Lys Val Lys Pro Ile
Tyr Phe His Thr Gln 900 905 910 15895PRTPapio hamadryas 15Met Glu
Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1 5 10 15
Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu 20
25 30 Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala
Ala 35 40 45 Pro Pro Gly Ala Ser Leu Gln Gln Gln Gln Gln Gln Gln
Gln Gln Glu 50 55 60 Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly
Glu Asp Gly Ser Pro 65 70 75 80 Gln Ala His Arg Arg Gly Pro Thr Gly
Tyr Leu Val Leu Asp Glu Glu
85 90 95 Gln Gln Pro Ser Gln Pro Gln Ser Ala Pro Glu Cys His Pro
Glu Arg 100 105 110 Gly Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala
Gly Lys Gly Leu 115 120 125 Pro Gln Gln Leu Pro Ala Pro Pro Asp Glu
Asp Asp Ser Ala Ala Pro 130 135 140 Ser Thr Leu Ser Leu Leu Gly Pro
Thr Phe Pro Gly Leu Ser Ser Cys 145 150 155 160 Ser Ala Asp Leu Lys
Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu 165 170 175 Leu Gln Gln
Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly 180 185 190 Arg
Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr 195 200
205 Leu Gly Gly Thr Ser Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys
210 215 220 Ala Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu
His Leu 225 230 235 240 Ser Pro Gly Glu Gln Leu Arg Gly Asp Cys Met
Tyr Ala Pro Val Leu 245 250 255 Gly Val Pro Pro Ala Val Arg Pro Thr
Pro Cys Ala Pro Leu Ala Glu 260 265 270 Cys Lys Gly Ser Leu Leu Asp
Asp Ser Ala Gly Lys Ser Thr Glu Asp 275 280 285 Thr Ala Glu Tyr Ser
Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu 290 295 300 Gly Glu Ser
Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly 305 310 315 320
Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu 325
330 335 Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro
Leu 340 345 350 Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His
Pro His Ala 355 360 365 Arg Ile Lys Leu Glu Asn Pro Leu Asp Tyr Gly
Ser Ala Trp Ala Ala 370 375 380 Ala Ala Ala Gln Cys Arg Tyr Gly Glu
Leu Ala Ser Leu His Gly Ala 385 390 395 400 Gly Ala Ala Gly Pro Gly
Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser 405 410 415 Ser Trp His Thr
Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro 420 425 430 Cys Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly 435 440 445
Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro Gln Gly 450
455 460 Leu Ala Gly Gln Glu Gly Asp Phe Thr Ala Pro Asp Val Trp Tyr
Pro 465 470 475 480 Gly Gly Met Val Ser Arg Val Pro Tyr Pro Ser Pro
Thr Cys Val Lys 485 490 495 Ser Glu Met Gly Pro Trp Met Asp Ser Tyr
Ser Gly Pro Tyr Gly Asp 500 505 510 Met Arg Leu Glu Thr Ala Arg Asp
His Val Leu Pro Ile Asp Tyr Tyr 515 520 525 Phe Pro Pro Gln Lys Thr
Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly 530 535 540 Cys His Tyr Gly
Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys 545 550 555 560 Arg
Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp 565 570
575 Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu
580 585 590 Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Lys
Leu Lys 595 600 605 Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu
Ala Ser Ser Thr 610 615 620 Thr Ser Pro Thr Glu Glu Thr Ala Gln Lys
Leu Thr Val Ser His Ile 625 630 635 640 Glu Gly Tyr Glu Cys Gln Pro
Ile Phe Leu Asn Val Leu Glu Ala Ile 645 650 655 Glu Pro Gly Val Val
Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser 660 665 670 Phe Ala Ala
Leu Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu 675 680 685 Val
His Val Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu 690 695
700 His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu
705 710 715 720 Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val
Asn Ser Arg 725 730 735 Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn
Glu Tyr Arg Met His 740 745 750 Lys Ser Arg Met Tyr Ser Gln Cys Val
Arg Met Arg His Leu Ser Gln 755 760 765 Glu Phe Gly Trp Leu Gln Ile
Thr Pro Gln Glu Phe Leu Cys Met Lys 770 775 780 Ala Leu Leu Leu Phe
Ser Ile Ile Pro Val Asp Gly Leu Lys Asn Gln 785 790 795 800 Lys Phe
Phe Asp Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg 805 810 815
Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe 820
825 830 Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro Ile Ala Arg
Glu 835 840 845 Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser His
Met Val Ser 850 855 860 Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile
Ser Val Gln Val Pro 865 870 875 880 Lys Ile Leu Ser Gly Lys Val Lys
Pro Ile Tyr Phe His Thr Gln 885 890 895 16896PRTSus scrofa 16Met
Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser 1 5 10
15 Lys Thr Phe Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu
20 25 30 Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser
Ala Ala 35 40 45 Pro Pro Gly Ala Arg Leu Gln Gln Gln Gln Leu Gln
Gln Gln Glu Thr 50 55 60 Ser Pro Arg Arg Gln Gln Gln Gln Gln Gln
Gln Pro Ser Glu Asp Gly 65 70 75 80 Ser Pro Gln Val Gln Ser Arg Gly
Pro Thr Gly Tyr Leu Ala Leu Asp 85 90 95 Glu Lys Gln Gln Pro Ser
Gln Gln Gln Ser Ala Pro Glu Cys His Pro 100 105 110 Glu Ser Gly Cys
Thr Pro Glu Pro Gly Ala Ala Ser Ala Ala Ser Lys 115 120 125 Gly Leu
Gln Gln Gln Pro Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala 130 135 140
Ala Pro Ser Thr Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser 145
150 155 160 Ser Cys Ser Thr Asp Leu Lys Asp Ile Leu Ser Glu Ala Gly
Thr Met 165 170 175 Gln Leu Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Glu Ala Val 180 185 190 Ser Glu Gly Asn Ser Ser Gly Arg Ala Arg
Glu Ala Thr Gly Ala Pro 195 200 205 Ile Ser Ser Lys Asp Ser Tyr Leu
Gly Gly Ser Ser Thr Ile Ser Asp 210 215 220 Ser Ala Lys Glu Leu Cys
Lys Ala Val Ser Val Ser Met Gly Leu Gly 225 230 235 240 Val Glu Ala
Leu Glu His Leu Ser Pro Gly Glu Gln Leu Arg Gly Asp 245 250 255 Cys
Met Tyr Ala Pro Leu Leu Thr Gly Pro Pro Ser Val Arg Pro Thr 260 265
270 Pro Cys Ala Pro Leu Ala Glu Cys Lys Gly Ser Leu Leu Asp Asp Gly
275 280 285 Pro Gly Lys Ser Asn Glu Glu Thr Ala Glu Tyr Ser Pro Phe
Lys Ala 290 295 300 Gly Tyr Thr Lys Gly Leu Asp Ser Glu Ser Leu Gly
Cys Ser Ser Gly 305 310 315 320 Gly Glu Ala Gly Gly Ser Gly Thr Leu
Glu Leu Pro Ser Ala Leu Ser 325 330 335 Leu Tyr Lys Ser Gly Ala Leu
Asp Asp Val Ala Ala Tyr Pro Ser Arg 340 345 350 Asp Tyr Tyr Asn Phe
Pro Leu Ala Leu Ala Gly Pro Pro Pro Pro Pro 355 360 365 Pro Pro Pro
His Pro His Ala Arg Ile Lys Leu Glu Asn Pro Leu Asp 370 375 380 Tyr
Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg Tyr Gly Asp 385 390
395 400 Leu Ala Ser Leu His Gly Gly Gly Ala Pro Gly Pro Gly Ser Gly
Ser 405 410 415 Pro Ser Ala Thr Ser Ser Ser Ser Trp His Thr Leu Phe
Thr Ala Glu 420 425 430 Glu Ser Gln Leu Tyr Gly Pro Cys Gly Gly Gly
Gly Gly Gly Ser Ala 435 440 445 Gly Glu Ala Gly Ala Val Ala Pro Tyr
Gly Tyr Thr Arg Pro Pro Gln 450 455 460 Gly Leu Ala Gly Gln Glu Gly
Asp Leu Ala Ile Pro Asp Ile Trp Tyr 465 470 475 480 Pro Gly Gly Val
Val Ser Arg Val Pro Tyr Pro Ser Pro Ser Cys Val 485 490 495 Lys Ser
Glu Met Gly Pro Trp Met Glu Ser Tyr Ser Gly Pro Tyr Gly 500 505 510
Asp Met Arg Leu Glu Pro Thr Arg Asp His Val Leu Pro Ile Asp Tyr 515
520 525 Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala
Ser 530 535 540 Gly Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys
Val Phe Phe 545 550 555 560 Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr
Leu Cys Ala Ser Arg Asn 565 570 575 Asp Cys Thr Ile Asp Lys Phe Arg
Arg Lys Asn Cys Pro Ser Cys Arg 580 585 590 Leu Arg Lys Cys Tyr Glu
Ala Gly Met Thr Leu Gly Ala Arg Lys Leu 595 600 605 Lys Lys Leu Gly
Asn Leu Lys Leu Gln Glu Glu Gly Glu Ala Ser Ser 610 615 620 Ala Thr
Ser Pro Thr Glu Glu Pro Ala Gln Lys Leu Thr Val Ser His 625 630 635
640 Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val Leu Glu Ala
645 650 655 Ile Glu Pro Gly Val Val Cys Ala Gly His Asp Asn Asn Gln
Pro Asp 660 665 670 Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu
Gly Glu Arg Gln 675 680 685 Leu Val His Val Val Lys Trp Ala Lys Ala
Leu Pro Gly Phe Arg Asn 690 695 700 Leu His Val Asp Asp Gln Met Ala
Val Ile Gln Tyr Ser Trp Met Gly 705 710 715 720 Leu Met Val Phe Ala
Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser 725 730 735 Arg Met Leu
Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr Arg Met 740 745 750 His
Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg His Leu Ser 755 760
765 Gln Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln Glu Phe Leu Cys Met
770 775 780 Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro Val Asp Gly Leu
Lys Asn 785 790 795 800 Gln Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr
Ile Lys Glu Leu Asp 805 810 815 Arg Ile Ile Ala Cys Lys Arg Lys Asn
Pro Thr Ser Cys Ser Arg Arg 820 825 830 Phe Tyr Gln Leu Thr Lys Leu
Leu Asp Ser Val Gln Pro Ile Ala Arg 835 840 845 Glu Leu His Gln Phe
Thr Phe Asp Leu Leu Ile Lys Ser His Met Val 850 855 860 Ser Val Asp
Phe Pro Glu Met Met Ala Glu Ile Ile Ser Val Gln Val 865 870 875 880
Pro Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe His Thr Gln 885
890 895 17709PRTOryctolagus cuniculus 17His His Gln Gln Gln Gln Asp
Ala Ala Thr Glu Gly Ser Ser Ser Gly 1 5 10 15 Arg Ala Arg Arg Pro
Ser Gly Ala Ser Thr Ser Ser Lys Asp Ser Tyr 20 25 30 Leu Gly Ser
Thr Ser Val Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys 35 40 45 Ala
Val Ser Val Ser Leu Gly Leu Gly Val Glu Ala Leu Glu His Leu 50 55
60 Ser Ser Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu
65 70 75 80 Gly Gly Pro Pro Val Val Arg Pro Thr Pro Cys Leu Pro Leu
Val Glu 85 90 95 Cys Lys Gly Ser Leu Leu Asp Asp Gly Pro Gly Lys
Gly Thr Glu Glu 100 105 110 Thr Ala Glu Tyr Thr Pro Phe Lys Gly Gly
Tyr Asn Lys Gly Leu Glu 115 120 125 Ala Glu Ser Leu Gly Cys Ser Gly
Ser Gly Glu Ala Gly Ser Ser Gly 130 135 140 Thr Leu Glu Leu Pro Ser
Thr Leu Ser Leu Tyr Lys Ser Gly Thr Leu 145 150 155 160 Asp Glu Ala
Ala Ala Tyr Gln Thr Arg Asp Tyr Tyr Asn Phe Pro Leu 165 170 175 Ala
Leu Ala Gly Gln Pro Pro Pro Pro His Pro Arg Arg Ile Lys Leu 180 185
190 Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln
195 200 205 Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Gly Gly Ala
Ala Gly 210 215 220 Pro Gly Ser Gly Ser Pro Ser Thr Ala Ala Ser Ser
Ser Trp His Thr 225 230 235 240 Leu Phe Thr Thr Glu Glu Gly Gln Leu
Tyr Gly Leu Cys Gly Gly Gly 245 250 255 Gly Gly Ser Gly Pro Gly Glu
Ala Gly Ala Val Ala Pro Tyr Gly Tyr 260 265 270 Thr Arg Pro Pro Gln
Gly Leu Thr Gly Gln Glu Gly Asp Phe Pro Ala 275 280 285 Pro Glu Val
Trp Tyr Pro Gly Gly Val Val Ser Arg Val Pro Tyr Pro 290 295 300 Asn
Pro Ser Cys Val Lys Ser Glu Met Gly Pro Trp Met Glu Ser Tyr 305 310
315 320 Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg Asp His
Val 325 330 335 Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys
Leu Ile Cys 340 345 350 Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala
Leu Thr Cys Gly Ser 355 360 365 Cys Lys Val Phe Phe Lys Arg Ala Ala
Glu Gly Lys Gln Lys Tyr Leu 370 375 380 Cys Ala Ser Arg Asn Asp Cys
Thr Ile Asp Lys Phe Arg Arg Lys Asn 385 390 395 400 Cys Pro Ser Cys
Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu 405 410 415 Gly Ala
Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu 420 425 430
Gly Glu Ser Ser Ser Ala Ser Ser Pro Thr Glu Asp Thr Thr Gln Lys 435
440 445 Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe
Leu 450 455 460 Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala
Gly His Asp 465 470 475 480 Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu
Leu Ser Ser Leu Asn Glu 485 490 495 Leu Gly Glu Arg Gln Leu Val His
Val Val Lys Trp Ala Lys Ala Leu 500 505 510 Pro Gly Phe Arg Asn Leu
His Val Asp Asp Gln Met Ala Val Ile Gln 515 520 525 Tyr Ser Trp Met
Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe 530 535 540 Thr Asn
Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe 545 550 555
560 Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys Val Arg
565 570 575 Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile Thr
Pro Gln 580
585 590 Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro
Val 595 600 605 Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg
Met Asn Tyr 610 615 620 Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys
Arg Lys Asn Pro Thr 625 630 635 640 Ser Cys Ser Arg Arg Phe Tyr Gln
Leu Thr Lys Leu Leu Asp Ser Val 645 650 655 Gln Pro Ile Ala Arg Glu
Leu His Gln Phe Thr Phe Asp Leu Leu Ile 660 665 670 Lys Ser His Met
Val Ser Val Asp Phe Pro Glu Met Met Ala Glu Ile 675 680 685 Ile Ser
Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys Pro Ile 690 695 700
Tyr Phe His Thr Gln 705 182760DNAHomo sapiens 18atggaagtgc
agttagggct gggaagggtc taccctcggc cgccgtccaa gacctaccga 60ggagctttcc
agaatctgtt ccagagcgtg cgcgaagtga tccagaaccc gggccccagg
120cacccagagg ccgcgagcgc agcacctccc ggcgccagtt tgctgctgct
gcagcagcag 180cagcagcagc agcagcagca gcagcagcag cagcagcagc
agcagcagca gcagcagcaa 240gagactagcc ccaggcagca gcagcagcag
cagggtgagg atggttctcc ccaagcccat 300cgtagaggcc ccacaggcta
cctggtcctg gatgaggaac agcaaccttc acagccgcag 360tcggccctgg
agtgccaccc cgagagaggt tgcgtcccag agcctggagc cgccgtggcc
420gccagcaagg ggctgccgca gcagctgcca gcacctccgg acgaggatga
ctcagctgcc 480ccatccacgt tgtccctgct gggccccact ttccccggct
taagcagctg ctccgctgac 540cttaaagaca tcctgagcga ggccagcacc
atgcaactcc ttcagcaaca gcagcaggaa 600gcagtatccg aaggcagcag
cagcgggaga gcgagggagg cctcgggggc tcccacttcc 660tccaaggaca
attacttagg gggcacttcg accatttctg acaacgccaa ggagttgtgt
720aaggcagtgt cggtgtccat gggcctgggt gtggaggcgt tggagcatct
gagtccaggg 780gaacagcttc ggggggattg catgtacgcc ccacttttgg
gagttccacc cgctgtgcgt 840cccactcctt gtgccccatt ggccgaatgc
aaaggttctc tgctagacga cagcgcaggc 900aagagcactg aagatactgc
tgagtattcc cctttcaagg gaggttacac caaagggcta 960gaaggcgaga
gcctaggctg ctctggcagc gctgcagcag ggagctccgg gacacttgaa
1020ctgccgtcta ccctgtctct ctacaagtcc ggagcactgg acgaggcagc
tgcgtaccag 1080agtcgcgact actacaactt tccactggct ctggccggac
cgccgccccc tccgccgcct 1140ccccatcccc acgctcgcat caagctggag
aacccgctgg actacggcag cgcctgggcg 1200gctgcggcgg cgcagtgccg
ctatggggac ctggcgagcc tgcatggcgc gggtgcagcg 1260ggacccggtt
ctgggtcacc ctcagccgcc gcttcctcat cctggcacac tctcttcaca
1320gccgaagaag gccagttgta tggaccgtgt ggtggtggtg ggggtggtgg
cggcggcggc 1380ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
aggcgggagc tgtagccccc 1440tacggctaca ctcggccccc tcaggggctg
gcgggccagg aaagcgactt caccgcacct 1500gatgtgtggt accctggcgg
catggtgagc agagtgccct atcccagtcc cacttgtgtc 1560aaaagcgaaa
tgggcccctg gatggatagc tactccggac cttacgggga catgcgtttg
1620gagactgcca gggaccatgt tttgcccatt gactattact ttccacccca
gaagacctgc 1680ctgatctgtg gagatgaagc ttctgggtgt cactatggag
ctctcacatg tggaagctgc 1740aaggtcttct tcaaaagagc cgctgaaggg
aaacagaagt acctgtgcgc cagcagaaat 1800gattgcacta ttgataaatt
ccgaaggaaa aattgtccat cttgtcgtct tcggaaatgt 1860tatgaagcag
ggatgactct gggagcccgg aagctgaaga aacttggtaa tctgaaacta
1920caggaggaag gagaggcttc cagcaccacc agccccactg aggagacaac
ccagaagctg 1980acagtgtcac acattgaagg ctatgaatgt cagcccatct
ttctgaatgt cctggaagcc 2040attgagccag gtgtagtgtg tgctggacac
gacaacaacc agcccgactc ctttgcagcc 2100ttgctctcta gcctcaatga
actgggagag agacagcttg tacacgtggt caagtgggcc 2160aaggccttgc
ctggcttccg caacttacac gtggacgacc agatggctgt cattcagtac
2220tcctggatgg ggctcatggt gtttgccatg ggctggcgat ccttcaccaa
tgtcaactcc 2280aggatgctct acttcgcccc tgatctggtt ttcaatgagt
accgcatgca caagtcccgg 2340atgtacagcc agtgtgtccg aatgaggcac
ctctctcaag agtttggatg gctccaaatc 2400accccccagg aattcctgtg
catgaaagca ctgctactct tcagcattat tccagtggat 2460gggctgaaaa
atcaaaaatt ctttgatgaa cttcgaatga actacatcaa ggaactcgat
2520cgtatcattg catgcaaaag aaaaaatccc acatcctgct caagacgctt
ctaccagctc 2580accaagctcc tggactccgt gcagcctatt gcgagagagc
tgcatcagtt cacttttgac 2640ctgctaatca agtcacacat ggtgagcgtg
gactttccgg aaatgatggc agagatcatc 2700tctgtgcaag tgcccaagat
cctttctggg aaagtcaagc ccatctattt ccacacccag 2760192760DNAHomo
sapiens 19atggaagtgc agttagggct gggaagggtc taccctcggc cgccgtccaa
gacctaccga 60ggagctttcc agaatctgtt ccagagcgtg cgcgaagtga tccagaaccc
gggccccagg 120cacccagagg ccgcgagcgc agcacctccc ggcgccagtt
tgctgctgct gcagcagcag 180cagcagcagc agcagcagca gcagcagcag
cagcagcagc agcagcagca gcagcagcaa 240gagactagcc ccaggcagca
gcagcagcag cagggtgagg atggttctcc ccaagcccat 300cgtagaggcc
ccacaggcta cctggtcctg gatgaggaac agcaaccttc acagccgcag
360tcggccctgg agtgccaccc cgagagaggt tgcgtcccag agcctggagc
cgccgtggcc 420gccagcaagg ggctgccgca gcagctgcca gcacctccgg
acgaggatga ctcagctgcc 480ccatccacgt tgtccctgct gggccccact
ttccccggct taagcagctg ctccgctgac 540cttaaagaca tcctgagcga
ggccagcacc atgcaactcc ttcagcaaca gcagcaggaa 600gcagtatccg
aaggcagcag cagcgggaga gcgagggagg cctcgggggc tcccacttcc
660tccaaggaca attacttagg gggcacttcg accatttctg acaacgccaa
ggagttgtgt 720aaggcagtgt cggtgtccat gggcctgggt gtggaggcgt
tggagcatct gagtccaggg 780gaacagcttc ggggggattg catgtacgcc
ccacttttgg gagttccacc cgctgtgcgt 840cccactcctt gtgccccatt
ggccgaatgc aaaggttctc tgctagacga cagcgcaggc 900aagagcactg
aagatactgc tgagtattcc cctttcaagg gaggttacac caaagggcta
960gaaggcgaga gcctaggctg ctctggcagc gctgcagcag ggagctccgg
gacacttgaa 1020ctgccgtcta ccctgtctct ctacaagtcc ggagcactgg
acgaggcagc tgcgtaccag 1080agtcgcgact actacaactt tccactggct
ctggccggac cgccgccccc tccgccgcct 1140ccccatcccc acgctcgcat
caagctggag aacccgctgg actacggcag cgcctgggcg 1200gctgcggcgg
cgcagtgccg ctatggggac ctggcgagcc tgcatggcgc gggtgcagcg
1260ggacccggtt ctgggtcacc ctcagccgcc gcttcctcat cctggcacac
tctcttcaca 1320gccgaagaag gccagttgta tggaccgtgt ggtggtggtg
ggggtggtgg cggcggcggc 1380ggcggcggcg gcggcggcgg cggcggcggc
ggcggcggcg aggcgggagc tgtagccccc 1440tacggctaca ctcggccccc
tcaggggctg gcgggccagg aaagcgactt caccgcacct 1500gatgtgtggt
accctggcgg catggtgagc agagtgccct atcccagtcc cacttgtgtc
1560aaaagcgaaa tgggcccctg gatggatagc tactccggac cttacgggga
catgcgtttg 1620gagactgcca gggaccatgt tttgcccatt gactattact
ttccacccca gaagacctgc 1680ctgatctgtg gagatgaagc ttctgggtgt
cactatggag ctctcacatg tggaagctgc 1740aaggtcttct tcaaaagagc
cgctgaaggg aaacagaagt acctgtgcgc cagcagaaat 1800gattgcacta
ttgataaatt ccgaaggaaa aattgtccat cttgtcgtct tcggaaatgt
1860tatgaagcag ggatgactct gggagcccgg aagctgaaga aacttggtaa
tctgaaacta 1920caggaggaag gagaggcttc cagcaccacc agccccactg
aggagacaac ccagaagctg 1980acagtgtcac acattgaagg ctatgaatgt
cagcccatct ttctgaatgt cctggaagcc 2040attgagccag gtgtagtgtg
tgctggacac gacaacaacc agcccgactc ctttgcagcc 2100ttgctctcta
gcctcaatga actgggagag agacagcttg tacacgtggt caagtgggcc
2160aaggccttgc ctggcttccg caacttacac gtggacgacc agatggctgt
cattcagtac 2220tcctggatgg ggctcatggt gtttgccatg ggctggcgat
ccttcaccaa tgtcaactcc 2280aggatgctct acttcgcccc tgatctggtt
ttcaatgagt accgcatgca caagtcccgg 2340atgtacagcc agtgtgtccg
aatgaggcac ctctctcaag agtttggatg gctccaaatc 2400accccccagg
aattcctgtg catgaaagca ctgctactct tcagcattat tccagtggat
2460gggctgaaaa atcaaaaatt ctttgatgaa cttcgaatga actacatcaa
ggaactcgat 2520cgtatcattg catgcaaaag aaaaaatccc acatcctgct
caagacgctt ctaccagctc 2580accaagctcc tggactccgt gcagcctatt
gcgagagagc tgcatcagct cacttttgac 2640ctgctaatca agtcacacat
ggtgagcgtg gactttccgg aaatgatggc agagatcatc 2700tctgtgcaag
tgcccaagat cctttctggg aaagtcaagc ccatctattt ccacacccag
27602020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20agagactcag aggcgaccat 202120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21atcagcaaac acagcagctc 202224DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 22agagctgttg gatgaggacc agaa
242324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 23aggctccaaa ggcacttgac tact 242421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24gaccaagcgg gttgttattg a 212523DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 25tgccttgtcg gtcatatttt tca
232620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26cggagaacca aacggaaagg 202719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27cttcgcccac agtgaatgc 192824DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 28acagcagccg gtttattgtg cttc
242924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29tggcattcag tctcacacca ctgt 243024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30accactcacc atcatctcaa ggca 243124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
31tgctcttctt tgccagatcc tcgt 243220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
32gccttaccct tgcagcttac 203320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 33gagcatgctg tccactctgt
203424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34cttgcgcatt cccaagtcag atgt 243524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
35tttcctctcc ttctcgtgct gctt 243620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36gagcctgcta cagatggtca 203720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 37tgtctaccag caggacgaag
203820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38cctcctgaag aatcgattcc 203920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39gaggtccaca cactgaagtt 204024DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 40tttctgggct ggccaaacat aagc
244124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41acacaaggta atgtgtgggt ccga 244224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42cggcaggtgt ttgtgtgtgg aaat 244324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
43agaagacaca cagcacagca gaca 244424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44tagtgaaacc agtgtgtctg ccca 244524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
45agcgttcagc acttctgagg tctt 244621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
46cgctctggtt catctgctct g 214722DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47tcatcaaagg tgctctcgtc tg
224824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 48tggagaggaa gttcagccat caga 244924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49aggagagctg ctttcgctta gtct 245024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50acctgctcag cctttgtctc tgat 245124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
51agatccaggc ttgcttactg tcct 245224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
52attctgggtt gggagtgcaa ggaa 245324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53aggagacatg cccaggatga aaca 245424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
54actaggcagg acattgacat ccca 245524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
55acagtaaacc tctccacaca tggc 245624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56tatgacaccc agggctttcg ttca 245724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57taacgttccc tgcgcgttta caga 245818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
58tcccaaatcc tgacccca 185920DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 59accacacagc ccctaggaga
206021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 60acagggtggc ccaaatagaa c 216121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
61cctgtcttgg acaagcggag a 216224DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 62gggtcatttc caccacctca
aaca 246324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 63ggagaaaggc cttacagtag tctc 24
* * * * *