U.S. patent application number 17/562732 was filed with the patent office on 2022-08-04 for methods and uses for modulating bile acid homeostasis and treatment of bile acid disorders and diseases.
The applicant listed for this patent is NGM Biopharmaceuticals, Inc.. Invention is credited to Lei LING, Hui TIAN.
Application Number | 20220241373 17/562732 |
Document ID | / |
Family ID | 1000006289273 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220241373 |
Kind Code |
A1 |
LING; Lei ; et al. |
August 4, 2022 |
METHODS AND USES FOR MODULATING BILE ACID HOMEOSTASIS AND TREATMENT
OF BILE ACID DISORDERS AND DISEASES
Abstract
Provided herein are methods of modulating bile acid homeostasis
or treating a bile-acid related or associated disorder, comprising
using variants and fusions of fibroblast growth factor 19 (FGF19),
variants and fusions of fibroblast growth factor 21 (FGF21),
fusions of FGF19 and/or FGF21, and variants or fusions of FGF19
and/or FGF21 proteins and peptide sequences (and peptidomimetics),
in combination with agents effective in modulating bile acid
homeostasis or treating a bile-acid related or associated
disorder.
Inventors: |
LING; Lei; (Foster City,
CA) ; TIAN; Hui; (Foster City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NGM Biopharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000006289273 |
Appl. No.: |
17/562732 |
Filed: |
December 27, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16600139 |
Oct 11, 2019 |
11241481 |
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17562732 |
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15316108 |
Dec 2, 2016 |
10456449 |
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PCT/US15/35752 |
Jun 15, 2015 |
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16600139 |
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62012899 |
Jun 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61K 31/519 20130101; A61K 45/06 20130101; A61K 31/573 20130101;
C07K 14/50 20130101; A61K 31/155 20130101; A61K 31/575 20130101;
A61K 31/52 20130101; A61K 38/26 20130101; C07K 2319/00 20130101;
A61K 31/165 20130101; A61K 31/64 20130101; A61K 38/208 20130101;
A61K 38/28 20130101; A61K 38/13 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 45/06 20060101 A61K045/06; C07K 14/50 20060101
C07K014/50; A61K 38/13 20060101 A61K038/13; A61K 38/20 20060101
A61K038/20; A61K 38/28 20060101 A61K038/28; A61K 38/26 20060101
A61K038/26; A61K 31/519 20060101 A61K031/519; A61K 31/155 20060101
A61K031/155; A61K 31/52 20060101 A61K031/52; A61K 31/575 20060101
A61K031/575; A61K 31/165 20060101 A61K031/165; A61K 31/64 20060101
A61K031/64; A61K 31/573 20060101 A61K031/573 |
Claims
1.-69. (canceled)
70. A method of reducing bile acid synthesis in a subject in need
thereof, comprising administering to the subject a therapeutically
effective amount of a polypeptide comprising or consisting of the
amino acid sequence set forth in SEQ ID NO:141, thereby reducing
bile acid synthesis in the subject without inducing hepatocellular
carcinoma (HCC) formation.
71. The method of claim 70, wherein the method further comprises
administering to the subject at least one additional agent, wherein
the additional agent is an anti-CD20 agent, an anti-CD80 agent, an
anti-cytokine antibody, an anti-retroviral therapy, an apical
sodium bile acid transporter (ASBT) inhibitor, an autoimmune agent,
azathioprine, colchicine, a CSCL10 neutralizing antibody, a CXCR3
ligand, cyclosporine, a cytokine anti-inflammatory drug (CSAID), a
cytokine, a growth factor, a fibrate, a fish oil, an immune
checkpoint inhibitor, a non-steroidal anti-inflammatory drug
(NSAID), a farnesoid X receptor (FXR) agonist, a steroid, a
chenodeoxycholic acid (CDCA), an obeticholic acid (OCA), or an
ursodeoxycholic acid (UDCA).
72. The method of claim 71, wherein the cytokine is IL-12.
73. The method of claim 71, wherein the steroid is a
glucocorticoid.
74. The method of claim 70, wherein the subject has nonalcoholic
steatohepatitis (NASH).
75. The method of claim 70, wherein the subject has primary biliary
cirrhosis (PBC).
76. The method of claim 70, wherein the subject has
cholestasis.
77. The method of claim 70, wherein the subject has primary
sclerosing cholangitis (PSC).
78. The method of claim 70, wherein the subject has bile acid
diarrhea (BAD) or bile acid malabsorption.
79. The method of claim 70, wherein the subject has pregnancy
intrahepatic cholestasis (PIC).
80. The method of claim 70, wherein the subject has liver
fibrosis.
81. The method of claim 70, wherein the subject has nonalcoholic
fatty liver disease (NAFLD).
82. The method of claim 70, wherein the subject has cirrhosis.
83. The method of claim 70, wherein the polypeptide comprises the
amino acid sequence set forth in SEQ ID NO:141.
84. The method of claim 70, wherein the polypeptide consists of the
amino acid sequence set forth in SEQ ID NO:141.
85. The method of claim 71, wherein the method comprises
administration of SEQ ID NO:141 and an ASBT inhibitor.
86. The method of claim 85, wherein the ASBT inhibitor is selected
from the group consisting of LUM001 and SC-435.
87. A method of reducing bile acid synthesis in a subject in need
thereof, comprising administering to the subject a therapeutically
effective amount of (i) a polypeptide comprising or consisting of
the amino acid sequence set forth in SEQ ID NO:69, and (ii) at
least one additional agent, wherein the additional agent is an
anti-CD20 agent, an anti-CD80 agent, an anti-cytokine antibody, an
anti-retroviral therapy, an ASBT inhibitor, an autoimmune agent,
azathioprine, colchicine, a CSCL10 neutralizing antibody, a CXCR3
ligand, cyclosporine, a CSAID, a cytokine, a growth factor, a
fibrate, a fish oil, an immune checkpoint inhibitor, a NSAID, a FXR
agonist, a steroid, a CDCA, an OCA, or an UDCA; thereby reducing
bile acid synthesis in the subject without inducing HCC
formation.
88. The method of claim 87, wherein the cytokine is IL-12.
89. The method of claim 87, wherein the steroid is a
glucocorticoid.
90. The method of claim 87, wherein the subject has NASH, PBC,
cholestasis, PSC, BAD or bile acid malabsorption, PIC, liver
fibrosis, NAFLD or liver cirrhosis.
91. The method of claim 87, wherein the polypeptide comprises the
amino acid sequence set forth in SEQ ID NO:69.
92. The method of claim 87, wherein the polypeptide consists of the
amino acid sequence set forth in SEQ ID NO:69.
93. A method of reducing bile acid synthesis in a subject in need
thereof, comprising administering to the subject a therapeutically
effective amount of (i) a polypeptide comprising or consisting of
the amino acid sequence set forth in SEQ ID NO:52, and (ii) at
least one additional agent, wherein the additional agent is an
anti-CD20 agent, an anti-CD80 agent, an anti-cytokine antibody, an
anti-retroviral therapy, an ASBT inhibitor, an autoimmune agent,
azathioprine, colchicine, a CSCL10 neutralizing antibody, a CXCR3
ligand, cyclosporine, a CSAID, a cytokine, a growth factor, a
fibrate, a fish oil, an immune checkpoint inhibitor, a NSAID, a FXR
agonist, a steroid, a CDCA, an OCA, or an UDCA; thereby reducing
bile acid synthesis in the subject without inducing HCC
formation.
94. The method of claim 93, wherein the cytokine is IL-12.
95. The method of claim 93, wherein the steroid is a
glucocorticoid.
96. The method of claim 93, wherein the subject has NASH, PBC,
cholestasis, PSC, BAD or bile acid malabsorption, PIC, liver
fibrosis, NAFLD or liver cirrhosis.
97. The method of claim 93, wherein the polypeptide comprises the
amino acid sequence set forth in SEQ ID NO:52.
98. The method of claim 93, wherein the polypeptide consists of the
amino acid sequence set forth in SEQ ID NO:52.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
16/600,139 filed Oct. 11, 2019, which is a continuation of U.S.
Ser. No. 15/316,108 filed Dec. 2, 2016 (now U.S. Pat. No.
10,456,449), which is a 371 national stage application of
international application Serial No. PCT/US2015/035752 filed Jun.
15, 2015, which claims the benefit of priority to U.S. Ser. No.
62/012,899 filed Jun. 16, 2014, the contents of each of which is
incorporated herein by reference in its entirety.
FIELD
[0002] The invention relates, in part, to the treatment or
prevention of bile acid-related and associated disorders with
variants of fibroblast growth factor 19 (FGF19) proteins and
peptide sequences (and peptidomimetics) and fusions of FGF19 and/or
fibroblast growth factor 21 (FGF21) proteins and peptide sequences
(and peptidomimetics), and variants of fusions of FGF19 and/or
FGF21 proteins and peptide sequences (and peptidomimetics) in
combination with one or more additional therapeutic agents.
Introduction
[0003] Bile acids, steroid acids that are found predominantly in
the bile of mammals, regulate cholesterol, triglyceride, glucose
and energy homeostasis, and facilitate digestion and absorption of
lipids in the small intestine. In humans, bile acid production
occurs primarily in the perivenous hepatocytes through a series of
enzymatic reactions that convert cholesterol into the two primary
bile acids, cholic acid and chenodeoxycholic acid.
[0004] The primary bile acids are synthesized by two distinct
pathways. In the "classic" or "neutral" pathway, the primary bile
acids are produced by hydroxylation of cholesterol through
catalysis by the cytochrome P450 enzyme cholesterol
7.alpha.-hydroxylase (CYP7A1), which catalyzes the first and
rate-limiting step. The conversion of cholesterol to bile acids is
primarily effected by this pathway. See, e.g., Inagaki et al., Cell
Metabolism 2:217-25 (October 2005). CYP7A1 activity is
down-regulated by cholic acid and up-regulated by cholesterol;
thus, CYP7A1 is regulated by bile acids themselves. Thus,
repression of CYP7A1 results in the decreased synthesis of bile
acids from intrahepatic cholesterol in response to the daily
feeding-fasting cycle. In addition, in most individuals
approximately 6% of bile acids are synthesized by an "alternative"
or "acidic" pathway. This pathway is regulated by the enzyme
CYP27A1, which converts oxysterols to bile acids. In contrast to
CYP7A1, CYP27A1 is not regulated by bile acids.
[0005] When cholic acid and chenodeoxycholic acid are secreted into
the lumen of the intestine, intestinal bacteria dehydroxylate a
portion of each to form the secondary bile acids, deoxycholic acid
(derived from cholic acid) and lithocholic acid (derived from
chenodeoxycholic acid). Enterohepatic circulation enables
.about.90-95% of all four bile acids to be reabsorbed from the
distal ileum and transported back to the liver. The approximately
5% of bile acids that are not reabsorbed are eliminated in the
feces, and that amount of loss is subsequently replaced by de novo
bile acid synthesis in the liver See, e.g., Rose et al., Cell
Metabolism, 14:1, pp 123-130 (6 Jul. 2011).
[0006] As surfactants or detergents, bile acids are potentially
toxic to cells, and the size of the bile acid pool is tightly
regulated within the liver and intestine to prevent cytotoxic
accumulation. When the bile acid pool size increases, a feedback
mechanism involving the interplay of several nuclear receptors,
including FXR, is activated to inhibit de novo bile acid synthesis.
See, e.g., Fiorucci et al., Prog Lipid Res. 2010 April;
49(2):171-85. Epub 2009 Dec. 2. In one signaling pathway,
intestinal FXR activation due to transintestinal bile acid flux
after a meal induces the expression of the hormone FGF19, which is
released by small intestinal epithelial cells and circulates to
bind to hepatocyte FGF receptor 4 (FGFR4) receptors. The FGFR4
receptors signal a reduction in bile acid synthesis via c-Jun
NH.sub.2-terminal kinase (JNK) pathway activation.
[0007] Cholestasis, one of the most common bile acid-related
disorders, is a condition characterized by a reduction or cessation
of bile flow from the liver to the small intestine (principally the
duodenum). Primary biliary cirrhosis (PBC) is the most common
cholestatic liver disease and is the fifth most common cause of
liver transplant in the United States. PBC is a progressive hepatic
disease that primarily results from autoimmune destruction of the
bile ducts that transport bile acids out of the liver. As the
disease progresses, persistent toxic build-up of bile acids causes
progressive liver damage marked by chronic inflammation and
fibrosis. A majority of PBC patients are asymptomatic at the time
of initial diagnosis, but most develop symptoms, such as fatigue
and pruritus, over time. Jaundice may result from advanced
disease.
[0008] Though several therapeutic modalities exist for the
treatment and prevention of bile acid-related disorders in general,
and primary biliary cirrhosis in particular, many patients are
inadequately treated with current agents as monotherapy, and such
patients would benefit from new treatment regimens.
SUMMARY
[0009] The invention is based, in part, on the use of variants of
FGF19 peptide sequences, fusions of FGF19 and/or FGF21 peptide
sequences and variants of fusions (chimeras) of FGF19 and/or FGF21
peptide sequences having one or more activities associated with the
treatment and/or prevention of bile acid-related disorders, in
combination with other therapeutic agents and/or treatment
modalities. Such variants and fusions (chimeras) of FGF19 and/or
FGF21 peptide sequences include sequences that do not substantially
increase or induce hepatocellular carcinoma (HCC) formation or HCC
tumorigenesis and/or do not induce a substantial elevation or
increase in lipid profile. Examples of such variants and fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences further include
those sequences disclosed in PCT Pub. No. WO 2013/006486 and US
Pub. No. 2013/0023474, published Jan. 20, 2013 and Jan. 24, 2013,
respectively; as well as PCT Publ. No. WO 2014/085365, published
Jun. 5, 2014.
[0010] Provided herein are compositions and mixtures comprising
certain peptide sequences, including subsequences, variants and
modified forms of the exemplified peptide sequences (including the
FGF19 and FGF21 variants and subsequences listed in the Sequence
Listing or Table 1, and the FGF19/FGF21 fusions and chimeras listed
in the Sequence Listing or Table 1), and one or more
pharmaceutically acceptable carriers or excipients. Combinations,
such as one or more peptide sequences in a pharmaceutically
acceptable carrier or excipient, with one or more therapeutic
agents or treatment modalities useful in the treatment and/or
prevention of a bile acid-related disease, disorder, or condition
are also provided. Such combinations of peptide sequence(s)
provided herein with one or more additional agents or modalities
are useful in accordance with the methods and uses provided
herein.
[0011] Uses and methods of treatment that include administration or
delivery of a chimeric peptide or peptide sequence in combination
with an agent that improves bile acid homeostasis are also provided
herein. In particular embodiments, a use or method of treatment of
a subject includes administering a chimeric peptide or peptide
sequence provided herein to a subject having, or at risk of having,
a disorder of bile acid homeostasis treatable by a peptide sequence
provided herein, in an amount effective for treating the disorder,
in combination with at least one additional agent or treatment
modality having an additive, synergistic or complementary effect.
The additional agent or treatment modality may also confer one or
more further benefits, such as, but not limited to, the ability to
lower the dose of one or more of the peptide sequence(s) provided
herein or the additional agent(s) in order to favorably impact one
or more of the adverse effects experienced by the subject (e.g.,
decreasing the frequency or severity of an adverse effect).
[0012] In one embodiment, a method or use of modulating bile acid
homeostasis or treating a bile-acid related or associated disorder
includes: a) administering a chimeric peptide sequence, comprising:
i) an N-terminal region comprising at least seven amino acid
residues, the N-terminal region having a first amino acid position
and a last amino acid position, wherein the N-terminal region
comprises DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122), and ii) a
C-terminal region comprising a portion of SEQ ID NO:99 (FGF19), the
C-terminal region having a first amino acid position and a last
amino acid position, wherein the C-terminal region comprises amino
acid residues 16-29 of SEQ ID NO:99 (FGF19), WGDPIRLRHLYTSG (SEQ ID
NO:169), wherein the W residue corresponds to the first amino acid
position of the C-terminal region; and b) administering at least
one additional agent effective in modulating bile acid homeostasis
or treating a bile-acid related or associated disorder; to modulate
bile acid homeostasis or treat the bile-acid related or associated
disorder.
[0013] In another embodiment, a method or use of modulating bile
acid homeostasis or treating a bile-acid related or associated
disorder includes: a) administering a chimeric peptide sequence,
comprising: i) an N-terminal region comprising a portion of SEQ ID
NO:100 (FGF21), the N-terminal region having a first amino acid
position and a last amino acid position, wherein the N-terminal
region comprises amino acid residues GQV, and wherein the V residue
corresponds to the last amino acid position of the N-terminal
region, and ii) a C-terminal region comprising a portion of SEQ ID
NO:99 (FGF19), the C-terminal region having a first amino acid
position and a last amino acid position, wherein the C-terminal
region comprises amino acid residues 21-29 of SEQ ID NO:99 (FGF19),
RLRHLYTSG (SEQ ID NO:185), and wherein the R residue corresponds to
the first position of the C-terminal region; and b) administering
at least one additional agent effective in modulating bile acid
homeostasis or treating a bile-acid related or associated disorder;
to modulate bile acid homeostasis or treat the bile-acid related or
associated disorder.
[0014] In a further embodiment, a method or use of modulating bile
acid homeostasis or treating a bile-acid related or associated
disorder includes: a) administering a chimeric peptide sequence,
comprising: ii) an N-terminal region comprising a portion of SEQ ID
NO:100 (FGF21), the N-terminal region having a first amino acid
position and a last amino acid position, wherein the N-terminal
region comprises at least 5 contiguous amino acids of SEQ ID NO:100
(FGF21) including the amino acid residues GQV, and wherein the V
residue corresponds to the last amino acid position of the
N-terminal region, and ii) a C-terminal region comprising a portion
of SEQ ID NO:99 (FGF19), the C-terminal region having a first amino
acid position and a last amino acid position, wherein the
C-terminal region comprises amino acid residues 21-29 of SEQ ID
NO:99 (FGF19), RLRHLYTSG (SEQ ID NO:185), and wherein the R residue
corresponds to the first position of the C-terminal region; and b)
administering at least one additional agent effective in modulating
bile acid homeostasis or treating a bile-acid related or associated
disorder; to modulate bile acid homeostasis or treat the bile-acid
related or associated disorder.
[0015] In an additional embodiment, a method or use of modulating
bile acid homeostasis or treating a bile-acid related or associated
disorder includes: administering a) peptide sequence, comprising or
consisting of any of: i) a FGF19 sequence variant having one or
more amino acid substitutions, insertions or deletions compared to
a reference or wild type FGF19, ii) a FGF21 sequence variant having
one or more amino acid substitutions, insertions or deletions
compared to a reference or wild type FGF21, iii) a portion of an
FGF19 sequence fused to a portion of an FGF21 sequence, or iv) a
portion of an FGF19 sequence fused to a portion of an FGF21
sequence, wherein the FGF19 and/or FGF21 sequence portion(s) have
one or more amino acid substitutions, insertions or deletions
compared to a reference or wild type FGF19 and/or FGF21; and b)
administering at least one additional agent effective in modulating
bile acid homeostasis or treating a bile-acid related or associated
disorder; to modulate bile acid homeostasis or treat the bile-acid
related or associated disorder.
[0016] In various particular embodiments, a chimeric peptide
sequence has an N-terminal region with at least 6 contiguous amino
acids of SEQ ID NO:100 (FGF21) including the amino acid residues
GQ; or has an N-terminal region with at least 7 contiguous amino
acids of SEQ ID NO:100 (FGF21) including the amino acid residues
GQV.
[0017] In various additional embodiments, a peptide sequence has
amino-terminal amino acids 1-16 of SEQ ID NO:100 (FGF21) fused to
carboxy-terminal amino acids 21-194 of SEQ ID NO:99 (FGF19), or the
peptide sequence has amino-terminal amino acids 1-147 of SEQ ID
NO:99 (FGF19) fused to carboxy-terminal amino acids 147-181 of SEQ
ID NO:100 (FGF21) (M41), or the peptide sequence has amino-terminal
amino acids 1-20 of SEQ ID NO:99 (FGF19) fused to carboxy-terminal
amino acids 17-181 of SEQ ID NO:100 (FGF21) (M44), or the peptide
sequence has amino-terminal amino acids 1-146 of SEQ ID NO:100
(FGF21) fused to carboxy-terminal amino acids 148-194 of SEQ ID
NO:99 (FGF19) (M45), or the peptide sequence has amino-terminal
amino acids 1-20 of SEQ ID NO:99 (FGF19) fused to internal amino
acids 17-146 of SEQ ID NO:100 (FGF21) or fused to carboxy-terminal
amino acids 148-194 of SEQ ID NO:99 (FGF19) (M46).
[0018] In various further embodiments, a peptide sequence has at
least one amino acid substitution to amino acid residues 125-129 of
SEQ ID NO:99 (FGF19), EIRPD; at least one amino acid substitution
to amino acid residues 126-128 of SEQ ID NO:99 (FGF19), IRP; or at
least one amino acid substitution to amino acid residues 127-128 of
SEQ ID NO:99 (FGF19), RP, or at least one amino acid substitution
to amino acid residues 1-124 of SEQ ID NO:99 (FGF19) and/or to
amino acid residues 130-194 of SEQ ID NO:99 (FGF19). More
specifically, for example, a peptide sequence with a substitution
to one of amino acid residues 127-128 of SEQ ID NO:99 (FGF19), IRP,
wherein at least one amino acid substitution is R127L or P128E. In
certain embodiments, the amino acid sequence of the peptide
comprises at least one amino acid substitution in the Loop-8 region
of FGF19, or the corresponding FGF19 sequence thereof in a variant
peptide provided herein. In certain embodiments, the amino acid
sequence of the peptide comprises one amino acid substitution to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In other embodiments, the amino acid
sequence of the peptide comprises three amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises four amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises five amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises one amino acid substitution to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In other embodiments, the amino acid
sequence of the peptide comprises three amino acid substitutions to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises one amino acid substitution to
the RP (amino acids 4-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the RP (amino acids 4-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
substitution to the RP (amino acids 4-5 of SEQ ID NO:190) amino
acid sequence in the Loop-8 region of FGF19 is an Arg (R) to Leu
(L) substitution. In other embodiments, the substitution to the RP
(amino acids 4-5 of SEQ ID NO:190) amino acid sequence in the
Loop-8 region of FGF19 is a Pro (P) to Glu (E) substitution. In
some embodiments, the substitutions to the RP (amino acids 4-5 of
SEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19 is
an Arg (R) to Leu (L) substitution and a Pro (P) to Glu (E)
substitution. In specific embodiments, the foregoing
substitution(s) in the Loop-8 region of FGF19 is in the
corresponding FGF19 sequence thereof in a variant peptide provided
herein. That is, said substitutions within a corresponding FGF19
sequence (e.g., EIRPD, IRP or RP) of a peptide variant provided
herein is also contemplated.
[0019] Methods and uses provided herein can be practiced using a
peptide or chimeric sequence, as set forth herein. For example, a
sequence that includes or consists of any peptide sequence set
forth herein as M1-M98, M101 to M160, or M200 to M207, or SEQ ID
NOs:1 to 98, or 101 to 135, or 138 to 212. In other embodiments,
the peptide sequence includes or consists of any sequence set forth
in Table 1. In yet other embodiments, the peptide sequence that
includes or consists of any sequence set forth in the Sequence
Listing herein.
[0020] Methods and uses provided herein can be practiced using a
peptide or chimeric sequence of any suitable length. In particular
embodiments, the N-terminal or C-terminal region of the peptide or
chimeric sequence is from about 20 to about 200 amino acid residues
in length. In other particular aspects, a peptide or chimeric
sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more amino acid deletions from the amino
terminus, the carboxy-terminus or internally. In further particular
embodiments, a peptide or chimeric sequence has an N-terminal
region, or a C-terminal region that includes or consists of an
amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40,
40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino
acids. In additional more particular embodiments, a peptide or
chimeric sequence has an FGF19 sequence portion, or an FGF21
sequence portion that includes or consists of an amino acid
sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50,
50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino
acids of FGF19 or FGF21.
[0021] In yet additional embodiments, a peptide sequence or a
chimeric peptide sequence has a WGDPI (SEQ ID NO:170) sequence
motif corresponding to the WGDPI (SEQ ID NO:170) sequence of amino
acids 16-20 of SEQ ID NO:99 (FGF19); has a substituted, mutated or
absent WGDPI (SEQ ID NO:170) sequence motif corresponding to FGF19
WGDPI (SEQ ID NO:170) sequence of amino acids 16-20 of FGF19; has a
WGDPI (SEQ ID NO:170) sequence with one or more amino acids
substituted, mutated or absent. In various other further aspects,
the peptide sequence is distinct from an FGF19 variant sequence
having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID
NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI
(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),
WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID
NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID
NO:183) or FGDPI (SEQ ID NO:184) substituted for the FGF19 WGDPI
(SEQ ID NO:170) sequence at amino acids 16-20.
[0022] In yet further embodiments, a peptide sequence or a chimeric
peptide sequence has N-terminal region comprises amino acid
residues VHYG (SEQ ID NO:101), wherein the N-terminal region
comprises amino acid residues DASPHVHYG (SEQ ID NO:102), or the
N-terminal region comprises amino acid residues DSSPLVHYG (SEQ ID
NO:103). More particularly, in one aspect the G corresponds to the
last position of the N-terminal region.
[0023] In various additional aspects, the N-terminal region
comprises amino acid residues DSSPLLQ (SEQ ID NO:104), where the Q
residue is the last amino acid position of the N-terminal region,
or comprises amino acid residues DSSPLLQFGGQV (SEQ ID NO:105),
where the V residue corresponds to the last position of the
N-terminal region.
[0024] In certain embodiments, an N-terminal region comprises or
consists of (or further comprises or consists of): RHPIP (SEQ ID
NO:106), where R is the first amino acid position of the N-terminal
region; or HPIP (SEQ ID NO:107), where H is the first amino acid
position of the N-terminal region; or RPLAF (SEQ ID NO:108), where
R is the first amino acid position of the N-terminal region; or
PLAF (SEQ ID NO:109), where P is the first amino acid position of
the N-terminal region; or R, where R is the first amino acid
position of the N-terminal region.
[0025] In various other aspects, a peptide or chimeric sequence
has: amino acid residues HPIP (SEQ ID NO:107), which are the first
4 amino acid residues of the N-terminal region. In various still
further aspects, a peptide or chimeric sequence has: an R residue
at the first position of the N-terminal region, or the first
position of the N-terminal region is an M residue, or the first and
second positions of the N-terminal region is an MR sequence, or the
first and second positions of the N-terminal region is an RM
sequence, or the first and second positions of the N-terminal
region is an RD sequence, or the first and second positions of the
N-terminal region is an DS sequence, or the first and second
positions of the N-terminal region is an MD sequence, or the first
and second positions of the N-terminal region is an MS sequence, or
the first through third positions of the N-terminal region is an
MDS sequence, or the first through third positions of the
N-terminal region is an RDS sequence, or the first through third
positions of the N-terminal region is an MSD sequence, or the first
through third positions of the N-terminal region is an MSS
sequence, or the first through third positions of the N-terminal
region is an DSS sequence, or the first through fourth positions of
the N-terminal region is an RDSS (SEQ ID NO:115), sequence, or the
first through fourth positions of the N-terminal region is an MDSS
(SEQ ID NO:116), sequence, or the first through fifth positions of
the N-terminal region is an MRDSS (SEQ ID NO:117), sequence, or the
first through fifth positions of the N-terminal region is an MSSPL
(SEQ ID NO:113) sequence, or the first through sixth positions of
the N-terminal region is an MDSSPL (SEQ ID NO:110) sequence, or the
first through seventh positions of the N-terminal region is an
MSDSSPL (SEQ ID NO:111) sequence.
[0026] In various other particular aspects, a peptide or chimeric
sequence has at the N-terminal region first amino acid position an
"M" residue, an "R" residue, a "S" residue, a "H" residue, a "P"
residue, a "L" residue or an "D" residue. In various alternative
particular aspects, a peptide or chimeric sequence peptide sequence
does not have a "M" residue or an "R" residue at the first amino
acid position of the N-terminal region.
[0027] In further various other aspects, a peptide or chimeric
sequence has an N-terminal region with any one of the following
sequences: MDSSPL (SEQ ID NO:110), MSDSSPL (SEQ ID NO:111), SDSSPL
(SEQ ID NO:112), MSSPL (SEQ ID NO:113) or SSPL (SEQ ID NO:114).
[0028] In various still additional aspects, a peptide or chimeric
sequence has a residue at the last position of the C-terminal
region that corresponds to about residue 194 of SEQ ID NO:99
(FGF19). In still other embodiments, a peptide sequence or a
chimeric peptide sequence an addition of amino acid residues 30-194
of SEQ ID NO:99 (FGF19) at the C-terminus, resulting in a chimeric
polypeptide having at the last position of the C-terminal region
that corresponds to about residue 194 of SEQ ID NO:99 (FGF19). In
further other embodiments, a chimeric peptide sequence or peptide
sequence comprises all or a portion of an FGF19 sequence (e.g., SEQ
ID NO:99), positioned at the C-terminus of the peptide, or where
the amino terminal "R" residue is deleted from the peptide.
[0029] In more particular embodiments, a chimeric peptide sequence
or peptide sequence comprises or consists of any of M1-M98 variant
peptide sequences, or a subsequence or fragment of any of the
M1-M98 variant peptide sequences. Methods and uses provided herein
can also be practiced using a peptide or chimeric sequence, as set
forth herein. For example, a sequence that comprises or consists of
any peptide sequence set forth herein as M1 to M98, M101 to M160,
or M200 to M207 or SEQ ID NOs:1 to 98, 101 to 135, 138 to 212, or a
peptide sequence that comprises of consists of any sequence set
forth in Table 1, or a peptide sequence that comprises or consists
of any sequence set forth in the Sequence Listing herein.
[0030] In various more particular aspects, a peptide sequence
comprises or consists of any one of the following sequences:
TABLE-US-00001 (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF
GLVTGLEAVRSPSFEK (M3); (SEQ ID NO: 194)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M140); (SEQ ID NO: 196)
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M160); (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVS
LSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVT
GLEAVRSPSFEK (M69); (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS
AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL
EAVRSPSFEK (M52); (SEQ ID NO: 5)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV
TGLEAVRSPSFEK (M5); (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVS
LSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVT
GLEAVRSPSFEK (M5-R); (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALK
PGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLP
GNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYA S
(M71); (SEQ ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALK
PGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLP
GNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYA S
(M72); (SEQ ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALK
PGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLP
GNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGG
EGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73); (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M1); (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M2); (SEQ ID NO: 48 or 6 or 148)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS
AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL
EAVRSPSFEK (M48); (SEQ ID NO: 49 or 7 or 149)
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLP
VSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGL
VTGLEAVRSPSFEK (M49); (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV
TGLEAVRSPSFEK (M50); (SEQ ID NO: 51 or 36 or 155)
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV
TGLEAVRSPSFEK (M51); (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS
AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL
EAVRSPSFEK (M53); (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV
TGLEAVRSPSFEK (M70); (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF
GLVTGLEAVRSPSFEK (M139); or (SEQ ID NO: 195)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M141);
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK (M160);
or a subsequence or fragment thereof of any of the foregoing
peptide sequences. In certain embodiments of any of the foregoing
peptide sequences, the R terminal residue (R residue at the
N-terminus) is deleted.
[0031] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00002 (SEQ ID NO: 197)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIL
EDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M200);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0032] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00003 (SEQ ID NO: 198)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVD
CARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYS
EEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLS
HFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK (M201);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0033] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00004 (SEQ ID NO: 199)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVD
CARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYS
EEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLS
HFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK (M202);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0034] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00005 (SEQ ID NO: 200)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M203);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0035] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00006 (SEQ ID NO: 201)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M204);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0036] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00007 (SEQ ID NO: 202)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M205);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0037] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00008 (SEQ ID NO: 203)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M206);
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0038] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00009 (SEQ ID NO: 204)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
LEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M207);
or a subsequence or fragment thereof.
[0039] In some embodiments, the peptide is a variant peptide
designated M139. In some embodiments, the peptide comprises an
amino acid sequence set forth in SEQ ID NO:193. In other
embodiments, the peptide consists of an amino acid sequence set
forth in SEQ ID NO:193. In some embodiments, the peptide is a
variant peptide designated M140. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:194. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:194. In some embodiments, the peptide is a
variant peptide designated M141. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:195. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:195. In some embodiments, the peptide is a
variant peptide designated M160. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:196. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:196. In some embodiments, the peptide is a
variant peptide designated M200. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:197. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:197. In some embodiments, the peptide is a
variant peptide designated M201. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:198. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:198. In other embodiments, the peptide is a
variant peptide designated M202. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:199. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:199. In certain embodiments, the peptide is
a variant peptide designated M203. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:200. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:200. In some embodiments, the peptide is a
variant peptide designated M204. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:201. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:201. In another embodiment, the peptide is a
variant peptide designated M205. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:202. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:202. In other embodiments, the peptide is a
variant peptide designated M206. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:203. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:203. In yet other embodiments, the peptide
is a variant peptide designated M207. In some embodiments, the
peptide comprises an amino acid sequence set forth in SEQ ID
NO:204. In other embodiments, the peptide consists of an amino acid
sequence set forth in SEQ ID NO:204.
[0040] In various additional particular aspects, the N-terminus of
the peptide sequence includes or consists of any of:
TABLE-US-00010 (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R); (amino acids 2-22 of SEQ ID NO:
6) DSSPLLQFGGQVRLRHLYTSG (M6-R); (amino acids 1-27 of SEQ ID NO: 7)
RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7); (amino acids 2-26 of SEQ ID NO:
8) HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R); (amino acids 2-28 of SEQ ID
NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R); (amino acids 2-28 of SEQ
ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R); (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11); (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13);
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG
(M14-R); (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15); (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16); (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17); (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18); (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19); (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20); (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21); (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22);
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG
(M23); (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHEYWGDPIRLRHLYTSG (M24); (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25); (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26); (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27); (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG (M28); (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29); (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30); (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG (M31);
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32); (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33); (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG (M34); (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG (M35); (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG (M36); (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG (M37); (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG (M38); (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG (M39); (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40);
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG
(M74-R); (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG
(M75-R); (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG (M77-R);
(amino acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9); (amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8); (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10); (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13); (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG (M14); amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43); or (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG (M6);
or any of the foregoing peptide sequences where the amino terminal
R residue is deleted.
[0041] In certain embodiments, the peptide comprises or consists of
any of:
TABLE-US-00011 (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R); (amino acids 2-22 of SEQ ID NO:
6) DSSPLLQFGGQVRLRHLYTSG (M6-R); (amino acids 1-27 of SEQ ID NO: 7)
RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7); (amino acids 2-26 of SEQ ID NO:
8) HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R); (amino acids 2-28 of SEQ ID
NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R); (amino acids 2-28 of SEQ
ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R); (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11); (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13);
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG
(M14-R); (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15); (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16); (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17); (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18); (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19); (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20); (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21); (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22);
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG
(M23); (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24); (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25); (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26); (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27); (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG (M28); (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29); (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30); (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG (M31);
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32); (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33); (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG (M34); (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG (M35); (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG (M36); (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG (M37); (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG (M38); (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG (M39); (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40);
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG
(M74-R); (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG
(M75-R); (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG (M77-R);
(amino acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9); (amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8); (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10);) (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13); (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG (M14); amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43); or (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG (M6).
In some embodiments, the peptide comprise one of the foregoing
sequences. In another embodiment, the peptide consists of one of
the foregoing sequences. In some embodiments, the peptide comprises
a C-terminal region comprising a portion of SEQ ID NO:99 (FGF19),
the C-terminal region having a first amino acid position and a last
amino acid position,
[0042] wherein the C-terminal region comprises amino acid residues
16-29 of SEQ ID NO:99 (FGF19), WGDPIRLRHLYTSG (SEQ ID NO:169),
wherein the W residue corresponds to the first amino acid position
of the C-terminal region.
[0043] In various further particular aspects. a peptide seauence
includes or consists of:
TABLE-US-00012 (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 138 or 161)
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLR
GHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or (SEQ ID NO: 141)
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPED
LRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof of any of the foregoing
peptide sequences. In certain embodiments of any of the foregoing
peptide sequences, the R terminal residue is deleted.
[0044] In further embodiments, a peptide sequence comprises or
consists of:
TABLE-US-00013 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70),
or a subsequence or fragment thereof. In certain embodiments, a
peptide sequence includes the addition of amino acid residues
30-194 of SEQ ID NO:99 (FGF19) at the C-terminus, resulting in a
chimeric polypeptide.
[0045] In some embodiments, a peptide sequence has at least one
amino acid substitution to amino acid residues 125-129 of SEQ ID
NO:99 (FGF19), EIRPD. In other embodiments, the peptide sequence
has at least one amino acid substitution to amino acid residues
126-128 of SEQ ID NO:99 (FGF19), IRP. In other embodiments, the
peptide sequence has at least one amino acid substitution to amino
acid residues 127-128 of SEQ ID NO:99 (FGF19), RP. In other
embodiments, the peptide sequence has at least one amino acid
substitution to amino acid residues 1-124 of SEQ ID NO:99 (FGF19)
and/or to amino acid residues 130-194 of SEQ ID NO:99 (FGF19). For
example, in certain embodiments, a peptide sequence comprises
substitution to one of amino acid residues 127-128 of SEQ ID NO:99
(FGF19), RP, wherein at least one amino acid substitution is R127L
or P128E. Said substitutions within a corresponding FGF19 sequence
(e.g., EIRPD, IRP or RP) of a peptide variant provided herein is
also contemplated. In certain embodiments, the peptide comprises
both a R127L and P128E substitution to amino acid residues 127-128
of SEQ ID NO:99 (FGF19), RP, or the corresponding FGF19 sequence
thereof in a variant peptide provided herein. In certain
embodiments, the amino acid sequence of the peptide comprises at
least one amino acid substitution in the Loop-8 region of FGF19, or
the corresponding FGF19 sequence thereof in a variant peptide
provided herein. In certain embodiments, the amino acid sequence of
the peptide comprises one amino acid substitution to the EIRPD
(amino acids 2-6 of SEQ ID NO:190) amino acid sequence in the
Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In other embodiments, the amino acid
sequence of the peptide comprises three amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises four amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises five amino acid substitutions to
the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises one amino acid substitution to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In other embodiments, the amino acid
sequence of the peptide comprises three amino acid substitutions to
the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
sequence of the peptide comprises one amino acid substitution to
the RP (amino acids 4-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In some embodiments, the amino acid
sequence of the peptide comprises two amino acid substitutions to
the RP (amino acids 4-5 of SEQ ID NO:190) amino acid sequence in
the Loop-8 region of FGF19. In certain embodiments, the amino acid
substitution to the RP (amino acids 4-5 of SEQ ID NO:190) amino
acid sequence in the Loop-8 region of FGF19 is an Arg (R) to Leu
(L) substitution. In other embodiments, the substitution to the RP
(amino acids 4-5 of SEQ ID NO:190) amino acid sequence in the
Loop-8 region of FGF19 is a Pro (P) to Glu (E) substitution. In
some embodiments, the substitutions to the RP (amino acids 4-5 of
SEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19 is
an Arg (R) to Leu (L) substitution and a Pro (P) to Glu (E)
substitution. In specific embodiments, the foregoing
substitution(s) in the Loop-8 region of FGF19 is in the
corresponding FGF19 sequence thereof in a variant peptide provided
herein. That is, said substitutions within a corresponding FGF19
sequence (e.g., EIRPD, IRP or RP) of a peptide variant provided
herein is also contemplated.
[0046] Peptide or chimeric sequences provided herein can be of any
suitable length. In particular embodiments, the N-terminal or
C-terminal region of the peptide or chimeric sequence is from about
20 to about 200 amino acid residues in length. In further
particular embodiments, a chimeric peptide sequence or peptide
sequence has at least one amino acid deletion. In other particular
aspects, a peptide or chimeric sequence has 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid
deletions from the amino terminus, the carboxy-terminus or
internally. In one embodiment, the amino acid substitution, or
deletion is at any of amino acid positions 8-20 of FGF19
(AGPHVHYGWGDPI) (SEQ ID NO:187). In further particular embodiments,
a peptide or chimeric sequence has an N-terminal region, or a
C-terminal region that comprises or consists of an amino acid
sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50,
60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids. In
additional more particular embodiments, a peptide or chimeric
sequence has an FGF19 sequence portion, or an FGF21 sequence
portion that comprises or consists of an amino acid sequence of
about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60
to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids of FGF19
or FGF21.
[0047] In various further embodiments, a peptide or chimeric
sequence has an amino acid substitution, an addition, insertion or
is a subsequence that has at least one amino acid deleted. Such
amino acid substitutions, additions, insertions and deletions of a
peptide sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino
acid residues (10-20, 20-30, 30-40, 40-50, etc.), for example, at
the N- or C-terminus, or internal. For example, a subsequence that
has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more amino acid deletions from the amino terminus, the
carboxy-terminus or internally. In a particular aspect, the amino
acid substitution, or deletion is at any of amino acid positions
8-20 of FGF19 (AGPHVHYGWGDPI) (SEQ ID NO:187).
[0048] In various still more particular aspects, a peptide or
chimeric sequence includes all or a portion of an FGF19 sequence
set forth as:
TABLE-US-00014 (SEQ ID NO: 188)
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYL
CMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG
LVTGLEAVRSPSFEK
positioned at the C-terminus of the peptide, or the amino terminal
"R" residue is deleted from the sequence.
[0049] In various embodiments, a peptide or chimeric sequence has a
function or activity greater or less than a comparison sequence. In
further particular embodiments, chimeric peptide sequences and
peptide sequences have particular functions or activities. In one
aspect, a chimeric peptide sequence or peptide sequence maintains
or increases a fibroblast growth factor receptor 4 (FGFR4) mediated
activity. In additional aspects, a chimeric peptide sequence or
peptide sequence binds to FGFR4 or activates FGFR4, or does not
detectably bind to FGFR4 or activate FGFR4, or binds to FGFR4 with
an affinity less than, comparable to or greater than FGF19 binding
affinity for FGFR4, or activates FGFR4 to an extent or amount less
than, comparable to or greater than FGF19 activates FGFR4. In some
embodiments, a chimeric peptide sequence or peptide sequence
provided herein activates FGFR4 to an extent or amount less than
the extent or amount that FGF19 activates FGFR4. In some
embodiments, a chimeric peptide sequence or peptide sequence
provided herein activates FGFR4 to an extent or amount comparable
to the extent or amount that FGF19 activates FGFR4. In some
embodiments, a chimeric peptide sequence or peptide sequence
provided herein activates FGFR4 to an extent or amount greater than
the extent or amount that FGF19 activates FGFR4.
[0050] In one embodiment, a chimeric peptide sequence or peptide
sequence provided herein maintains an FGFR4 mediated activity. In
one embodiment, a chimeric peptide sequence or peptide sequence
provided herein increases an FGFR4 mediated activity. In some
embodiments, a chimeric peptide sequence or peptide sequence
provided herein binds to FGFR4 with an affinity less than FGF19
binding affinity for FGFR4. In some embodiments, a chimeric peptide
sequence or peptide sequence provided herein binds to FGFR4 with an
affinity comparable to FGF19 binding affinity for FGFR4. In some
embodiments, a chimeric peptide sequence or peptide sequence
provided herein binds to FGFR4 with an affinity greater than FGF19
binding affinity for FGFR4. In some embodiments, a chimeric peptide
sequence or peptide sequence provided herein does not detectably
bind to FGFR4.
[0051] In further aspects, a chimeric peptide sequence or peptide
sequence has reduced HCC formation compared to FGF19, or an FGF19
variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),
WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174),
GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID
NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ
ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ
ID NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ
ID NO:170) sequence at amino acids 16-20 of FGF19; or has greater
glucose lowering activity compared to FGF19, or an FGF19 variant
sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ
ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI
(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),
WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID
NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID
NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID
NO:170) sequence at amino acids 16-20 of FGF19; has less lipid
increasing activity compared to FGF19, or an FGF19 variant sequence
having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID
NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI
(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),
WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID
NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID
NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID
NO:170) sequence at amino acids 16-20 of FGF19; or has less
triglyceride, cholesterol, non-HDL or HDL increasing activity
compared to FGF19, or an FGF19 variant sequence having any of GQV,
GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID
NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI
(SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178),
WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181),
WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184)
substituted for the WGDPI (SEQ ID NO:170) sequence at amino acids
16-20 of FGF19; or the peptide sequence has less lean mass reducing
activity compared to FGF21. Such functions and activities can be
ascertained in vitro or in vivo, for example, in a db/db mouse.
[0052] In one embodiment, a peptide or chimeric sequence has a
function or activity greater or less than a comparison sequence. In
some embodiments, the comparison sequence is FGF19. In another
embodiment, the comparison sequence is FGF19 variant sequence
having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID
NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI
(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),
WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID
NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID
NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID
NO:170) sequence at amino acids 16-20 of FGF19. In one embodiment,
a peptide or chimeric peptide sequence provided herein has greater
glucose lowering activity compared to a comparison sequence. In
another embodiment, a peptide or chimeric peptide sequence provided
herein has less lipid increasing activity compared to a comparison
sequence. In other embodiment, a peptide or chimeric peptide
sequence provided herein has lower or reduced lipid (e.g.,
triglyceride, cholesterol, non-HDL) activity compared to a
comparison sequence. In other embodiments, a peptide or chimeric
peptide sequence provided herein has more HDL increasing activity
as compared to a comparison sequence. In other embodiment, a
peptide or chimeric peptide sequence provided herein has less lean
mass reducing activity compared to a comparison sequence or
FGF21.
[0053] In further additional various embodiments, a peptide or
chimeric sequence includes one or more L-amino acids, D-amino
acids, non-naturally occurring amino acids, or amino acid mimetic,
derivative or analogue. In still further various embodiments, a
peptide or chimeric sequence has an N-terminal region, or a
C-terminal region, or a FGF19 sequence portion, or an FGF21
sequence portion, joined by a linker or spacer.
[0054] Non-limiting exemplary bile acid related or associated
disorders treatable according to the methods and uses provided
herein include: cholestasis, including, for example diseases of
intrahepatic cholestasis (e.g., primary biliary cirrhosis (PBC),
primary familial intrahepatic cholestasis (PFIC) (e.g., progressive
PFIC), primary sclerosing choangitis (PSC), pregnancy intrahepatic
cholestasis (PIC), neonatal cholestasis, and drug-induced
cholestasis (e.g., estrogen)), and diseases of extrahepatic
cholestasis (e.g., bile cut compression from tumor, bile duct
blockade by gall stones); bile acid malabsorption and other
disorders involving the distal small intestine, including ileal
resection, inflammatory bowel diseases (e.g., Crohn's disease and
ulcerative colitis), short bowel syndrome, disorders impairing
absorption of bile acids not otherwise characterized (idiopathic))
leading to diarrhea (e.g., bile acid diarrhea (BAD)) and GI
symptoms, and GI, liver, and/or biliary cancers (e.g., colon cancer
and hepatocellular cancer); and/or bile acid synthesis
abnormalities, such as those contributing to non-alcoholic
steatohepatitis (NASH), cirrhosis and portal hypertension; e.g., in
mammals, such as humans. Additional bile acid-related disorders
include metabolic syndrome; a lipid or glucose disorder;
cholesterol or triglyceride metabolism; type 2 diabetes.
[0055] In one particular embodiment, the bile acid related or
associated disorder is bile acid malabsorption. In another
particular embodiment, the bile acid related or associated disorder
is diarrhea. In a still further particular embodiment, the bile
acid related or associated disorder is cholestasis (e.g.,
intrahepatic or extrahepatic cholestasis), and in another further
particular embodiment, the bile acid related or associated disorder
is primary billiary cirrhosis (PBC). In other particular
embodiments, the bile acid related or associated disorder is
primary sclerosing cholangitis. In another embodiment, the bile
acid related or associated disorder is PFIC (e.g., progressive
PFIC).
[0056] In still further embodiments, the at least one additional
agent effective in modulating bile acid homeostasis or treating a
bile-acid related or associated disorder is: a glucocorticoid;
CDCA; UDCA; insulin, an insulin secretagogues, an insulin mimetic,
a sulfonylurea and a meglitinide; a biguanide; an alpha-glucosidase
inhibitors; a DPP-IV inhibitor, GLP-1, a GLP-1 agonists and a GLP-1
analog; a DPP-IV-resistant analogue; a PPAR gamma agonist, a
dual-acting PPAR agonist, a pan-acting PPAR agonist; a PTP1B
inhibitor; an SGLT inhibitor; an RXR agonist; a glycogen synthase
kinase-3 inhibitor; an immune modulator; a beta-3 adrenergic
receptor agonist; an 11beta-HSD1 inhibitor; amylin and an amylin
analogue; a bile acid sequestrant; or an SGLT-2 inhibitor.
[0057] In certain embodiments, the at least one additional agent
effective in modulating PBC is UDCA, an FXR agonist, OCA, an ASBT
inhibitor, an autoimmune agent, an anti-IL-12 agent, an anti-CD80
agent, an anti-CD20 agent, a CXCL10 neutralizing antibody, a ligand
for CXCR3, a fibrate, fish oil, colchicine, methotrexate,
azathioprine, cyclosporine, or an anti-retroviral therapy. In
particular embodiments, the at least one additional agent effective
in modulating PBC is UDCA, OCA, an ASBT inhibitor, an anti-IL-12
agent, an anti-CD20 agent, or a fibrate.
[0058] In some embodiments, the combination of a chimeric peptide
sequence or a peptide sequence described herein and at least one
additional therapeutic agent or treatment modality is assessed to
ensure that it does not cause untoward adverse effects in the
subject. In a particular aspect, the combination of a chimeric
peptide sequence or a peptide sequence described herein and at
least one additional therapeutic agent or treatment modality is
assessed to ensure that it does not induce HCC in the subject. Such
assessments may be performed before initiation of therapy (e.g., in
a dose escalation study), during therapy, (e.g., by evaluating a
marker correlating with HCC activity), or subsequent to termination
of therapy (e.g., by performing a liver biopsy). In some aspects,
the assessment is performed in a suitable test environment (e.g., a
validated animal model). One of ordinary skill in the art is
familiar with additional means for ensuring that the combination
therapy described herein is suitable for the particular subject, or
a subject population representative of the particular subject,
taking into consideration all relevant factors including, for
example, the severity of the subject's bile acid-related disorder
(e.g., PBC) and the other medications be taken by the subject.
[0059] A detailed description of PBC, including its diagnosis and
treatment, are described elsewhere herein. Absent effective
therapy, the median time to develop extensive fibrosis is around 2
years. The median survival of untreated patients with PBC is
approximately 9-10 years from presentation, with 26% developing
liver failure during this time (Trivedi, P. et al., Ther. Adv.
Chronic Dis. 4(3):119-41 (2013)). Thus, alternative treatment
regimens such as those described herein have the potential of
dramatically delaying disease progression and improving patient
survival.
DETAILED DESCRIPTION
[0060] Provided herein, in certain embodiments, are uses of
chimeric and peptide sequences that modulate bile acid homeostasis
in combination with one or more additional therapeutic agents or
treatment modalities that are useful in the treatment and/or
prevention of bile acid-related or associated disorders. The
invention is based, in part, on the use of variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of a bile acid-related disorder (e.g.,
PBC), in combination with other therapeutic agents and/or treatment
modalities. Such variants and fusions (chimeras) of FGF19 and/or
FGF21 peptide sequences include sequences that do not substantially
increase or induce HCC formation or HCC tumorigenesis and/or do not
induce a substantial elevation or increase in lipid profile.
[0061] In one embodiment, a chimeric peptide sequence includes or
consists of an N-terminal region having at least seven amino acid
residues and the N-terminal region having a first amino acid
position and a last amino acid position, where the N-terminal
region has a DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122)
sequence; and a C-terminal region having a portion of FGF19 and the
C-terminal region having a first amino acid position and a last
amino acid position, where the C-terminal region includes amino
acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG; SEQ ID NO:169) and
the W residue corresponds to the first amino acid position of the
C-terminal region. In particular embodiments, the variant is
M70:
TABLE-US-00015 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK.
[0062] In another embodiment, a chimeric peptide sequence includes
or consists of an N-terminal region having a portion of FGF21 and
the N-terminal region having a first amino acid position and a last
amino acid position, where the N-terminal region has a GQV sequence
and the V residue corresponds to the last amino acid position of
the N-terminal region; and a C-terminal region having a portion of
FGF19 and the C-terminal region having a first amino acid position
and a last amino acid position where the C-terminal region includes
amino acid residues 21-29 of FGF19 (RLRHLYTSG; SEQ ID NO: 185) and
the R residue corresponds to the first position of the C-terminal
region.
[0063] In particular aspects, modifications to the Loop-8 region of
FGF19 are disclosed herein that possess favorable metabolic
parameters without exhibiting substantial tumorigenicity. Herein,
FGF19 residues 127-129 are defined as constituting the Loop-8
region, although in the literature the Loop-8 region is sometimes
defined as including or consisting of other residues (e.g.,
residues 125-129). Certain combinations of R127L and P128E
substitutions to the FGF19 framework had an unexpectedly positive
effect on HCC formation. Even more surprisingly, a combination of
R127L and P128E substitutions and a substitution of Gln (Q) for Leu
(L) in the FGF19 core region had an even more significant effect on
preventing HCC formation. Accordingly, variants of FGF19 Loop-8
region are included since they can reduce or eliminate substantial,
measurable or detectable HCC formation. Furthermore, the effect of
reducing HCC formation may be enhanced by modifications to amino
acid residues outside of the Loop 8 region (e.g., substitutions of
amino acid residues in the core region).
[0064] In further embodiments, a peptide sequence includes or
consists of a FGF19 variant having one or more amino acid
substitutions, insertions or deletions compared to a reference or
wild type FGF19. In additional embodiments, a peptide sequence
includes or consists of a FGF21 sequence variant having one or more
amino acid substitutions, insertions or deletions compared to a
reference or wild type FGF21. In yet additional embodiments, a
peptide sequence includes or consists of a portion of an FGF19
sequence fused to a portion of an FGF21 sequence. In still
additional embodiments, a peptide sequence includes or consists of
a portion of an FGF19 sequence fused to a portion of an FGF21
sequence, where the FGF19 and/or FGF21 sequence portion(s) have one
or more amino acid substitutions, insertions or deletions compared
to a reference or wild type FGF19 and/or FGF21. Examples of such
sequences are disclosed in PCT Pub. No. WO 2013/006486 and US Pub.
No. 2013/0023474, as well as PCT Publ. No. WO 2014/085365,
published Jun. 5, 2014. Table 1 and the Sequence Listing also sets
forth representative sequences that may be used in the methods
provided herein.
Peptide Molecules
[0065] The terms "peptide," "protein," and "polypeptide" sequence
are used interchangeably herein to refer to two or more amino
acids, or "residues," including chemical modifications and
derivatives of amino acids, covalently linked by an amide bond or
equivalent. The amino acids forming all or a part of a peptide may
be from among the known 21 naturally occurring amino acids, which
are referred to by both their single letter abbreviations and their
common three-letter abbreviation. In the peptide sequences provided
herein, conventional amino acid residues have their conventional
meaning. Thus, "Leu" is leucine, "Ile" is isoleucine, "Nle" is
norleucine, and so on. To assist the reader, conventional amino
acids and their corresponding three letter and single letter
abbreviations are as follows:
TABLE-US-00016 alanine Ala (A) arginine Arg (R) asparagine Asn (N)
aspartic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E)
glutamine Gln (Q) glycine Gly (G) histidine His (H) isoleucine Ile
(I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine
Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan
Trp (W) tyrosine Tyr (Y) valine Val (V)
[0066] In various particular aspects, a peptide or chimeric
sequence provided herein has at the N-terminal region first amino
acid position an "M" residue, an "R" residue, a "S" residue, a "H"
residue, a "P" residue, a "L" residue or an "D" residue. In various
alternative particular aspects, a peptide or chimeric sequence
peptide sequence does not have a "M" residue or an "R" residue at
the first amino acid position of the N-terminal region.
[0067] Typically, the number of amino acids or residues in a
peptide sequence provided herein will total less than about 250
(e.g., amino acids or mimetics thereof). In various particular
embodiments, the number of residues comprise from about 20 up to
about 200 residues (e.g., amino acids or mimetics thereof). In
additional embodiments, the number of residues comprise from about
50 up to about 200 residues (e.g., amino acids or mimetics
thereof). In further embodiments, the number of residues comprise
from about 100 up to about 195 residues (e.g., amino acids or
mimetics thereof) in length.
[0068] Amino acids or residues can be linked by amide or by
non-natural and non-amide chemical bonds including, for example,
those formed with glutaraldehyde, N-hydroxysuccinimide esters,
bifunctional maleimides, or N, N'-dicyclohexylcarbodiimide (DCC).
Non-amide bonds include, for example, ketomethylene,
aminomethylene, olefin, ether, thioether and the like (see, e.g.,
Spatola in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, Vol. 7, pp 267-357 (1983), "Peptide and Backbone
Modifications," Marcel Decker, NY). Thus, when a peptide provided
herein includes a portion of an FGF19 sequence and a portion of an
FGF21 sequence, the two portions need not be joined to each other
by an amide bond, but can be joined by any other chemical moiety or
conjugated together via a linker moiety.
[0069] Also provided herein are subsequences, variants and modified
forms of the exemplified peptide sequences (including the FGF19 and
FGF21 variants and subsequences listed in the Sequence Listing, or
Table 1), so long as the foregoing retains at least a detectable or
measureable activity or function. Also, certain exemplified variant
peptides, for example, those having all or a portion of FGF21
sequence at the amino-terminus, have an "R" residue positioned at
the N-terminus, which can be omitted. Similarly, certain
exemplified variant peptides, include an "M" residue positioned at
the N-terminus, which can be appended to or further substituted for
an omitted residue, such as an "R" residue. More particularly, in
various embodiments peptide sequences at the N-terminus include any
of: RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID NO:116) or MRDSS (SEQ
ID NO:117). Furthermore, when a "M" residue is adjacent to a "S"
residue, the "M" residue may be cleaved such that the "M" residue
is deleted from the peptide sequence, whereas when the "M" residue
is adjacent to a "D" residue, the "M" residue may not be cleaved.
Thus, by way of example, in various embodiments peptide sequences
include those with the following residues at the N-terminus: MDSSPL
(SEQ ID NO:119), MSDSSPL (SEQ ID NO:120) (cleaved to SDSSPL (SEQ ID
NO:112)) and MSSPL (SEQ ID NO:113) (cleaved to SSPL (SEQ ID
NO:114)).
[0070] As used herein, the term "modify" and grammatical variations
thereof, means that the composition deviates relative to a
reference composition, such as a peptide sequence. Such modified
peptide sequences, nucleic acids and other compositions may have
greater or less activity or function, or have a distinct function
or activity compared with a reference unmodified peptide sequence,
nucleic acid, or other composition, or may have a property
desirable in a protein formulated for therapy (e.g. serum
half-life), to elicit antibody for use in a detection assay, and/or
for protein purification. For example, a peptide sequence provided
herein can be modified to increase serum half-life, to increase in
vitro and/or in vivo stability of the protein, etc.
[0071] Particular examples of such subsequences, variants and
modified forms of the peptide sequences exemplified herein (e.g., a
peptide sequence listed in the Sequence Listing or Table 1) include
substitutions, deletions and/or insertions/additions of one or more
amino acids, to or from the amino-terminus, the carboxy-terminus or
internally. One example is a substitution of an amino acid residue
for another amino acid residue within the peptide sequence. Another
is a deletion of one or more amino acid residues from the peptide
sequence, or an insertion or addition of one or more amino acid
residues into the peptide sequence.
[0072] The number of residues substituted, deleted or
inserted/added are one or more amino acids (e.g., 1-3, 3-5, 5-10,
10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,
100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,
170-180, 180-190, 190-200, 200-225, 225-250, or more) of a peptide
sequence. Thus, an FGF19 or FGF21 sequence can have few or many
amino acids substituted, deleted or inserted/added (e.g., 1-3, 3-5,
5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,
90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160,
160-170, 170-180, 180-190, 190-200, 200-225, 225-250, or more). In
addition, an FGF19 amino acid sequence can include or consist of an
amino acid sequence of about 1-3, 3-5, 5-10, 10-20, 20-30, 30-40,
40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,
120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,
190-200, 200-225, 225-250, or more amino acids from FGF21; or an
FGF21 amino acid or sequence can include or consist of an amino
acid sequence of about 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,
50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130,
130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200,
200-225, 225-250, or more amino acids from FGF19.
[0073] Specific examples of substitutions include substituting a D
residue for an L-residue. Accordingly, although residues are listed
in the L-isomer configuration, D-amino acids at any particular or
all positions of the peptide sequences provided herein are
included, unless a D-isomer leads to a sequence that has no
detectable or measurable function.
[0074] Additional specific examples are non-conservative and
conservative substitutions. A "conservative substitution" is a
replacement of one amino acid by a biologically, chemically or
structurally similar residue. Biologically similar means that the
substitution is compatible with a biological activity, e.g.,
activity that improves PBC and/or the manifestations thereof.
Structurally similar means that the amino acids have side chains
with similar length, such as alanine, glycine and serine, or having
similar size, or the structure of a first, second or additional
peptide sequence is maintained. Chemical similarity means that the
residues have the same charge or are both hydrophilic and
hydrophobic. Particular examples include the substitution of one
hydrophobic residue, such as isoleucine, valine, leucine or
methionine, for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acids, or glutamine for asparagine, serine
for threonine, etc. Routine assays can be used to determine whether
a subsequence, variant or modified form has activity, e.g.,
activity that improves PBC and/or the manifestations thereof.
[0075] Particular examples of subsequences, variants and modified
forms of the peptide sequences exemplified herein have 50%-60%,
60%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 96%, 97%,
98%, or 99% identity to a reference peptide sequence. The term
"identity" and "homology" and grammatical variations thereof mean
that two or more referenced entities are the same. Thus, where two
amino acid sequences are identical, they have the identical amino
acid sequence. "Areas, regions or domains of identity" mean that a
portion of two or more referenced entities are the same. Thus,
where two amino acid sequences are identical or homologous over one
or more sequence regions, they share identity in those regions.
[0076] The extent of identity between two sequences can be
ascertained using a computer program and mathematical algorithm
known in the art. Such algorithms that calculate percent sequence
identity (homology) generally account for sequence gaps and
mismatches over the comparison region. For example, a BLAST (e.g.,
BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol.
Biol. 215:403 (1990), publicly available through NCBI) has
exemplary search parameters as follows: Mismatch -2; gap open 5;
gap extension 2. For peptide sequence comparisons, a BLASTP
algorithm is typically used in combination with a scoring matrix,
such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g.,
FASTA2 and FASTA3) and SSEARCH sequence comparison programs are
also used to quantitate the extent of identity (Pearson et al.,
Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol
Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195
(1981)). Programs for quantitating protein structural similarity
using Delaunay-based topological mapping have also been developed
(Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
[0077] In the peptide sequences, including subsequences, variants
and modified forms of the peptide sequences exemplified herein, an
"amino acid" or "residue" includes conventional alpha-amino acids
as well as beta-amino acids; alpha, alpha disubstituted amino
acids; and N-substituted amino acids, wherein at least one side
chain is an amino acid side chain moiety as defined herein. An
"amino acid" further includes N-alkyl alpha-amino acids, wherein
the N-terminus amino group has a C.sub.1 to C.sub.6 linear or
branched alkyl substituent. The term "amino acid" therefore
includes stereoisomers and modifications of naturally occurring
protein amino acids, non-protein amino acids, post-translationally
modified amino acids (e.g., by glycosylation, phosphorylation,
ester or amide cleavage, etc.), enzymatically modified or
synthesized amino acids, derivatized amino acids, constructs or
structures designed to mimic amino acids, amino acids with a side
chain moiety modified, derivatized from naturally occurring
moieties, or synthetic, or not naturally occurring, etc. Modified
and unusual amino acids are included in the peptide sequences
provided herein (see, for example, in Synthetic Peptides: A User's
Guide; Hruby et al., Biochem. J. 268:249 (1990); and Toniolo C.,
Int. J. Peptide Protein Res. 35:287 (1990)).
[0078] In addition, protecting and modifying groups of amino acids
are included. The term "amino acid side chain moiety" as used
herein includes any side chain of any amino acid, as the term
"amino acid" is defined herein. This therefore includes the side
chain moiety in naturally occurring amino acids. It further
includes side chain moieties in modified naturally occurring amino
acids as set forth herein and known to one of skill in the art,
such as side chain moieties in stereoisomers and modifications of
naturally occurring protein amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically modified
or synthesized amino acids, derivatized amino acids, constructs or
structures designed to mimic amino acids, etc. For example, the
side chain moiety of any amino acid disclosed herein or known to
one of skill in the art is included within the definition.
[0079] A "derivative of an amino acid side chain moiety" is
included within the definition of an amino acid side chain moiety.
Non-limiting examples of derivatized amino acid side chain moieties
include, for example: (a) adding one or more saturated or
unsaturated carbon atoms to an existing alkyl, aryl, or aralkyl
chain; (b) substituting a carbon in the side chain with another
atom, such as oxygen or nitrogen; (c) adding a terminal group to a
carbon atom of the side chain, including methyl (--CH.sub.3),
methoxy (--OCH.sub.3), nitro (--NO.sub.2), hydroxyl (--OH), or
cyano (--C.dbd.N); (d) for side chain moieties including a hydroxy,
thiol or amino groups, adding a suitable hydroxy, thiol or amino
protecting group; or (e) for side chain moieties including a ring
structure, adding one or more ring substituents, including
hydroxyl, halogen, alkyl, or aryl groups attached directly or
through, e.g., an ether linkage. For amino groups, suitable
protecting groups are known to the skilled artisan. Provided such
derivatization provides a desired activity in the final peptide
sequence (e.g., activity that improves PBC and/or the
manifestations thereof).
[0080] An "amino acid side chain moiety" includes all such
derivatization, and particular non-limiting examples include:
gamma-amino butyric acid, 12-amino dodecanoic acid,
alpha-aminoisobutyric acid, 6-amino hexanoic acid,
4-(aminomethyl)-cyclohexane carboxylic acid, 8-amino octanoic acid,
biphenylalanine, Boc-t-butoxycarbonyl, benzyl, benzoyl, citrulline,
diaminobutyric acid, pyrrollysine, diaminopropionic acid,
3,3-diphenylalanine, orthonine, citrulline,
1,3-dihydro-2H-isoindolecarboxylic acid, ethyl,
Fmoc-fluorenylmethoxycarbonyl, heptanoyl
(CH.sub.3--(CH.sub.2).sub.5--C(.dbd.O)--), hexanoyl
(CH.sub.3--(CH.sub.2).sub.4--C(.dbd.O)--), homoarginine,
homocysteine, homolysine, homophenylalanine, homoserine, methyl,
methionine sulfoxide, methionine sulfone, norvaline (NVA),
phenylglycine, propyl, isopropyl, sarcosine (SAR),
tert-butylalanine, and benzyloxycarbonyl.
[0081] A single amino acid, including stereoisomers and
modifications of naturally occurring protein amino acids,
non-protein amino acids, post-translationally modified amino acids,
enzymatically-synthesized amino acids, non-naturally occurring
amino acids including derivatized amino acids, an alpha, alpha
disubstituted amino acid derived from any of the foregoing (i.e.,
an alpha, alpha disubstituted amino acid, wherein at least one side
chain is the same as that of the residue from which it is derived),
a beta-amino acid derived from any of the foregoing (i.e., a
beta-amino acid which, other than for the presence of a
beta-carbon, is the same as the residue from which it is derived)
etc., including all of the foregoing can be referred to herein as a
"residue." Suitable substituents, in addition to the side chain
moiety of the alpha-amino acid, include C.sub.1 to C.sub.6 linear
or branched alkyl. Aib is an example of an alpha, alpha
disubstituted amino acid. While alpha, alpha disubstituted amino
acids can be referred to using conventional L- and D-isomeric
references, it is to be understood that such references are for
convenience, and that where the substituents at the alpha-position
are different, such amino acid can interchangeably be referred to
as an alpha, alpha disubstituted amino acid derived from the L- or
D-isomer, as appropriate, of a residue with the designated amino
acid side chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can
be referred to as either an alpha, alpha disubstituted amino acid
derived from L-Nle (norleucine) or as an alpha, alpha disubstituted
amino acid derived from D-Ala. Similarly, Aib can be referred to as
an alpha, alpha disubstituted amino acid derived from Ala. Whenever
an alpha, alpha disubstituted amino acid is provided, it is to be
understood as including all (R) and (S) configurations thereof.
[0082] An "N-substituted amino acid" includes any amino acid
wherein an amino acid side chain moiety is covalently bonded to the
backbone amino group, optionally where there are no sub stituents
other than H in the alpha-carbon position. Sarcosine is an example
of an N-substituted amino acid. By way of example, sarcosine can be
referred to as an N-substituted amino acid derivative of Ala, in
that the amino acid side chain moiety of sarcosine and Ala is the
same, i.e., methyl.
[0083] In certain embodiments, covalent modifications of the
peptide sequences, including subsequences, variants and modified
forms of the peptide sequences exemplified herein are provided. An
exemplary type of covalent modification includes reacting targeted
amino acid residues with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues of the peptide. Derivatization with
bifunctional agents is useful, for instance, for cross-linking
peptide to a water-insoluble support matrix or surface for use in
the method for purifying anti-peptide antibodies, and vice-versa.
Commonly used cross linking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0084] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the alpha-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 (1983)), acetylation of the N-terminal amine, amidation of
any C-terminal carboxyl group, etc.
[0085] Exemplified peptide sequences, and subsequences, variants
and modified forms of the peptide sequences exemplified herein can
also include alterations of the backbone for stability,
derivatives, and peptidomimetics. The term "peptidomimetic"
includes a molecule that is a mimic of a residue (referred to as a
"mimetic"), including but not limited to piperazine core molecules,
keto-piperazine core molecules and diazepine core molecules. Unless
otherwise specified, an amino acid mimetic of a peptide sequence
provided herein includes both a carboxyl group and amino group, and
a group corresponding to an amino acid side chain, or in the case
of a mimetic of Glycine, no side chain other than hydrogen.
[0086] By way of example, these would include compounds that mimic
the sterics, surface charge distribution, polarity, etc. of a
naturally occurring amino acid, but need not be an amino acid,
which would impart stability in the biological system. For example,
Proline may be substituted by other lactams or lactones of suitable
size and substitution; Leucine may be substituted by an alkyl
ketone, N-substituted amide, as well as variations in amino acid
side chain length using alkyl, alkenyl or other substituents,
others may be apparent to the skilled artisan. The essential
element of making such substitutions is to provide a molecule of
roughly the same size and charge and configuration as the residue
used to design the molecule. Refinement of these modifications will
be made by analyzing the compounds in a functional (e.g., glucose
lowering) or other assay, and comparing the structure-activity
relationship. Such methods are within the scope of the skilled
artisan working in medicinal chemistry and drug development.
[0087] The term "bind," or "binding," when used in reference to a
peptide sequence, means that the peptide sequence interacts at the
molecular level. Specific and selective binding can be
distinguished from non-specific binding using assays known in the
art (e.g., competition binding, immunoprecipitation, ELISA, flow
cytometry, Western blotting).
[0088] Peptides and peptidomimetics can be produced and isolated
using methods known in the art. Peptides can be synthesized, in
whole or in part, using chemical methods (see, e.g., Caruthers
(1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga,
A. K., Therapeutic Peptides and Proteins, Formulation, Processing
and Delivery Systems (1995) Technomic Publishing Co., Lancaster,
Pa.). Peptide synthesis can be performed using various solid-phase
techniques (see, e.g., Roberge Science 269:202 (1995); Merrifield,
Methods Enzymol. 289:3 (1997)) and automated synthesis may be
achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin
Elmer) in accordance with the manufacturer's instructions. Peptides
and peptide mimetics can also be synthesized using combinatorial
methodologies. Synthetic residues and polypeptides incorporating
mimetics can be synthesized using a variety of procedures and
methodologies known in the art (see, e.g., Organic Syntheses
Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons,
Inc., NY). Modified peptides can be produced by chemical
modification methods (see, for example, Belousov, Nucleic Acids
Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995);
and Blommers, Biochemistry 33:7886 (1994)). Peptide sequence
variations, derivatives, substitutions and modifications can also
be made using methods such as oligonucleotide-mediated
(site-directed) mutagenesis, alanine scanning, and PCR-based
mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids
Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487
(1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)),
restriction selection mutagenesis (Wells et al., Philos. Trans. R.
Soc. London SerA 317:415 (1986)) and other techniques can be
performed on cloned DNA to produce peptide sequences, variants,
fusions and chimeras provided herein, and variations, derivatives,
substitutions and modifications thereof.
[0089] A "synthesized" or "manufactured" peptide sequence is a
peptide made by any method involving manipulation by the hand of
man. Such methods include, but are not limited to, the
aforementioned, such as chemical synthesis, recombinant DNA
technology, biochemical or enzymatic fragmentation of larger
molecules, and combinations of the foregoing.
[0090] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Table
1), can also be modified to form a chimeric molecule. In certain
embodiments, provided herein are peptide sequences that include a
heterologous domain. Such domains can be added to the
amino-terminus or at the carboxyl-terminus of the peptide sequence.
Heterologous domains can also be positioned within the peptide
sequence, and/or alternatively flanked by FGF19 and/or FGF21
derived amino acid sequences.
[0091] The term "peptide" also includes dimers or multimers
(oligomers) of peptides. In certain embodiments, dimers or
multimers (oligomers) of the exemplified peptide sequences are
provided herein, as well as subsequences, variants and modified
forms of the exemplified peptide sequences, including sequences
listed in the Sequence Listing or Table 1.
[0092] In certain embodiments, a peptide sequence provided herein
comprises an amino acid sequence set forth in Table 1. In other
embodiments, a peptide sequence provided herein consists of an
amino acid sequence set forth in Table 1.
TABLE-US-00017 TABLE 1 SEQ ID NO. Amino Acid Sequence 1.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 2.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 3.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 4.
RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 5.
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 6.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 7.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 8.
RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 9.
RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 10.
RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 11.
RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 12.
RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 13.
RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 14.
RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 15.
RPLAFSDAGPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 16.
RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 17.
RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 18.
RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 19.
RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 20.
RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 21.
RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 22.
RPLAFSDAGPHVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 23.
RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 24.
RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 25.
RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 26.
RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 27.
RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 28.
RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 29.
RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 30.
RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 31.
RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 32.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 33.
RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 34.
RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 35.
RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 36.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 37.
RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 38.
RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 39.
RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 40.
RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 41.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQPPDVGSSDPL
SMVGPSQGRSPSYAS 42.
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQPPDVGSSDPLSMVGP
SQGRSPSYAS 43.
RPLAFSDAGPHVHYGGDIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 44.
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY
QSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDP
LSMVGPSQGRSPSYAS 45.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLK
ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH
GLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 46.
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY
QSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDP
LSMVGPSQGRSPSYASPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV RSPSFEK
47. HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 48.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 49.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 50.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 51.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 52.
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 53.
MDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 54.
RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 55.
RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 56.
RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 57.
RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 58.
RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 59.
RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 60.
RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 61.
RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 62.
RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 63.
RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 64.
RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 65.
RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 66.
RPLAFSDAGPHLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 67.
RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 68.
RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 69.
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 70.
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 71.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLK
ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH
SLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVG
PSQGRSPSYAS 72.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLK
ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH
GLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMV
GPSQGRSPSYAS 73.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLK
ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH
GLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMV
VQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE 74.
RDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 75.
RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL
RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL
PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSM
DPFGLVTGLEAVRSPSFEK 76.
RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIK
GVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA
KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVT
GLEAVRSPSFEK 77.
RRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHS
VRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK
78. RAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 79.
RGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEK
HRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLET
DSMDPFGLVTGLEAVRSPSFEK 80.
RPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 81.
RHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS
MDPFGLVTGLEAVRSPSFEK 82.
RPLAFSAAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 83.
RPLAFSDAAPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 84.
RPLAFSDAGAHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 85.
RPLAFSDAGPHVHYGAGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 86.
RPLAFSDAGPHVHYGWGAPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 87.
RPLAFSDAGPHVHYGWGDAICARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCM
GADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRG
FLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K 88.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 89.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 90.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 91.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 92.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 93.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 94.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 95.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 96.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 97.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 98.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDM
FSSPLETDSMDPFGLVTGLEAVRSPSFEK 138.
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS
MDPFGLVTGLEAVRSPSFEK 139.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 140.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 141.
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEK
HRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLET
DSMDPFGLVTGLEAVRSPSFEK 142.
RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 143.
RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 144.
RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 145.
RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 146.
RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 147.
RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 148.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 149.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 150.
RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 151.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 152.
RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 153.
RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 154.
RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 155.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 156.
RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 157.
RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 158.
RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 159.
RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 160.
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 161.
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR
LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS
MDPFGLVTGLEAVRSPSFEK 162.
HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 163.
HPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 164.
HPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVY
RSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 165.
HPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 166.
DAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 167.
VHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDP
FGLVTGLEAVRSPSFEK 168.
RLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSV
RYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQL
YKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV RSPSFEK
188. PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGAD
GKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLP
LSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 192.
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 193.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 194.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 195.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 196.
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMF
SSPLETDSMDPFGLVTGLEAVRSPSFEK 197.
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSE
KHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 198.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 199.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFS
SPLETDSMDPFGLVTGLEAVRSPSFEK 200.
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 201.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 202.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETD
SMDPFGLVTGLEAVRSPSFEK 203.
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK 204.
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRS
EKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL
ETDSMDPFGLVTGLEAVRSPSFEK
[0093] In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:1. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:2. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:3. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:4. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:5. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:6. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:7. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:8. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:9. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:10. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:11. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:12. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:13. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:14. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:15. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:16. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:17. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:18. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:19. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:20. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:21. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:22. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:23. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:24. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:25. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:26. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:27. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:28. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:29. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:30. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:31. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:32. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:33. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:34. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:35. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:36. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:37. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:38. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:39. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:40. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:41. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:42. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:43. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:44. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:45. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:46. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:47. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:48. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:49. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:50. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:51. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:52. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:53. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:54. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:55. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:56. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:57. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:58. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:59. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:60. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:61. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:62. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:63. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:64. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:65. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:66. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:67. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:68. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:69. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:70. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:71. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:72. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:73. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:74. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:75. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:76. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:77. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:78. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:79. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:80. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:81. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:82. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:83. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:84. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:85. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:86. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:87. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:88. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:89. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:90. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:91. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:92. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:93. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:94. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:95. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:96. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:97. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:98. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:138. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:139. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:140. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:141. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:142. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:143. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:144. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:145. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:146. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:147. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:148. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:149. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:150. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:151. In another embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:152. In
other embodiments, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:153. In one embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:154. In another embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:155. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:156. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:157. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:158. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:159. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:160. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:161. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:162. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:163. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:164. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:165. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:166. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:167. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:168. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:192. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:193. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:194. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:195. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:196. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:197. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:198. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:199. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:200. In another embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:201. In
other embodiments, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:202. In one embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:203. In another embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:204. In certain
embodiments of the various peptide sequences provided herein, the R
residue at the N-terminus is deleted.
[0094] In yet other embodiments, the peptide sequence consists of
an amino acid sequence set forth in SEQ ID NO:1. In another
embodiment, the peptide sequence consists of an amino acid sequence
set forth in SEQ ID NO:2. In other embodiments, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:3. In one embodiment, the peptide sequence consists of an amino
acid sequence set forth in SEQ ID NO:4. In another embodiment, the
peptide sequence consists of an amino acid sequence set forth in
SEQ ID NO:5. In other embodiments, the peptide sequence consists of
an amino acid sequence set forth in SEQ ID NO:6. In one embodiment,
the peptide sequence consists of an amino acid sequence set forth
in SEQ ID NO:7. In another embodiment, the peptide sequence
consists of an amino acid sequence set forth in SEQ ID NO:8. In
other embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:9. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:10. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:11. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:12. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:13. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:14. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:15. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:16. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:17. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:18. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:19. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:20. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:21. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:22. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:23. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:24. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:25. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:26. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:27. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:28. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:29. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:30. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:31. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:32. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:33. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:34. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:35. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:36. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:37. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:38. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:39. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:40. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:41. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:42. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:43. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:44. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:45. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:46. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:47. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:48. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:49. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:50. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:51. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:52. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:53. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:54. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:55. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:56. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:57. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:58. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:59. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:60. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:61. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:62. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:63. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:64. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:65. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:66. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:67. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:68. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:69. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:70. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:71. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:72. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:73. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:74. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:75. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:76. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:77. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:78. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:79. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:80. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:81. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:82. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:83. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:84. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:85. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:86. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:87. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:88. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:89. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:90. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:91. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:92. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:93. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:94. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:95. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:96. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:97. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:98. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:138. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:139. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:140. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:141. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:142. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:143. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:144. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:145. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:146. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:147. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:148. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:149. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:150. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:151. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:152. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:153. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:154. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:155. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:156. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:157. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:158. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:159. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:160. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:161. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:162. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:163. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:164. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:165. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:166. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:167. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:168. In another embodiment, the
peptide sequence consists of an amino acid sequence set forth in
SEQ ID NO:192. In other embodiments, the peptide sequence consists
of an amino acid sequence set forth in SEQ ID NO:193. In one
embodiment, the peptide sequence consists of an amino acid sequence
set forth in SEQ ID NO:194. In another embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:195. In other embodiments, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:196. In one embodiment,
the peptide sequence consists of an amino acid sequence set forth
in SEQ ID NO:197. In another embodiment, the peptide sequence
consists of an amino acid sequence set forth in SEQ ID NO:198. In
other embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:199. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:200. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:201. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:202. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:203. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:204. In certain
embodiments of the various peptide sequences provided herein, the R
residue at the N-terminus is deleted.
Nucleic Acid Molecules
[0095] Also provided are nucleic acid molecules encoding peptide
sequences provided herein, including subsequences, sequence
variants and modified forms of the sequences listed in the Sequence
Listing (and in PCT Pub. No. WO 2013/006486 and US Pub. No.
2013/0023474, as well as PCT Publ. No. WO 2014/085365) or Table 1,
and vectors that include nucleic acid encoding the peptides used in
the methods described herein. Accordingly, "nucleic acids" include
those that encode the exemplified peptide sequences disclosed
herein, as well as those encoding functional subsequences, sequence
variants and modified forms of the exemplified peptide sequences,
so long as the foregoing retain at least detectable or measureable
activity or function useful in the treatment or prevention of a
bile acid-related disorder (e.g., PBC).
[0096] Nucleic acid, which can also be referred to herein as a
gene, polynucleotide, nucleotide sequence, primer, oligonucleotide
or probe, refers to natural or modified purine- and
pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotides or mixed
polyribo-polydeoxyribo nucleotides and .alpha.-anomeric forms
thereof. The two or more purine- and pyrimidine-containing polymers
are typically linked by a phosphoester bond or analog thereof. The
terms can be used interchangeably to refer to all forms of nucleic
acid, including deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). The nucleic acids can be single strand, double, or triplex,
linear or circular. Nucleic acids include genomic DNA and cDNA. RNA
nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA or
antisense. Nucleic acids include naturally occurring, synthetic, as
well as nucleotide analogs and derivatives.
[0097] As a result of the degeneracy of the genetic code, the
nucleic acid molecules provided herein include sequences degenerate
with respect to nucleic acid molecules encoding the peptide
sequences useful in the methods provided herein. Thus, degenerate
nucleic acid sequences encoding peptide sequences, including
subsequences, variants and modified forms of the peptide sequences
exemplified herein (e.g., in the Sequence Listing or Table 1), are
provided. The term "complementary," when used in reference to a
nucleic acid sequence, means the referenced regions are 100%
complementary, i.e., exhibit 100% base pairing with no
mismatches.
[0098] Nucleic acid can be produced using any of a variety of known
standard cloning and chemical synthesis methods, and can be altered
intentionally by site-directed mutagenesis or other recombinant
techniques known to one skilled in the art. Purity of
polynucleotides can be determined through, for example, sequencing,
gel electrophoresis, and UV spectrometry.
[0099] Nucleic acids may be inserted into a nucleic acid construct
in which expression of the nucleic acid is influenced or regulated
by an "expression control element," referred to herein as an
"expression cassette." The term "expression control element" refers
to one or more nucleic acid sequence elements that regulate or
influence expression of a nucleic acid sequence to which it is
operatively linked. An expression control element can include, as
appropriate, promoters, enhancers, transcription terminators, gene
silencers, a start codon (e.g., ATG) in front of a protein-encoding
gene, etc.
[0100] An expression control element operatively linked to a
nucleic acid sequence controls transcription and, as appropriate,
translation of the nucleic acid sequence. The term "operatively
linked" refers to a juxtaposition wherein the referenced components
are in a relationship permitting them to function in their intended
manner. Typically, expression control elements are juxtaposed at
the 5' or the 3' ends of the genes but can also be intronic.
[0101] Expression control elements include elements that activate
transcription constitutively, that are inducible (i.e., require an
external signal or stimuli for activation), or derepressible (i.e.,
require a signal to turn transcription off; when the signal is no
longer present, transcription is activated or "derepressed"). Also
included in the expression cassettes provided herein are control
elements sufficient to render gene expression controllable for
specific cell types or tissues (i.e., tissue-specific control
elements). Typically, such elements are located upstream or
downstream (i.e., 5' or 3') of the coding sequence. Promoters are
generally positioned 5' of the coding sequence. Promoters, produced
by recombinant DNA or synthetic techniques, can be used to provide
for transcription of the polynucleotides provided herein. A
"promoter" typically means a minimal sequence element sufficient to
direct transcription.
[0102] Nucleic acids may be inserted into a plasmid for
transformation into a host cell and for subsequent expression
and/or genetic manipulation. A plasmid is a nucleic acid that can
be stably propagated in a host cell; plasmids may optionally
contain expression control elements in order to drive expression of
the nucleic acid. For purposes of this invention, a vector is
synonymous with a plasmid. Plasmids and vectors generally contain
at least an origin of replication for propagation in a cell and a
promoter. Plasmids and vectors may also include an expression
control element for expression in a host cell, and are therefore
useful for expression and/or genetic manipulation of nucleic acids
encoding peptide sequences, expressing peptide sequences in host
cells and organisms, or producing peptide sequences, for
example.
[0103] As used herein, the term "transgene" means a polynucleotide
that has been introduced into a cell or organism by artifice. For
example, in a cell having a transgene, the transgene has been
introduced by genetic manipulation or "transformation" of the cell.
A cell or progeny thereof into which the transgene has been
introduced is referred to as a "transformed cell" or
"transformant." Typically, the transgene is included in progeny of
the transformant or becomes a part of the organism that develops
from the cell. Transgenes may be inserted into the chromosomal DNA
or maintained as a self-replicating plasmid, YAC, minichromosome,
or the like.
[0104] Bacterial system promoters include T7 and inducible
promoters such as pL of bacteriophage plac, ptrp, ptac (ptrp-lac
hybrid promoter) and tetracycline-responsive promoters. Insect cell
system promoters include constitutive or inducible promoters (e.g.,
ecdysone). Mammalian cell constitutive promoters include SV40, RSV,
bovine papilloma virus (BPV) and other virus promoters, or
inducible promoters derived from the genome of mammalian cells
(e.g., metallothionein IIA promoter; heat shock promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the
inducible mouse mammary tumor virus long terminal repeat).
Alternatively, a retroviral genome can be genetically modified for
introducing and directing expression of a peptide sequence in
appropriate host cells.
[0105] As methods and uses provided herein include in vivo
delivery, expression systems further include vectors designed for
in vivo use. Particular non-limiting examples include adenoviral
vectors (U.S. Pat. Nos. 5,700,470 and 5,731,172), adeno-associated
vectors (U.S. Pat. No. 5,604,090), herpes simplex virus vectors
(U.S. Pat. No. 5,501,979), retroviral vectors (U.S. Pat. Nos.
5,624,820, 5,693,508 and 5,674,703), BPV vectors (U.S. Pat. No.
5,719,054), CMV vectors (U.S. Pat. No. 5,561,063) and parvovirus,
rotavirus, Norwalk virus and lentiviral vectors (see, e.g., U.S.
Pat. No. 6,013,516). Vectors include those that deliver genes to
cells of the intestinal tract, including the stem cells (Croyle et
al., Gene Ther. 5:645 (1998); S. J. Henning, Adv. Drug Deliv. Rev.
17:341 (1997), U.S. Pat. Nos. 5,821,235 and 6,110,456). Many of
these vectors have been approved for human studies.
[0106] Yeast vectors include constitutive and inducible promoters
(see, e.g., Ausubel et al., In: Current Protocols in Molecular
Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. & Wiley
Interscience, 1988; Grant et al. Methods in Enzymology, 153:516
(1987), eds. Wu & Grossman; Bitter Methods in Enzymology,
152:673 (1987), eds. Berger & Kimmel, Acad. Press, N.Y.; and,
Strathern et al., The Molecular Biology of the Yeast Saccharomyces
(1982) eds. Cold Spring Harbor Press, Vols. I and II). A
constitutive yeast promoter such as ADH or LEU2 or an inducible
promoter such as GAL may be used (R. Rothstein In: DNA Cloning, A
Practical Approach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press,
Wash., D.C., 1986). Vectors that facilitate integration of foreign
nucleic acid sequences into a yeast chromosome, via homologous
recombination for example, are known in the art. Yeast artificial
chromosomes (YAC) are typically used when the inserted
polynucleotides are too large for more conventional vectors (e.g.,
greater than about 12 Kb).
[0107] Expression vectors also can contain a selectable marker
conferring resistance to a selective pressure or identifiable
marker (e.g., beta-galactosidase), thereby allowing cells having
the vector to be selected for, grown and expanded. Alternatively, a
selectable marker can be on a second vector that is co-transfected
into a host cell with a first vector containing a nucleic acid
encoding a peptide sequence. Selection systems include, but are not
limited to, herpes simplex virus thymidine kinase gene (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad.
Sci. USA 48:2026 (1962)), and adenine phosphoribosyltransferase
(Lowy et al., Cell 22:817 (1980)) genes that can be employed in
tk-, hgprt- or aprt-cells, respectively. Additionally,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, which
confers resistance to mycophenolic acid (Mulligan et al., Proc.
Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers
resistance to aminoglycoside G-418 (Colberre-Garapin et al., J.
Mol. Biol. 150:1(1981)); puromycin; and hygromycin gene, which
confers resistance to hygromycin (Santerre et al., Gene 30:147
(1984)). Additional selectable genes include trpB, which allows
cells to utilize indole in place of tryptophan; hisD, which allows
cells to utilize histinol in place of histidine (Hartman et al.,
Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine
decarboxylase), which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue (1987) In: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory).
Cell Lines and Animal Models
[0108] In certain embodiments, also provided is a transformed
cell(s) (in vitro, ex vivo and in vivo) and host cells that produce
a variant or fusion of FGF19 and/or FGF21 as set forth herein,
where expression of the variant or fusion of FGF19 and/or FGF21 is
conferred by a nucleic acid encoding the variant or fusion of FGF19
and/or FGF21. As used herein, a "transformed" or "host" cell is a
cell into which a nucleic acid is introduced that can be propagated
and/or transcribed for expression of an encoded peptide sequence.
The term also includes any progeny or subclones of the host cell.
Transformed and host cells that express peptide sequences provided
herein typically include a nucleic acid that encodes the peptide
sequence. In one embodiment, a transformed or host cell is a
prokaryotic cell. In another embodiment, a transformed or host cell
is a eukaryotic cell. In various aspects, the eukaryotic cell is a
yeast or mammalian (e.g., human, primate, etc.) cell.
[0109] Transformed and host cells include but are not limited to
microorganisms such as bacteria and yeast; and plant, insect and
mammalian cells. For example, bacteria transformed with recombinant
bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic
acid expression vectors; yeast transformed with recombinant yeast
expression vectors; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid); insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus); and
animal cell systems infected with recombinant virus expression
vectors (e.g., retroviruses, adenovirus, vaccinia virus), or
transformed animal cell systems engineered for transient or stable
propagation or expression.
[0110] For gene therapy uses and methods, a transformed cell can be
in a subject. A cell in a subject can be transformed with a nucleic
acid that encodes a peptide sequence as set forth herein in vivo.
Alternatively, a cell can be transformed in vitro with a transgene
or polynucleotide, and then transplanted into a tissue of subject
in order to effect treatment. Alternatively, a primary cell isolate
or an established cell line can be transformed with a transgene or
polynucleotide that encodes a variant of FGF19 and/or FGF21 or a
fusion/chimeric sequence (or variant) thereof, such as a chimeric
peptide sequence including all or a portion of FGF19, or including
all or a portion of FGF21, and then optionally transplanted into a
tissue of a subject.
[0111] Non-limiting target cells for expression of peptide
sequences, particularly for expression in vivo, include pancreas
cells (islet cells), muscle cells, mucosal cells and endocrine
cells. Such endocrine cells can provide inducible production
(secretion) of a variant of FGF19 and/or FGF21, or a
fusion/chimeric sequence (or variant) thereof, such as a chimeric
peptide sequence including all or a portion of FGF19, or including
all or a portion of FGF21. Additional cells to transform include
stem cells or other multipotent or pluripotent cells, for example,
progenitor cells that differentiate into the various pancreas cells
(islet cells), muscle cells, mucosal cells and endocrine cells.
Targeting stem cells provides longer term expression of peptide
sequences provided herein.
[0112] As used herein, the term "cultured," when used in reference
to a cell, means that the cell is grown in vitro. A particular
example of such a cell is a cell isolated from a subject, and grown
or adapted for growth in tissue culture. Another example is a cell
genetically manipulated in vitro, and transplanted back into the
same or a different subject.
[0113] The term "isolated," when used in reference to a cell, means
a cell that is separated from its naturally occurring in vivo
environment. "Cultured" and "isolated" cells may be manipulated by
the hand of man, such as genetically transformed. These terms
include any progeny of the cells, including progeny cells that may
not be identical to the parental cell due to mutations that occur
during cell division. The terms do not include an entire human
being.
[0114] Nucleic acids encoding peptide sequences provided herein can
be introduced for stable expression into cells of a whole organism.
Such organisms, including non-human transgenic animals, are useful
for studying the effect of peptide expression in a whole animal and
therapeutic benefit. For example, as disclosed herein, production
of a variant of FGF19 and/or FGF21 or a fusion/chimeric sequence
(or variant) thereof, such as a chimeric peptide sequence including
all or a portion of FGF19, or including all or a portion of FGF21
as set forth herein, in mice.
[0115] Mice strains that develop or are susceptible to developing a
particular disease (e.g., diabetes, degenerative disorders, cancer,
etc.) are also useful for introducing therapeutic proteins as
described herein in order to study the effect of therapeutic
protein expression in the disease-susceptible mouse. Transgenic and
genetic animal models that are susceptible to particular disease or
physiological conditions, such as streptozotocin (STZ)-induced
diabetic (STZ) mice, are appropriate targets for expressing
variants of FGF19 and/or FGF21, fusions/chimeric sequences (or
variant) thereof, such as a chimeric peptide sequence including all
or a portion of FGF19, or including all or a portion of FGF21, as
set forth herein. Thus, in certain embodiments, there are provided
non-human transgenic animals that produce a variant of FGF19 and/or
FGF21, or a fusion/chimeric sequence (or variant) thereof, such as
a chimeric peptide sequence including all or a portion of FGF19, or
including all or a portion of FGF21, the production of which is not
naturally occurring in the animal which is conferred by a transgene
present in somatic or germ cells of the animal.
[0116] The term "transgenic animal" refers to an animal whose
somatic or germ line cells bear genetic information received,
directly or indirectly, by deliberate genetic manipulation at the
subcellular level, such as by microinjection or infection with
recombinant virus. The term "transgenic" further includes cells or
tissues (i.e., "transgenic cell," "transgenic tissue") obtained
from a transgenic animal genetically manipulated as described
herein. In the present context, a "transgenic animal" does not
encompass animals produced by classical crossbreeding or in vitro
fertilization, but rather denotes animals in which one or more
cells receive a nucleic acid molecule. Transgenic animals provided
herein can be either heterozygous or homozygous with respect to the
transgene. Methods for producing transgenic animals, including
mice, sheep, pigs and frogs, are well known in the art (see, e.g.,
U.S. Pat. Nos. 5,721,367, 5,695,977, 5,650,298, and 5,614,396) and,
as such, are additionally included.
[0117] Peptide sequences, nucleic acids encoding peptide sequences,
vectors and transformed host cells expressing peptide sequences
include isolated and purified forms. The term "isolated," when used
as a modifier of a composition provided herein, means that the
composition is separated, substantially, completely, or at least in
part, from one or more components in an environment. Generally,
compositions that exist in nature, when isolated, are substantially
free of one or more materials with which they normally associate
with in nature, for example, one or more protein, nucleic acid,
lipid, carbohydrate or cell membrane. The term "isolated" does not
exclude alternative physical forms of the composition, such as
variants, modifications or derivatized forms, fusions and chimeras,
multimers/oligomers, etc., or forms expressed in host cells. The
term "isolated" also does not exclude forms (e.g., pharmaceutical
compositions, combination compositions, etc.) in which there are
combinations therein, any one of which is produced by the hand of
man. An "isolated" composition can also be "purified" when free of
some, a substantial number of, or most or all of one or more other
materials, such as a contaminant or an undesired substance or
material.
[0118] As used herein, the term "recombinant," when used as a
modifier of peptide sequences, nucleic acids encoding peptide
sequences, etc., means that the compositions have been manipulated
(i.e., engineered) in a fashion that generally does not occur in
nature (e.g., in vitro). A particular example of a recombinant
peptide would be where a peptide sequence provided herein is
expressed by a cell transfected with a nucleic acid encoding the
peptide sequence. A particular example of a recombinant nucleic
acid would be a nucleic acid (e.g., genomic or cDNA) encoding a
peptide sequence cloned into a plasmid, with or without 5', 3' or
intron regions that the gene is normally contiguous within the
genome of the organism. Another example of a recombinant peptide or
nucleic acid is a hybrid or fusion sequence, such as a chimeric
peptide sequence comprising a portion of FGF19 and a portion of
FGF21.
Particular Modifications to Enhance Peptide Function
[0119] It is frequently beneficial, and sometimes imperative, to
improve one of more physical properties of the treatment modalities
disclosed herein and/or the manner in which they are administered.
Improvements of physical properties include, for example,
modulating immunogenicity; methods of increasing solubility,
bioavailability, serum half-life, and/or therapeutic half-life;
and/or modulating biological activity. Certain modifications may
also be useful to, for example, raise of antibodies for use in
detection assays (e.g., epitope tags) and to provide for ease of
protein purification. Such improvements must generally be imparted
without adversely impacting the bioactivity of the treatment
modality and/or increasing its immunogenicity.
[0120] Pegylation of is one particular modification contemplated
herein, while other modifications include, but are not limited to,
glycosylation (N- and O-linked); polysialylation; albumin fusion
molecules comprising serum albumin (e.g., human serum albumin
(HSA), cyno serum albumin, or bovine serum albumin (BSA)); albumin
binding through, for example a conjugated fatty acid chain
(acylation); and Fc-fusion proteins.
[0121] Pegylation: The clinical effectiveness of protein
therapeutics is often limited by short plasma half-life and
susceptibility to protease degradation. Studies of various
therapeutic proteins (e.g., filgrastim) have shown that such
difficulties may be overcome by, for example, conjugating or
linking the protein to any of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes. This is frequently effected by a linking moiety
covalently bound to both the protein and the nonproteinaceous
polymer, e.g., a PEG. Such PEG-conjugated biomolecules have been
shown to possess clinically useful properties, including better
physical and thermal stability, protection against susceptibility
to enzymatic degradation, increased solubility, longer in vivo
circulating half-life and decreased clearance, reduced
immunogenicity and antigenicity, and reduced toxicity. In addition
to the beneficial effects of pegylation on pharmacokinetic
parameters, pegylation itself may enhance activity.
[0122] PEGs suitable for conjugation to a polypeptide sequence are
generally soluble in water at room temperature, and have the
general formula R(O--CH.sub.2--CH.sub.2).sub.nO--R, where R is
hydrogen or a protective group such as an alkyl or an alkanol
group, and where n is an integer from 1 to 1000. When R is a
protective group, it generally has from 1 to 8 carbons. The PEG
conjugated to the polypeptide sequence can be linear or branched.
Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are
contemplated by the present disclosure. A molecular weight of the
PEG used in embodiments provided herein is not restricted to any
particular range, and examples are set forth elsewhere herein; by
way of example, certain embodiments have molecular weights between
5 kDa and 20 kDa, while other embodiments have molecular weights
between 4 kDa and 10 kDa.
[0123] In other embodiments, provided herein are compositions of
conjugates wherein the PEGs have different n values, and thus the
various different PEGs are present in specific ratios. For example,
some compositions comprise a mixture of conjugates where n=1, 2, 3
and 4. In some compositions, the percentage of conjugates where n=1
is 18-25%, the percentage of conjugates where n=2 is 50-66%, the
percentage of conjugates where n=3 is 12-16%, and the percentage of
conjugates where n=4 is up to 5%. Such compositions can be produced
by reaction conditions and purification methods know in the art.
Cation exchange chromatography may be used to separate conjugates,
and a fraction is then identified which contains the conjugate
having, for example, the desired number of PEGs attached, purified
free from unmodified protein sequences and from conjugates having
other numbers of PEGs attached.
[0124] Pegylation most frequently occurs at the alpha amino group
at the N-terminus of the polypeptide, the epsilon amino group on
the side chain of lysine residues, and the imidazole group on the
side chain of histidine residues. Since most recombinant
polypeptides possess a single alpha and a number of epsilon amino
and imidazole groups, numerous positional isomers can be generated
depending on the linker chemistry.
[0125] General pegylation strategies known in the art can be
applied herein. PEG may be bound to a polypeptide provided herein
via a terminal reactive group (a "spacer" or "linker") which
mediates a bond between the free amino or carboxyl groups of one or
more of the polypeptide sequences and polyethylene glycol. The PEG
having the spacer which may be bound to the free amino group
includes N-hydroxysuccinylimide polyethylene glycol which may be
prepared by activating succinic acid ester of polyethylene glycol
with N-hydroxysuccinylimide. Another activated polyethylene glycol
which may be bound to a free amino group is
2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which may
be prepared by reacting polyethylene glycol monomethyl ether with
cyanuric chloride. The activated polyethylene glycol which is bound
to the free carboxyl group includes polyoxyethylenediamine.
[0126] Conjugation of one or more of the polypeptide sequences
provided herein to PEG having a spacer may be carried out by
various conventional methods. For example, the conjugation reaction
can be carried out in solution at a pH of from 5 to 10, at
temperature from 4.degree. C. to room temperature, for 30 minutes
to 20 hours, utilizing a molar ratio of reagent to protein of from
4:1 to 30:1. Reaction conditions may be selected to direct the
reaction towards producing predominantly a desired degree of
substitution. In general, low temperature, low pH (e.g., pH=5), and
short reaction time tend to decrease the number of PEGs attached,
whereas high temperature, neutral to high pH (e.g., pH>7), and
longer reaction time tend to increase the number of PEGs attached.
Various means known in the art may be used to terminate the
reaction. In some embodiments, the reaction is terminated by
acidifying the reaction mixture and freezing at, e.g., -20.degree.
C. Pegylation of various molecules is discussed in, for example,
U.S. Pat. Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and
5,985,263.
[0127] In some embodiments, also provided herein are uses of PEG
mimetics. Recombinant PEG mimetics have been developed that retain
the attributes of PEG (e.g., enhanced serum half-life) while
conferring several additional advantageous properties. By way of
example, simple polypeptide chains (comprising, for example, Ala,
Glu, Gly, Pro, Ser and Thr) capable of forming an extended
conformation similar to PEG can be produced recombinantly already
fused to the peptide or protein drug of interest (e.g., XTEN
technology; Amunix; Mountain View, Calif.). This obviates the need
for an additional conjugation step during the manufacturing
process. Moreover, established molecular biology techniques enable
control of the side chain composition of the polypeptide chains,
allowing optimization of immunogenicity and manufacturing
properties.
[0128] Glycosylation: As used herein, "glycosylation" is meant to
broadly refer to the enzymatic process by which glycans are
attached to proteins, lipids or other organic molecules. The use of
the term "glycosylation" herein is generally intended to mean
adding or deleting one or more carbohydrate moieties (either by
removing the underlying glycosylation site or by deleting the
glycosylation by chemical and/or enzymatic means), and/or adding
one or more glycosylation sites that may or may not be present in
the native sequence. In addition, the phrase includes qualitative
changes in the glycosylation of the native proteins involving a
change in the nature and proportions of the various carbohydrate
moieties present.
[0129] Glycosylation can dramatically affect the physical
properties (e.g., solubility) of polypeptides and can also be
important in protein stability, secretion, and subcellular
localization. Glycosylated polypeptides may also exhibit enhanced
stability or may improve one or more pharmacokinetic properties,
such as half-life. In addition, solubility improvements can, for
example, enable the generation of formulations more suitable for
pharmaceutical administration than formulations comprising the
non-glycosylated polypeptide.
[0130] Addition of glycosylation sites can be accomplished by
altering the amino acid sequence. The alteration to the polypeptide
may be made, for example, by the addition of, or substitution by,
one or more serine or threonine residues (for O-linked
glycosylation sites) or asparagine residues (for N-linked
glycosylation sites). The structures of N-linked and O-linked
oligosaccharides and the sugar residues found in each type may be
different. One type of sugar that is commonly found on both is
N-acetylneuraminic acid (hereafter referred to as sialic acid).
Sialic acid is usually the terminal residue of both N-linked and
O-linked oligosaccharides and, by virtue of its negative charge,
may confer acidic properties to the glycoprotein. A particular
embodiment comprises the generation and use of N-glycosylation
variants.
[0131] The polypeptide sequences provided herein may optionally be
altered through changes at the nucleic acid level, particularly by
mutating the nucleic acid encoding the polypeptide at preselected
bases such that codons are generated that will translate into the
desired amino acids.
[0132] Polysialylation: In certain embodiments, also provided
herein is the use of polysialylation, the conjugation of
polypeptides to the naturally occurring, biodegradable
.alpha.-(2.fwdarw.8) linked polysialic acid ("PSA") in order to
improve the polypeptides' stability and in vivo
pharmacokinetics.
[0133] Albumin Fusion: Additional suitable components and molecules
for conjugation include albumins such as human serum albumin (HSA),
cyno serum albumin, and bovine serum albumin (BSA).
[0134] In some embodiments, albumin is conjugated to a drug
molecule (e.g., a polypeptide described herein) at the carboxyl
terminus, the amino terminus, both the carboxyl and amino termini,
and internally (see, e.g., U.S. Pat. Nos. 5,876,969 and
7,056,701).
[0135] In the HSA-drug molecule conjugates embodiments provided
herein, various forms of albumin may be used, such as albumin
secretion pre-sequences and variants thereof, fragments and
variants thereof, and HSA variants. Such forms generally possess
one or more desired albumin activities. In additional embodiments,
fusion proteins are provided herein comprising a polypeptide drug
molecule fused directly or indirectly to albumin, an albumin
fragment, an albumin variant, etc., wherein the fusion protein has
a higher plasma stability than the unfused drug molecule and/or the
fusion protein retains the therapeutic activity of the unfused drug
molecule. In some embodiments, the indirect fusion is effected by a
linker, such as a peptide linker or modified version thereof.
[0136] As alluded to above, fusion of albumin to one or more
polypeptides provided herein can, for example, be achieved by
genetic manipulation, such that the nucleic acid coding for HSA, or
a fragment thereof, is joined to the nucleic acid coding for the
one or more polypeptide sequences.
[0137] Alternative Albumin Binding Strategies: Several
albumin--binding strategies have been developed as alternatives to
direct fusion and may be used with the agents described herein. By
way of example, in certain embodiments, provided herein is albumin
binding through a conjugated fatty acid chain (acylation) and
fusion proteins which comprise an albumin binding domain (ABD)
polypeptide sequence and the sequence of one or more of the
polypeptides described herein.
[0138] Conjugation with Other Molecules: Additional suitable
components and molecules for conjugation include, for example,
thyroglobulin; tetanus toxoid; Diphtheria toxoid; polyamino acids
such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of
rotaviruses; influenza virus hemagglutinin, influenza virus
nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B
virus core protein and surface antigen; or any combination of the
foregoing.
[0139] Thus, in certain embodiments, conjugation of one or more
additional components or molecules at the N- and/or C-terminus of a
polypeptide sequence, such as another polypeptide (e.g., a
polypeptide having an amino acid sequence heterologous to the
subject polypeptide), or a carrier molecule is also contemplated.
Thus, an exemplary polypeptide sequence can be provided as a
conjugate with another component or molecule.
[0140] A polypeptide may also be conjugated to large, slowly
metabolized macromolecules such as proteins; polysaccharides, such
as sepharose, agarose, cellulose, or cellulose beads; polymeric
amino acids such as polyglutamic acid, or polylysine; amino acid
copolymers; inactivated virus particles; inactivated bacterial
toxins such as toxoid from diphtheria, tetanus, cholera, or
leukotoxin molecules; inactivated bacteria; and dendritic cells.
Such conjugated forms, if desired, can be used to produce
antibodies against a polypeptide provided herein.
[0141] Fc-fusion Molecules: In certain embodiments, the amino- or
carboxyl-terminus of a polypeptide sequence provided herein can be
fused with an immunoglobulin Fc region (e.g., human Fc) to form a
fusion conjugate (or fusion molecule). Fc fusion conjugates have
been shown to increase the systemic half-life of
biopharmaceuticals, and thus the biopharmaceutical product may
require less frequent administration.
[0142] Fc binds to the neonatal Fc receptor (FcRn) in endothelial
cells that line the blood vessels, and, upon binding, the Fc fusion
molecule is protected from degradation and re-released into the
circulation, keeping the molecule in circulation longer. This Fc
binding is believed to be the mechanism by which endogenous IgG
retains its long plasma half-life. More recent Fc-fusion technology
links a single copy of a biopharmaceutical to the Fc region of an
antibody to optimize the pharmacokinetic and pharmacodynamic
properties of the biopharmaceutical as compared to traditional
Fc-fusion conjugates.
[0143] Purification: Additional suitable components and molecules
for conjugation include those suitable for isolation or
purification. Particular non-limiting examples include binding
molecules, such as biotin (biotin-avidin specific binding pair), an
antibody, a receptor, a ligand, a lectin, or molecules that
comprise a solid support, including, for example, plastic or
polystyrene beads, plates or beads, magnetic beads, test strips,
and membranes.
[0144] Purification methods such as cation exchange chromatography
may be used to separate conjugates by charge difference, which
effectively separates conjugates into their various molecular
weights. For example, the cation exchange column can be loaded and
then washed with .about.20 mM sodium acetate, pH .about.4, and then
eluted with a linear (0 M to 0.5 M) NaCl gradient buffered at a pH
from 3 to 5.5, such as at pH .about.4.5. The content of the
fractions obtained by cation exchange chromatography may be
identified by molecular weight using conventional methods, for
example, mass spectroscopy, SDS-PAGE, or other known methods for
separating molecular entities by molecular weight. A fraction is
then identified which contains the conjugate having the desired
number of PEGs attached, purified free from unmodified protein
sequences and from conjugates having other numbers of PEGs
attached.
[0145] Other Modifications: In certain embodiments, also provided
herein is the use of other modifications, currently known or
developed in the future, to improve one or more properties.
Examples include hesylation, various aspects of which are described
in, for example, U.S. Patent Appln. Nos. 2007/0134197 and
2006/0258607, and fusion molecules comprising SUMO as a fusion tag
(LifeSensors, Inc.; Malvern, Pa.).
[0146] In still other embodiments, a peptide sequence provided
herein is linked to a chemical agent (e.g., an immunotoxin or
chemotherapeutic agent), including, but are not limited to, a
cytotoxic agent, including taxol, cytochalasin B, gramicidin D,
mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin, daunorubicin, and analogs or homologs
thereof. Other chemical agents include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine); alkylating
agents (e.g., mechlorethamine, carmustine and lomustine,
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cisplatin); antibiotics (e.g., bleomycin); and
anti-mitotic agents (e.g., vincristine and vinblastine). Cytotoxins
can be conjugated to a peptide provided herein using linker
technology known in the art and described herein.
[0147] Further suitable components and molecules for conjugation
include those suitable for detection in an assay. Particular
non-limiting examples include detectable labels, such as a
radioisotope (e.g., .sup.125I, .sup.35S, .sup.32P, .sup.33P), an
enzyme which generates a detectable product (e.g., luciferase,
.beta.-galactosidase, horse radish peroxidase and alkaline
phosphatase), a fluorescent protein, a chromogenic protein, dye
(e.g., fluorescein isothiocyanate); fluorescence emitting metals
(e.g., .sup.152Eu); chemiluminescent compounds (e.g., luminol and
acridinium salts); bioluminescent compounds (e.g., luciferin); and
fluorescent proteins. Indirect labels include labeled or detectable
antibodies that bind to a peptide sequence, where the antibody may
be detected.
[0148] In certain embodiments, a peptide sequence provided herein
is conjugated to a radioactive isotope to generate a cytotoxic
radiopharmaceutical (radioimmunoconjugates) useful as a diagnostic
or therapeutic agent. Examples of such radioactive isotopes
include, but are not limited to, iodine.sup.131, indium.sup.111,
yttrium.sup.90 and lutetium.sup.177. Methods for preparing
radioimmunoconjugates are known to the skilled artisan. Examples of
radioimmunoconjugates that are commercially available include
ibritumomab, tiuxetan, and tositumomab.
[0149] Linkers: Linkers and their use have been described above.
Any of the foregoing components and molecules used to modify the
polypeptide sequences provided herein may optionally be conjugated
via a linker. Suitable linkers include "flexible linkers" which are
generally of sufficient length to permit some movement between the
modified polypeptide sequences and the linked components and
molecules. The linker molecules are generally about 6-50 atoms
long. The linker molecules may also be, for example, aryl
acetylene, ethylene glycol oligomers containing 2-10 monomer units,
diamines, diacids, amino acids, or combinations thereof. Suitable
linkers can be readily selected and can be of any suitable length,
such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,
10-20, 20-30, 30-50 or more than 50 amino acids.
[0150] Exemplary flexible linkers include glycine polymers
(G).sub.n, glycine-serine polymers (for example, (GS).sub.n,
GSGGS.sub.n (SEQ ID NO:206) and GGGS.sub.n (SEQ ID NO:207), where n
is an integer of at least one), glycine-alanine polymers,
alanine-serine polymers, and other flexible linkers. Glycine and
glycine-serine polymers are relatively unstructured, and therefore
may serve as neutral tethers between components. Exemplary flexible
linkers include, but are not limited to, GGSG (SEQ ID NO:208),
GGSGG (SEQ ID NO:209), GSGSG (SEQ ID NO:210), GSGGG (SEQ ID
NO:211), GGGSG (SEQ ID NO:189), and GSSSG (SEQ ID NO:212).
Bile Acid-Related Disorders and the Treatment or Prevention
Thereof
[0151] As used herein, the phrases "bile acid-related disorder,"
"bile acid-related or associated disorder," and the like, when used
in reference to a condition of a subject, means a disruption of
bile acid homeostasis, which may manifest itself as, for example,
an acute, transient or chronic abnormal level of a bile acid or one
or more bile acids. The condition can be caused by inhibition,
reduction or a delay in bile acid synthesis, metabolism or
absorption such that the subject exhibits a bile acid level not
typically found in normal subjects.
[0152] Also provided herein are in vitro, ex vivo and in vivo
(e.g., on or in a subject) methods and uses. Such methods and uses
can be practiced with any of the peptide sequences set forth herein
in combination with one or more additional therapeutic agents
and/or treatment modalities. In various embodiments, the methods
include administering a peptide sequence, such as an FGF19 or FGF21
variant, fusion or chimera disclosed herein (e.g., in the Sequence
Listing or Table 1), or a subsequence, a variant or modified form
of an FGF19 or FGF21 variant, fusion or chimera disclosed herein
(e.g., the Sequence Listing or Table 1), to a subject in an amount
effective for treating a bile acid-related disorder, in combination
with an additional therapeutic agent(s) and/or treatment modalities
(e.g., an agent useful in the treatment and/or prevention of PBC).
As set forth herein, the additional therapeutic agent(s) can be
administered before, with, or following administration of the
peptides described herein.
[0153] Also provided here are methods of preventing (e.g., in
subjects predisposed to having a particular disorder(s)), delaying,
slowing or inhibiting progression of, the onset of, or treating
(e.g., ameliorating) a bile-acid related or associated disorder
relative to an appropriate matched subject of comparable age,
gender, race, etc.). Thus, in various embodiments, a method
provided herein for, for example, modulating bile acid homeostasis
or treating a bile-acid related or associated disorder includes
contacting or administering i) one or more peptides provided herein
(e.g., a variant or fusion of FGF19 and/or FGF21 as set forth in
the Sequence Listing or Table 1) in an amount effective to modulate
bile acid homeostasis or treat a bile-acid related or associated
disorder, and ii) at least one additional therapeutic agent or
treatment modality that is useful in the treatment or prevention of
a bile acid related disorder (e.g., PBC).
[0154] The term "subject" refers to an animal. Typically, the
animal is a mammal that would benefit from treatment with a peptide
sequence provided herein. Particular examples include primates
(e.g., humans), dogs, cats, horses, cows, pigs, and sheep.
[0155] Subjects include those having a disorder, e.g., a bile acid
related or associated disorder, such as cholestasis, including, for
example diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC,
PIC, neonatal cholestasis, and drug induced cholestasis (e.g.,
estrogen)), and diseases of extrahepatic cholestasis (e.g., bile
cut compression from tumor, bile duct blockade by gall stones);
bile acid malabsorption and other disorders involving the distal
small intestine, including ileal resection, inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), short
bowel syndrome, disorders impairing absorption of bile acids not
otherwise characterized (idiopathic)) leading to diarrhea (e.g.,
BAD) and GI symptoms, and GI, liver, and/or biliary cancers (e.g.,
colon cancer and hepatocellular cancer); and/or bile acid synthesis
abnormalities, such as those contributing to NASH, cirrhosis and
portal hypertension; or subjects that do not have a disorder but
may be at risk of developing the disorder.
[0156] Additional bile acid-related disorders that may be treated
or prevented with the peptide sequences provided herein in
combination with one or more additional therapeutic agents or
treatment modalities include metabolic syndrome, a lipid or glucose
disorder, cholesterol or triglyceride metabolism, diabetes (e.g.,
type 2 diabetes), other hyperglycemic-related disorders, including
kidney damage (e.g., tubule damage or nephropathy), liver
degeneration, eye damage (e.g., diabetic retinopathy or cataracts),
and diabetic foot disorders, and dyslipidemias and their sequelae
such as, for example, atherosclerosis, coronary artery disease,
cerebrovascular disorders and the like.
[0157] Other conditions which may be associated with metabolic
syndrome, such as obesity and elevated body mass (including the
co-morbid conditions thereof such as, but not limited to,
nonalcoholic fatty liver disease (NAFLD), nonalcoholic
steatohepatitis (NASH), and polycystic ovarian syndrome (PCOS)),
and also include thromboses, hypercoagulable and prothrombotic
states (arterial and venous), hypertension (including portal
hypertension (defined as a hepatic venous pressure gradient (HVPG)
greater than 5 mm Hg), cardiovascular disease, stroke and heart
failure; Disorders or conditions in which inflammatory reactions
are involved, including atherosclerosis, chronic inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), asthma,
lupus erythematosus, arthritis, or other inflammatory rheumatic
disorders; Disorders of cell cycle or cell differentiation
processes such as adipose cell tumors, lipomatous carcinomas
including, for example, liposarcomas, solid tumors, and neoplasms;
Neurodegenerative diseases and/or demyelinating disorders of the
central and peripheral nervous systems and/or neurological diseases
involving neuroinflammatory processes and/or other peripheral
neuropathies, including Alzheimer's disease, multiple sclerosis,
Parkinson's disease, progressive multifocal leukoencephalopathy and
Guillian-Barre syndrome; Skin and dermatological disorders and/or
disorders of wound healing processes, including erythemato-squamous
dermatoses; and Other Disorders such as syndrome X, osteoarthritis,
and acute respiratory distress syndrome.
[0158] Treatment of a bile acid-related disorder (e.g., NASH) may
have the benefit of alleviating or abolishing a disorder secondary
thereto. By way of example, a subject suffering from NASH may also
have depression or anxiety due to NASH; thus, treating the
subject's NASH may also indirectly treat the depression or anxiety.
The use of the therapies disclosed herein to target such secondary
disorders is also contemplated in certain embodiments.
[0159] In particular embodiments, the subject has or is at risk of
having PBC. In other particular embodiments, the subject has or is
at risk of having NASH. In one embodiment, the subject has PBC. In
one embodiment, the subject is at risk of having PBC. In other
embodiments, the subject has NASH. In other embodiments, the
subject is at risk of having NASH.
[0160] Subjects at risk of developing a bile acid-related or
associated disorder (such as the disorders described above)
include, for example, those who may have a family history or
genetic predisposition toward such disorder, as well those whose
diet may contribute to development of such disorders.
[0161] As disclosed herein, treatment methods include contacting or
administering a peptide as set forth herein (e.g., a variant or
fusion of FGF19 and/or FGF21 as set forth in the Sequence Listing
or Table 1) in an amount effective to achieve a desired outcome or
result in a subject. A treatment that results in a desired outcome
or result includes decreasing, reducing or preventing the severity
or frequency of one or more symptoms of the condition in the
subject, e.g., an improvement in the subject's condition or a
"beneficial effect" or "therapeutic effect." Therefore, treatment
can decrease or reduce or prevent the severity or frequency of one
or more symptoms of the disorder, stabilize or inhibit progression
or worsening of the disorder, and in some instances, reverse the
disorder, transiently (e.g., for 1-6, 6-12, or 12-24 hours), for
medium term (e.g., 1-6, 6-12, 12-24 or 24-48 days) or long term
(e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greater than 24-48
weeks). Thus, in the case of a bile acid related or associated
disorder, treatment can lower or reduce one or more symptoms or
effects of the bile acid-related or associated disorders described
above.
[0162] Treatment methods also include contacting or administering
one or more additional agents or therapeutic modalities useful in
the treatment or prevention of a bile acid related disorder, such
as those agents or therapeutic modalities described herein, in an
amount effective to achieve a desired outcome or result in a
subject. A treatment that results in a desired outcome or result
includes decreasing, reducing or preventing the severity or
frequency of one or more symptoms of the condition in the subject,
e.g., an improvement in the subject's condition or a "beneficial
effect" or "therapeutic effect." Therefore, treatment can decrease
or reduce or prevent the severity or frequency of one or more
symptoms of the disorder, stabilize or inhibit progression or
worsening of the disorder, and in some instances, reverse the
disorder, transiently (e.g., for 1-6, 6-12, or 12-24 hours), for
medium term (e.g., 1-6, 6-12, 12-24 or 24-48 days) or long term
(e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greater than 24-48
weeks). Thus, in the case of a bile acid related or associated
disorder, treatment with a peptide provided herein in combination
with another therapeutic agent can lower or reduce one or more
symptoms or effects of the bile acid-related or associated
disorders described above.
[0163] An "effective amount" or a "sufficient amount" for use
and/or for treating a subject refers to an amount that provides, in
single or multiple doses, alone, or in combination with one or more
other agents, treatments, protocols, or therapeutic regimens, a
detectable response of any duration of time (transient, medium or
long term), a desired outcome in or an objective or subjective
benefit to a subject of any measurable or detectable degree or for
any duration of time (e.g., for hours, days, months, years, in
remission or cured). Such amounts typically are effective to
ameliorate a disorder, or one, multiple or all adverse symptoms,
consequences or complications of the disorder, to a measurable
extent, although reducing or inhibiting a progression or worsening
of the disorder, is considered a satisfactory outcome.
[0164] As used herein, the term "ameliorate" means an improvement
in the subject's disorder, a reduction in the severity of the
disorder, or an inhibition of progression or worsening of the
disorder (e.g., stabilizing the disorder). In the case of a bile
acid-related or associated disorder such as those described above,
including cholestasis (e.g., PBC), disorders impairing absorption
of bile acids leading to diarrhea (e.g., BAD) and bile acid
synthesis abnormalities (e.g., NASH), an improvement can be a
lowering or a reduction in one or more symptoms or effects of the
disorder.
[0165] A therapeutic benefit or improvement therefore need not be
complete ablation of any one, most or all symptoms, complications,
consequences or underlying causes associated with the disorder or
disease. Thus, a satisfactory endpoint is achieved when there is a
transient, medium or long term, incremental improvement in a
subject's condition, or a partial reduction in the occurrence,
frequency, severity, progression, or duration, or inhibition or
reversal, of one or more associated adverse symptoms or
complications or consequences or underlying causes, worsening or
progression (e.g., stabilizing one or more symptoms or
complications of the condition, disorder or disease), of the
disorder or disease, over a duration of time (hours, days, weeks,
months, etc.).
[0166] Thus, in the case of a disorder treatable by a peptide
sequence provided herein in combination with an additional agent,
the amount of the peptide and the additional agent sufficient to
ameliorate a disorder will depend on the type, severity and extent,
or duration of the disorder, the therapeutic effect or outcome
desired, and can be readily ascertained by the skilled artisan.
Appropriate amounts will also depend upon the individual subject
(e.g., the bioavailability within the subject, gender, age, etc.).
For example, a transient, or partial, restoration of normal bile
acid homeostasis in a subject can reduce the dosage amount or
frequency of the peptides and agents described herein in order to
treat the bile acid-related or associated disorders described
previously even though complete freedom from treatment has not
resulted. An effective amount can be ascertained, for example, by
measuring one or more relevant physiological effects.
[0167] Methods and uses provided herein for treating a subject are
applicable for prophylaxis to prevent or reduce the likelihood of a
disorder in a subject, such as a bile acid-related or associated
disorder. Accordingly, methods and uses provided herein for
treating a subject having, or at risk of developing, a bile
acid-related disorder or associated disorder can be practiced prior
to, substantially contemporaneously with, or following
administration or application of another agent useful for the
treatment or prevention of a bile acid-related or associated
disorder, and/or can be supplemented with other forms of therapy.
Supplementary therapies include other glucose lowering treatments,
such as insulin, an insulin sensitivity enhancer and other drug
treatments, a change in diet (low sugar, fats, etc.), weight loss
surgery-(reducing stomach volume by gastric bypass, gastrectomy),
gastric banding, gastric balloon, gastric sleeve, etc. For example,
a method or use provided herein for treating a hyperglycemic or
insulin resistance disorder can be used in combination with drugs
or other pharmaceutical compositions that lower glucose or increase
insulin sensitivity in a subject.
[0168] PBC and Combination Therapy with Agents Effective in the
Treatment or Prevention Thereof. Primary biliary cirrhosis (PBC),
the most common cholestatic liver disease, is a progressive hepatic
disease that primarily results from autoimmune destruction of the
bile ducts that transport bile acids out of the liver. As the
disease progresses, persistent toxic build-up of bile acids causes
progressive liver damage marked by chronic inflammation and
fibrosis. Because patients with PBC have an increased risk of HCC,
combination therapy with the variants of FGF19 peptide sequences,
fusions of FGF19 and/or FGF21 peptide sequences and variants of
fusions (chimeras) of FGF19 and/or FGF21 peptide sequences
described herein is of particular import, as such sequences do not
induce, or do not substantially increase, HCC formation or HCC
tumorigenesis.
[0169] Although patients with PBC are often asymptomatic at the
time of initial diagnosis, most present, or subsequently develop,
one or more of the following: pruritus; fatigue; jaundice;
xanthoma; disorders associated with an extrahepatic autoimmune
disorder (e.g., Sjogren's Syndrome and rheumatoid arthritis); and
complications that result from cirrhosis or portal hypertension
(e.g., ascites, esophageal varices and hepatic encephalopathy).
[0170] While a definitive cause of PBC has not been identified,
most research suggests that it is an autoimmune disorder. There
appears to be a genetic predisposition, and genetic studies have
indicated that part of the IL-12 signaling cascade, including
IL-12A and I-12RB2 polymorphisms, is important in the etiology of
the disease.
[0171] There is no definitive means of diagnosing PBC; rather,
assessment of a number of factors is generally required. Moreover,
diagnosis of PBC requires that other conditions with similar
symptoms (e.g., autoimmune hepatitis and primary sclerosing
cholangitis) by ruled out; by way of example, abdominal ultrasound
or CT scan is usually performed to rule out blockage of the bile
ducts.
[0172] Diagnostic blood tests include deranged liver function tests
(gamma-glutamyl transferase and alkaline phosphatase) and the
presence of particular antibodies (antimitochondrial antibody (AMA)
an antinuclear antibody (ANA)). Antinuclear antibodies are believed
to be prognostic indicators of PBC. When other tests and procedures
are indicative of PBC, a liver biopsy is frequently performed to
confirm disease. Endoscopic retrograde cholangiopancreatography
(ERCP), an endoscopic evaluation of the bile duct, may also be
employed to confirm disease.
[0173] PBC is classified into four stages marking the progression
of disease. Stage 1 (Portal Stage) is characterized by portal
inflammation and mild bile duct damage; Stage 2 (Periportal Stage)
is characterized by enlarged triads, periportal fibrosis or
inflammation; Stage 3 (Septal Stage) is characterized by active
and/or passive fibrous septa; and Stage 4 (Biliary Cirrhosis) is
characterized by the presence of hepatic nodules. Liver biopsy is
required to determine the stage of disease.
[0174] Serum bilirubin is an indicator of PBC progression and
prognosis. Patients with a serum bilirubin level of 2-6 mg/dL have
a mean survival time of 4.1 years, patients with a serum bilirubin
level of 6-10 mg/dL have a mean survival time of 2.1 years, and
patients with a serum bilirubin level above 10 mg/dL have a mean
survival time of 1.4 years. Liver transplantation is an option in
advanced cases of PBC, although the recurrence rate may be as high
as 18% at 5 years, and up to 30% at 10 years.
[0175] Although disease progression may be slowed, pharmaceutical
intervention with currently used therapies is neither curative nor
effective in all patient populations. In order to improve the
therapeutic outcome of pharmacological therapy, one aspect pertains
to the use of one or more current therapies in combination with
variants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21
peptide sequences and variants of fusions (chimeras) of FGF19
and/or FGF21 peptide sequences having one or more activities
associated with the treatment and/or prevention of PBC and
associated diseases, disorders and conditions. The most commonly
used and/or promising agents for combination therapy are set forth
hereafter, although it is to be understood that these agents are
illustrative, and not exclusionary.
[0176] PBC treatment most frequently involves the bile acid
ursodeoxycholic acid (Urosdiol, UDCA). UDCA therapy is helpful in
reducing the cholestasis and improving the liver function tests in
PBC patients; however, it does not demonstrably improve symptoms
and has a questionable impact on prognosis. UDCA has been shown to
reduce mortality, adverse events and the need for transplantation
in PBC. Although UDCA is considered the first-line therapy,
approximately one-third of patients may be non-responsive and
remain at risk of progressive liver disease and are candidates for
alternative or additive therapy.
[0177] There are several alternative and adjuvant therapies, some
of which are currently in clinical development, that can be used in
combination with variants of FGF19 peptide sequences, fusions of
FGF19 and/or FGF21 peptide sequences and variants of fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences having one or
more activities associated with the treatment and/or prevention of
PBC and associated diseases, disorders and conditions.
[0178] Farnesoid-X-receptor agonists represent a promising class of
agents that may be used in combination therapy. One of the primary
functions of agonists of FXR, a nuclear receptor expressed at high
levels in the liver and intestine, is the suppression of
cholesterol 7.alpha. hydroxylase-1 (CYP7A1), the rate-limiting
enzyme in the synthesis of bile acids from cholesterol. Obeticholic
acid (OCA; Intercept Pharmaceuticals, NY) is a bile acid analog and
FXR agonist derived from the primary human bile acid
chenodeoxycholic acid, or CDCA. OCA is currently being evaluated
for patients having an inadequate therapeutic response to ursodiol
or who are unable to tolerate ursodiol.
[0179] Inhibitors of the apical sodium-dependent bile acid
transporter (ASBT) represent another class of agents that may be
used in combination with the variants of FGF19 peptide sequences,
fusions of FGF19 and/or FGF21 peptide sequences and variants of
fusions (chimeras) of FGF19 and/or FGF21 peptide sequences
described herein for the treatment and/or prevention of PBC and
associated diseases. ASBT, a member of the sodium/bile-salt
co-transport family coded by gene SLC10A2, is currently thought to
be the primary mechanism for bile acid reabsorption in the
intestine. Examples of ABST inhibitors include LUM001 and SC-435,
both of which are being developed by Lumena Pharmaceuticals (San
Diego, Calif.).
[0180] Bile acid sequestrants also find use in the treatment of
PBC. Cholestyramine and colestipol are the best known bile acid
sequestrants. However, their use is sometimes limited because they
are only available in powder form and are not tolerated by many
patients, often because of the poor texture and taste of the resin
powder. The bile acid sequestrant colesevelam is available in
tablet form and is often better tolerated. All bile acid
sequestrants are capable of binding other compounds, including the
fat-soluble vitamins A, D, E and K, and deficiencies of these
vitamins many necessitate supplementation. Importantly, the PBC
patient population inherently has poor lipid-dependent absorption
of vitamins A, D, E and K, and thus patients taking bile acid
sequestrants are at particular risk for deficiency of those
vitamins.
[0181] Agents associated with immune and inflammatory function are
candidates for combination therapy with the variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of PBC and associated diseases,
disorders and conditions.
[0182] The interleukin IL-12 is linked with autoimmunity. Data
indicate that the IL-12 signaling pathway plays a key role in the
effector mechanisms that lead to biliary destruction. Targeting the
p40 subunit of IL-12 has also been shown to ameliorate experimental
immune-mediated cholangiopathy. Thus, anti-IL-12 agents (e.g.,
monoclonal Ab inhibitors) provide a promising treatment.
Furthermore, because polymorphisms in CD80 have been identified as
conferring an increased susceptibility to PBC, blockade of
co-stimulation between T cells and antigen-presenting cells through
CD80 by use of an anti-CD80 agent could represent an important
therapeutic approach for the treatment of PBC. In addition,
improvement in IgM titre and an increase in intrahepatic regulatory
T-cell number using the anti-CD20 antibody rituximab (RITUXAN) have
shown promise.
[0183] The immune-mediated destruction of small-sized bile ducts in
PBC is predominantly cell-mediated, characterized by Th1 cells,
CD8+ T cells, NK cells and NKT cells which express CXCR3.
Therefore, neutralizing antibodies to CXCL10, a ligand for CXCR3,
may offer the possibility to interfere with one of the key
inflammatory processes and contribute to immune-mediated biliary
destruction in PBC. Similarly, blockade of co-stimulatory signals
between T cells expressing CD28 and antigen-presenting cells
expressing CD80 (e.g. cholangiocytes, antibody-secreting B cells)
might represent an important approach for the treatment of
autoimmune diseases.
[0184] The variants of FGF19 peptide sequences, fusions of FGF19
and/or FGF21 peptide sequences and variants of fusions (chimeras)
of FGF19 and/or FGF21 peptide sequences described herein can be
used in combination with other agents for the treatment and/or
prevention of those bile acid-related disorders referenced herein
that have an immune and/or inflammatory component, including, but
not limited to, PBC and associated diseases, disorders and
conditions. Examples of such other agents include, for example,
non-steroidal anti-inflammatory drugs (NSAID); steroids; cytokine
suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to, or
antagonists of, other human cytokines or growth factors (e.g.,
IL-2, IL-6, or PDGF); TNF antagonists (e.g., agents such as
REMICADE, p75TNFRIgG (ENBREL) or p55TNFR1gG (LENERCEPT));
interferon-.beta.1a (AVONEX); interferon-.beta.1b (BETASERON); and
immune checkpoint inhibitors, including PD1 (associated agents
include the antibodies nivolumab and lambrolizumab), PDL1, BTLA,
CTLA4 (associated agents include the fully humanized CTLA4
monoclonal antibody ipilimumab (YERVOY), TIM3, LAGS, and A2aR.
[0185] Fibrates have been shown to improve various aspects of PBC,
including liver function tests, both as monotherapy and in
combination with UDCA non-responders. In certain embodiments, a
fibrate is a member selected from the group of bezafibrate
(BEZALIP), ciprofibrate (MODALIM), gemfibrozil (LOPID), clofibrate,
and fenofibrate (TRICOR). Fish oil has exhibited similar
benefits.
[0186] In PBC patients demonstrating certain characteristics of
hepatitis on biopsy, corticosteroids such as budesonide may improve
liver histology and biochemistry, particularly when used in
combination with UDCA. Colchicine has been shown to improve liver
function tests (e.g., AST and ALP) and represents another
alternative treatment for PBC.
[0187] Though not an exhaustive list, other drugs that have shown
promise include methotrexate as an immunomodulatory treatment,
azathioprine, cyclosporine, and certain agents used in
anti-retroviral therapy (e.g., combivir).
[0188] Various treatments exist for the sequelae associated with
PBC. For example, itching can be relieved by the bile acid
sequestrant cholestyramine, or alternatively naltrexone and
rifampicin. The fatigue associated with PBC may effectively be
treated with modafinil (Provigil; Teva (formerly Cephalon)) without
damaging the liver. As patients with PBC have increased risk of
developing osteoporosis and esophageal varices compared to the
general population (and others with liver disease), screening and
treatment of these complications is an important part of the
management of PBC. Combination therapy with the variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of PBC and associated diseases,
disorders and conditions, as taught herein, offer novel, promising
alternatives to the management of such sequelae.
[0189] NASH and NAFLD and Combination Therapy with Agents Effective
in the Treatment or Prevention Thereof. Non-alcoholic
steatohepatitis (NASH), considered part of a spectrum of
non-alcoholic fatty liver diseases (NAFLD), causes inflammation and
accumulation of fat and fibrous tissue in the liver. Although the
exact cause of NASH is unknown, risk factors include central
obesity, type-2 diabetes mellitus, insulin resistance (IR) and
dyslipidemia; combinations of the foregoing are frequently
described as the metabolic syndrome. In addition, certain drugs
have been linked to NASH, including tamoxifen, amiodarone and
steroids (e.g., prednisone and hydrocortisone). Non-alcoholic fatty
liver disease is the most common cause of chronic liver disease in
the United States, and the estimated prevalence of NAFLD is 20-30%
and for NASH it is estimated at 3.5-5%. (See, e.g., Abrams, G.A.,
et al., Hepatology, 2004. 40(2):475-83; Moreira, R. K., Arch Pathol
Lab Med, 2007. 131(11):1728-34).
[0190] NASH frequently presents with no overt symptoms,
complicating its diagnosis. Liver function tests generally begin
the diagnostic process, with levels of AST (aspartate
aminotransferase) and ALT (alanine aminotransferase) elevated in
about 90% percent of individuals with NASH. Other blood tests are
often used for ruling out other causes of liver disease, such as
hepatitis. Imaging tests (e.g., ultrasound, CT scan, or MRI) may
reveal fat accumulation in the liver but frequently cannot
differentiate NASH from other causes of liver disease that have a
similar appearance. A liver biopsy is required to confirm NASH.
[0191] The prognosis for individuals suffering from NASH is
difficult to predict, although features in the liver biopsy can be
helpful. The most serious complication of NASH is cirrhosis, which
occurs when the liver becomes severely scarred. It has been
reported that between 8 and 26 percent of individuals with NASH
develop cirrhosis, and it is predicted that NASH will be the
leading indication for liver transplantation by 2020.
[0192] At the present time, treatment of NASH focuses primarily on
pharmacological and non-pharmacological management of those medical
conditions associated with it, including hyperlipidemia, diabetes
and obesity. Although not curative, pharmacological intervention of
NASH itself includes treatment with vitamin E, pioglitazone,
metformin, statins, omega-3 fatty acids, and ursodeoxycholic acid
(UDCA (ursodiol)). Other agents being evaluated, currently approved
for different indications, include losartan and telisartan,
exenatide, GLP-1 agonists, DPP IV inhibitors, and
carbamazepine.
[0193] In view of the deficiencies of the aforementioned current
therapies, combination therapy with agents having distinct
mechanisms of action offers a promising new avenue for the
treatment and prevention of NASH and NAFLD. Addressing such
deficiencies is also contemplated, for example, by using the
current therapies in combination with the variants of FGF19 peptide
sequences, fusions of FGF19 and/or FGF21 peptide sequences and
variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences as taught herein. Also provided herein is the
prophylactic and/or therapeutic use of these variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences in combination with therapies developed in the future for
the treatment or prevention of NASH and NAFLD.
[0194] Combination Therapy for the Treatment or Prevention of Other
Bile acid-related Disorders and Associated Diseses, Disorders and
Conditions. Also provided herein is the use of variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of other bile acid-related disorders
and associated diseases, disorders and conditions besides PBC, in
combination with other therapeutic agents and/or treatment
modalities.
[0195] By way of example, patients with bile acid diarrhea
secondary to Crohn's ileitis will be helped with glucocorticoid
treatment. Microscopic colitis is also helped by steroids. In
patients with a short-bowel syndrome (a bile acid deficiency occurs
in the proximal intestine that leads to impaired micellar
solubilization), cholylsarcosine (cholyl-N-methylglycine), a
synthetic bile acid analogue, has been shown to increase lipid
absorption.
[0196] Administration of the primary bile acid chenodeoxycholic
Acid (CDCA) has been shown to decrease biliary cholesterol
secretion and gradual dissolution of gallstones. Because CDCA is
slightly hepatotoxic, it was gradually replaced by UDCA. Despite
the efficacy and safety of UDCA administration for cholesterol
gallstone dissolution, it is not frequently used today because of
the success of laparoscopic cholecystectomy, which provides a rapid
cure for symptomatic disease. Medical therapy, in contrast,
requires months of therapy, does not always dissolve stones, and is
followed by gradual recurrence in some patients.
[0197] Bile acid replacement is used in inborn errors of bile acid
biosynthesis, usually with a mixture of chenodeoxycholic Acid
(CDCA) or Ursodeoxycholic Acid (UDCA) and cholic acid, to suppress
the synthesis of cytotoxic bile acid precursors and restore the
input of primary bile acids into the enterohepatic circulation.
[0198] In addition to the agents and therapeutic modalities set
forth above, combination therapy with numerous additional agents
(and classes thereof) is also contemplated, including. but not
limited to, 1) insulin e.g., bolus and basal analogs), insulin
mimetics and agents that entail stimulation of insulin secretion,
including sulfonylureas (e.g., chlorpropamide, tolazamide,
acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and
meglitinides (e.g., repaglinide (PRANDIN) and nateglinide
(STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE)) and other
agents that act by promoting glucose utilization, reducing hepatic
glucose production and/or diminishing intestinal glucose output; 3)
alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and
other agents that slow down carbohydrate digestion and consequently
absorption from the gut and reduce postprandial hyperglycemia; 4)
thiazolidinediones (e.g., rosiglitazone (AVANDIA), troglitazone
(REZULIN), pioglitazone (ACTOS), glipizide, balaglitazone,
rivoglitazone, netoglitazone, troglitazone, englitazone,
ciglitazone, adaglitazone, darglitazone that enhance insulin action
(e.g., by insulin sensitization), thus promoting glucose
utilization in peripheral tissues; 5) glucagon-like-peptides
including DPP-IV inhibitors (e.g., vildagliptin (GALVUS) and
sitagliptin (JANUVIA)) and Glucagon-Like Peptide-1 (GLP-1) and
GLP-1 agonists and analogs (e.g., exenatide (BYETTA and ITCA 650
(an osmotic pump inserted subcutaneously that delivers an exenatide
analog over a 12-month period; Intarcia, Boston, MA)); 6) and
DPP-IV-resistant analogues (incretin mimetics), PPAR gamma
agonists, dual-acting PPAR agonists, pan-acting PPAR agonists,
PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXR
agonists, glycogen synthase kinase-3 inhibitors, immune modulators,
beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and
amylin analogues.
[0199] Other exemplary agents that can be used, in certain
embodiments, in combination with the chimeric peptides and methods
provided herein include dipeptidyl peptidase-4 (DPP-4) inhibitors,
bromocriptine formulations (e.g. and bile acid sequestrants (e.g.,
colesevelam), and SGLT-2 inhibitors. Appetite suppression drugs are
also well known and can be used in combination with the
compositions and methods provided herein. Supplementary therapies
can be administered prior to, contemporaneously with or following
methods and uses provided herein.
Dosing and Administration
[0200] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Table
1), may be formulated in a unit dose or unit dosage form. In a
particular embodiment, a peptide sequence is in an amount effective
to treat a subject in need of treatment, e.g., due to abnormal or
aberrant bile acid homeostasis, such as metabolic syndrome; a
lipid- or glucose-related disorder; cholesterol or triglyceride
metabolism; type 2 diabetes; cholestasis, including, for example
diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,
neonatal cholestasis, and drug induced cholestasis (e.g.,
estrogen)), and diseases of extrahepatic cholestasis (e.g., bile
cut compression from tumor, bile duct blockade by gall stones);
bile acid malabsorption and other disorders involving the distal
small intestine, including ileal resection, inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), disorders
impairing absorption of bile acids not otherwise characterized
(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and
GI, liver, and/or biliary cancers (e.g., colon cancer and
hepatocellular cancer); and/or bile acid synthesis abnormalities,
such as those contributing to NASH, cirrhosis and portal
hypertension. Exemplary unit doses range from about 25-250,
250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,
25,000-50,000 ng; from about 25-250, 250-500, 500-1000, 1000-2500
or 2500-5000, 5000-25,000, 25,000-50,000 .mu.g; and from about
25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,
25,000-50,000 mg.
[0201] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Table
1) can be administered to provide the intended effect as a single
dose or multiple dosages, for example, in an effective or
sufficient amount. Exemplary doses range from about 25-250,
250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,
25,000-50,000 pg/kg; from about 50-500, 500-5000, 5000-25,000 or
25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000,
1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000 pg/kg. Single or
multiple doses can be administered, for example, multiple times per
day, on consecutive days, alternating days, weekly or
intermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6,
7 or 8 weeks, or once every 2, 3, 4, 5 or 6 months).
[0202] Peptide sequences provided herein including subsequences,
variants and modified forms of the exemplified peptide sequences
(e.g., sequences listed in the Sequence Listing or Table 1) can be
administered and methods may be practiced via systemic, regional or
local administration, by any route. For example, a peptide sequence
can be administered parenterally (e.g., subcutaneously,
intravenously, intramuscularly, or intraperitoneally), orally
(e.g., ingestion, buccal, or sublingual), inhalation,
intradermally, intracavity, intracranially, transdermally
(topical), transmucosally or rectally. Peptide sequences provided
herein including subsequences, variants and modified forms of the
exemplified peptide sequences (e.g., sequences listed in the
Sequence Listing or Table 1) and methods provided herein including
pharmaceutical compositions can be administered via a
(micro)encapsulated delivery system or packaged into an implant for
administration.
[0203] A particular non-limiting example of parenteral (e.g.,
subcutaneous) administration entails the use of Intarcia's
subcutaneous delivery system (Intarcia Therapeutics, Inc.; Hayward,
Calif.). The system comprises a miniature osmotic pump that
delivers a consistent amount of a therapeutic agent over a desired
period of time. In addition to maintaining drug levels within an
appropriate therapeutic range, the system can be used with
formulations that maintain the stability of proteinaceous
therapeutic agents at human body temperature for extended periods
of time.
Compositions
[0204] Also provided herein are "pharmaceutical compositions,"
which include a peptide sequence (or sequences) provided herein,
including subsequences, variants and modified forms of the
exemplified peptide sequences (e.g., sequences listed in the
Sequence Listing or Table 1), and one or more pharmaceutically
acceptable or physiologically acceptable diluents, carriers or
excipients; in combination with, or separate from, one or more
additional agents for the treatment of a bile acid-related disease,
disorder or condition, or a composition comprising such one or more
additional agents and one or more pharmaceutically acceptable or
physiologically acceptable diluents, carriers or excipients. In
particular embodiments, a peptide sequence or sequences and an
additional agent(s) are present in a therapeutically acceptable
amount. The pharmaceutical compositions may be used in accordance
with the methods and uses provided herein. Thus, for example, the
pharmaceutical compositions can be administered ex vivo or in vivo
to a subject in order to practice treatment methods and uses
provided herein. Pharmaceutical compositions provided herein can be
formulated to be compatible with the intended method or route of
administration; exemplary routes of administration are set forth
herein.
[0205] In some aspects, the pharmaceutical compositions may further
comprise other therapeutically active agents or compounds disclosed
herein (e.g., bile acid stabilizing agents or drugs) or known to
the skilled artisan which can be used in the treatment or
prevention of various bile acid diseases and disorders as set forth
herein. As set forth above, the additional therapeutically active
agents or compounds may be present in a separate pharmaceutical
composition(s). Exemplary dosing parameters and regimens are
described herein.
[0206] Pharmaceutical compositions typically comprise a
therapeutically effective amount of at least one of the peptide
sequences provided herein, including subsequences, variants and
modified forms of the exemplified peptide sequences (e.g.,
sequences listed in the Sequence Listing or Table 1) and/or one or
more additional agents described herein, and one or more
pharmaceutically and physiologically acceptable formulation agents.
Suitable pharmaceutically acceptable or physiologically acceptable
diluents, carriers or excipients include, but are not limited to,
antioxidants (e.g., ascorbic acid and sodium bisulfate),
preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or
n-propyl, p-hydroxybenzoate), emulsifying agents, suspending
agents, dispersing agents, solvents, fillers, bulking agents,
buffers, vehicles, diluents, and/or adjuvants. For example, a
suitable vehicle may be physiological saline solution or citrate
buffered saline, possibly supplemented with other materials common
in pharmaceutical compositions for parenteral administration.
Neutral buffered saline or saline mixed with serum albumin are
further exemplary vehicles. Those skilled in the art will readily
recognize a variety of buffers that could be used in the
pharmaceutical compositions and dosage forms used herein. Typical
buffers include, but are not limited to pharmaceutically acceptable
weak acids, weak bases, or mixtures thereof. Buffer components also
include water soluble materials such as phosphoric acid, tartaric
acids, lactic acid, succinic acid, citric acid, acetic acid,
ascorbic acid, aspartic acid, glutamic acid, and salts thereof.
[0207] A primary solvent in a vehicle may be either aqueous or
non-aqueous in nature. In addition, the vehicle may contain other
pharmaceutically acceptable excipients for modifying or maintaining
the pH, osmolarity, viscosity, sterility or stability of the
pharmaceutical composition. In certain embodiments, the
pharmaceutically acceptable vehicle is an aqueous buffer. In other
embodiments, a vehicle comprises, for example, sodium chloride
and/or sodium citrate.
[0208] Pharmaceutical compositions provided herein may contain
still other pharmaceutically-acceptable formulation agents for
modifying or maintaining the rate of release of a peptide and/or an
additional agent, as described herein. Such formulation agents
include those substances known to artisans skilled in preparing
sustained-release formulations. For further reference pertaining to
pharmaceutically and physiologically acceptable formulation agents,
see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, The
Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse,
N.J.); and Pharmaceutical Principles of Solid Dosage Forms (1993,
Technonic Publishing Co., Inc., Lancaster, Pa.). Additional
pharmaceutical compositions appropriate for administration are
known in the art and are applicable in the methods and compositions
provided herein.
[0209] A pharmaceutical composition may be stored in a sterile vial
as a solution, suspension, gel, emulsion, solid, or dehydrated or
lyophilized powder. Such compositions may be stored either in a
ready to use form, a lyophilized form requiring reconstitution
prior to use, a liquid form requiring dilution prior to use, or
other acceptable form. In some embodiments, a pharmaceutical
composition is provided in a single-use container (e.g., a
single-use vial, ampoule, syringe, or autoinjector (similar to,
e.g., an EpiPen.RTM.)), whereas a multi-use container (e.g., a
multi-use vial) is provided in other embodiments. Any drug delivery
apparatus may be used to deliver peptides and the other agents
described herein, including implants (e.g., implantable pumps) and
catheter systems, both of which are known to the skilled artisan.
Depot injections, which are generally administered subcutaneously
or intramuscularly, may also be utilized to release peptides and/or
other agents described herein over a defined period of time. Depot
injections are usually either solid- or oil-based and generally
comprise at least one of the formulation components set forth
herein. The skilled artisan is familiar with possible formulations
and uses of depot injections.
[0210] A pharmaceutical composition can be formulated to be
compatible with its intended route of administration. Thus,
pharmaceutical compositions include carriers, diluents, or
excipients suitable for administration by routes including
parenteral (e.g., subcutaneous (s.c.), intravenous, intramuscular,
or intraperitoneal), intradermal, oral (e.g., ingestion),
inhalation, intracavity, intracranial, and transdermal
(topical).
[0211] Pharmaceutical compositions may be in the form of a sterile
injectable aqueous or oleagenous suspension. This suspension may be
formulated using suitable dispersing or wetting agents and
suspending agents disclosed herein or known to the skilled artisan.
The sterile injectable preparation may also be a sterile injectable
solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent, for example, as a solution in 1,3-butane diol.
Acceptable diluents, solvents and dispersion media that may be
employed include water, Ringer's solution, isotonic sodium chloride
solution, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate
buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene
glycol, and liquid polyethylene glycol), and suitable mixtures
thereof. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil may be employed, including synthetic mono- or
diglycerides. Moreover, fatty acids such as oleic acid find use in
the preparation of injectables. Prolonged absorption of particular
injectable formulations can be achieved by including an agent that
delays absorption (e.g., aluminum monostearate or gelatin).
[0212] Pharmaceutical compositions may be in a form suitable for
oral use, for example, as tablets, capsules, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules,
emulsions, hard or soft capsules, or syrups, solutions, microbeads
or elixirs. Pharmaceutical compositions intended for oral use may
be prepared according to any method known to the art for the
manufacture of pharmaceutical compositions. Such compositions may
contain one or more agents such as sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets
containing a peptide provided herein may be in admixture with
non-toxic pharmaceutically acceptable excipients suitable for the
manufacture of tablets. These excipients include, for example,
diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, corn starch, or alginic acid;
binding agents, for example starch, gelatin or acacia, and
lubricating agents, for example magnesium stearate, stearic acid or
talc.
[0213] Tablets, capsules and the like suitable for oral
administration may be uncoated or they may be coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed. They
may also be coated by techniques known in the art to form osmotic
therapeutic tablets for controlled release. Additional agents
include biodegradable or biocompatible particles or a polymeric
substance such as polyesters, polyamine acids, hydrogel, polyvinyl
pyrrolidone, polyanhydrides, polyglycolic acid,
ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,
protamine sulfate, or lactide/glycolide copolymers,
polylactide/glycolide copolymers, or ethylenevinylacetate
copolymers in order to control delivery of an administered
composition. For example, the oral agent can be entrapped in
microcapsules prepared by coacervation techniques or by interfacial
polymerization, by the use of hydroxymethylcellulose or
gelatin-microcapsules or poly (methylmethacrolate) microcapsules,
respectively, or in a colloid drug delivery system. Colloidal
dispersion systems include macromolecule complexes, nano-capsules,
microspheres, microbeads, and lipid-based systems, including
oil-in-water emulsions, micelles, mixed micelles, and liposomes.
Methods of preparing liposomes are described in, for example, U.S.
Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for the
preparation of the above-mentioned formulations will be apparent to
those skilled in the art.
[0214] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate, kaolin or microcrystalline cellulose, or as soft gelatin
capsules wherein the active ingredient is mixed with water or an
oil medium, for example peanut oil, liquid paraffin, or olive
oil.
[0215] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture thereof.
Such excipients are suspending agents, for example sodium
carboxymethylcellulose, methylcellulose,
hydroxy-propylmethylcellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxy-ethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyleneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives.
[0216] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation.
[0217] Dispersible powders and granules suitable for preparation of
an aqueous suspension by addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified herein.
[0218] Pharmaceutical compositions provided herein may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example, liquid paraffin, or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example,
gum acacia or gum tragacanth; naturally-occurring phosphatides, for
example, soy bean, lecithin, and esters or partial esters derived
from fatty acids; hexitol anhydrides, for example, sorbitan
monooleate; and condensation products of partial esters with
ethylene oxide, for example, polyoxyethylene sorbitan
monooleate.
[0219] Pharmaceutical compositions can also include carriers to
protect the composition against rapid degradation or elimination
from the body, such as a controlled release formulation, including
implants, liposomes, hydrogels, prodrugs and microencapsulated
delivery systems. For example, a time delay material such as
glyceryl monostearate or glyceryl stearate alone, or in combination
with a wax, may be employed. Prolonged absorption of injectable
pharmaceutical compositions can be achieved by including an agent
that delays absorption, for example, aluminum monostearate or
gelatin. Prevention of the action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like.
[0220] Also provided herein are peptides and/or one or more
additional agents described herein in the form of suppositories for
rectal administration. The suppositories can be prepared by mixing
a peptide and/or one or more additional agents described herein
with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include, but are not limited to, cocoa butter and polyethylene
glycols.
Methods of Identifying Modulators of Bile Acid-Related
Disorders
[0221] Also provided herein are methods of identifying a peptide
(or a subsequence, variant or modified form as set forth herein)
that modulates bile acid homeostasis without having substantial HCC
activity. In one embodiment, a method includes: providing a
candidate peptide sequence; administering the candidate peptide
sequence to a test animal; measuring bile acid levels of the animal
after administration of the candidate peptide sequence, to
determine if the candidate peptide sequence favorably modulates
bile acid homeostasis; and analyzing the candidate peptide sequence
for induction of HCC in the animal, or expression of a marker
correlating with HCC activity. A candidate peptide that modulates
bile acid homeostasis but does not have substantial HCC activity
thereby identifies a peptide sequence that modulates bile acid
homeostasis without substantial HCC activity.
[0222] The terms "assaying" and "measuring" and grammatical
variations thereof are used interchangeably herein and refer to
either qualitative or quantitative determinations, or both
qualitative and quantitative determinations. When the terms are
used in reference to detection, any means of assessing the relative
amount is contemplated, including the various methods set forth
herein and known in the art. For example, bile acids and
precursors, such as 7 alpha-hydroxy-4-cholesten-3-one, can be
assayed or measured in a sample (e.g., serum) from a subject.
Another non-limiting examples is a two reaction method (Randox
Laboratories, Ltd.) using serum or heparinized plasma. In the first
reaction bile acids are oxidized by 3-.alpha.-hydroxysteroid
dehydrogenase with the subsequent reduction of Thio-NAD to
Thio-NADH. In the second reaction, oxidized bile acids are reduced
by the same enzyme with the subsequent oxidation of NADH to NAD.
The rate of formation of Thio-NADH is determined by measuring the
specific absorbance change at 405 nm.
[0223] Risk factors for HCC, the most common type of liver cancer,
include type 2 diabetes (probably exacerbated by obesity). The risk
of HCC in type 2 diabetics is greater (from .about.2.5 to .about.7
times the non-diabetic risk) depending on the duration of diabetes
and treatment protocol.
[0224] Various methodologies can be used in the screening and
diagnosis of HCC and are well known to the skilled artisan.
Indicators for HCC include detection of a tumor maker such as
elevated alpha-fetoprotein (AFP) or des-gamma carboxyprothrombin
(DCP) levels. A number of different scanning and imaging techniques
are also helpful, including ultrasound, CT scans and Mill. In
certain embodiments, evaluation of whether a peptide (e.g., a
candidate peptide) exhibits evidence of inducing HCC may be
determined in vivo by, for example, quantifying HCC nodule
formation in an animal model, such as db/db mice, administered a
peptide, compared to HCC nodule formation by wild type FGF19.
Macroscopically, liver cancer may be nodular, where the tumor
nodules (which are round-to-oval, grey or green, well circumscribed
but not encapsulated) appear as either one large mass or multiple
smaller masses. Alternatively, HCC may be present as an
infiltrative tumor which is diffuse and poorly circumscribed and
frequently infiltrates the portal veins.
[0225] Pathological assessment of hepatic tissue samples is
generally performed after the results of one or more of the
aforementioned techniques indicate the likely presence of HCC.
Thus, methods provided herein may further include assessing a
hepatic tissue sample from an in vivo animal model (e.g., a db/db
mouse) useful in HCC studies in order to determine whether a
peptide sequence exhibits evidence of inducing HCC. By microscopic
assessment, a pathologist can determine whether one of the four
general architectural and cytological types (patterns) of HCC are
present (i.e., fibrolamellar, pseudoglandular (adenoid),
pleomorphic (giant cell) and clear cell).
[0226] It is to be understood that the techniques, assays and the
like described in this section are applicable to identifying an
additional agent described herein having desired properties and/or
characteristics. Moreover, the techniques, assays and the like
described in this section are applicable to identifying a peptide
in combination with an additional agent described herein, for
example, a composition comprising a peptide in combination with an
additional agent described herein that has at least one favorable
characteristic; or a treatment regimen comprising a peptide
provided herein in combination with an additional agent described
herein that has at least one favorable characteristic.
Antibodies
[0227] Also provided herein is the generation and use of
antibodies, and fragments thereof, that bind the peptide sequences
provided herein, including subsequences, sequence variants and
modified forms of the exemplified peptide sequences (including the
peptides listed in the Sequence Listing or Table 1), and/or one or
more additional agents as described herein.
[0228] As used herein, the terms "antibodies" (Abs) and
"immunoglobulins" (Igs) refer to glycoproteins having the same
structural characteristics. While antibodies exhibit binding
specificity to an antigen, immunoglobulins include both antibodies
and other antibody-like molecules which may lack antigen
specificity.
[0229] The term "antibody" includes intact monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies) formed from at least two intact antibodies, and
antibody binding fragments including Fab and F(ab)'.sub.2, provided
that they exhibit the desired biological activity. The basic
antibody structural unit comprises a tetramer, and each tetramer is
composed of two identical pairs of polypeptide chains, each pair
having one "light" chain (about 25 kDa) and one "heavy" chain
(about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. In contrast, the
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa and lambda light chains, whereas human heavy
chains are classified as mu, delta, gamma, alpha, or epsilon, and
define the antibody's isotype as IgM, IgD, IgA, and IgE,
respectively. Binding fragments are produced by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
antibodies. Binding fragments include Fab, Fab', F(ab').sub.2, Fv,
and single-chain antibodies.
[0230] Each heavy chain has at one end a variable domain (VH)
followed by a number of constant domains. Each light chain has a
variable domain at one end (VL) and a constant domain at its other
end; the constant domain of the light chain is aligned with the
first constant domain of the heavy chain, and the light chain
variable domain is aligned with the variable domain of the heavy
chain. Within light and heavy chains, the variable and constant
regions are joined by a "J" region of about 12 or more amino acids,
with the heavy chain also including a "D" region of about 10 more
amino acids. The antibody chains all exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hyper-variable regions, also called
complementarity-determining regions or CDRs. The CDRs from the two
chains of each pair are aligned by the framework regions, enabling
binding to a specific epitope. From N-terminal to C-terminal, both
light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,
FR3, CDR3 and FR4.
[0231] An intact antibody has two binding sites and, except in
bifunctional or bispecific antibodies, the two binding sites are
the same. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments.
[0232] As used herein, the term "monoclonal antibody" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, that is, the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. In contrast to polyclonal antibody preparations
which include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen.
[0233] A "neutralizing antibody" is an antibody molecule that is
able to eliminate or significantly reduce an effector function of a
target antigen to which it binds.
[0234] Antibody binding fragments may be produced by enzymatic or
chemical cleavage of intact antibodies. Digestion of antibodies
with the enzyme papain results in two identical antigen-binding
fragments, also known as "Fab" fragments, and an "Fc" fragment
which has no antigen-binding activity. Digestion of antibodies with
the enzyme pepsin results in a F(ab').sub.2 fragment in which the
two arms of the antibody molecule remain linked and comprise
two-antigen binding sites. The F(ab').sub.2 fragment has the
ability to crosslink antigen.
[0235] The term "Fab" refers to a fragment of an antibody that
comprises the constant domain of the light chain and the CH1 domain
of the heavy chain. The term "Fv" when used herein refers to the
minimum fragment of an antibody that retains both
antigen-recognition and antigen-binding sites. In a two-chain Fv
species, this region consists of a dimer of one heavy-chain and one
light-chain variable domain in non-covalent association. In a
single-chain Fv species, one heavy-chain and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three CDRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. While the six CDRs, collectively, confer
antigen-binding specificity to the antibody, even a single variable
domain (or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen.
[0236] The terms "complementarity determining regions" or "CDRs"
refer to parts of immunological receptors that make contact with a
specific ligand and determine its specificity. The term
"hypervariable region" refers to the amino acid residues of an
antibody which are responsible for antigen-binding. The
hypervariable region generally comprises amino acid residues from a
"complementarity determining region" or "CDR" and/or those residues
from a "hypervariable loop".
[0237] As used herein, the term "epitope" refers to binding sites
for antibodies on protein antigens. Epitopic determinants usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains, as well as specific three
dimensional structural and charge characteristics. An antibody is
said to bind an antigen when the dissociation constant is .ltoreq.1
.mu.M, such as .ltoreq.100 nM or .ltoreq.10 nM. An increased
equilibrium constant ("KD") means that there is less affinity
between the epitope and the antibody, whereas a decreased
equilibrium constant means that there is a higher affinity between
the epitope and the antibody. An antibody with a K.sub.D of "no
more than" a certain amount means that the antibody will bind to
the epitope with the given K.sub.D or more strongly. Whereas
K.sub.D describes the binding characteristics of an epitope and an
antibody, "potency" describes the effectiveness of the antibody
itself for a function of the antibody. There is not necessarily a
correlation between an equilibrium constant and potency; thus, for
example, a relatively low K.sub.D does not automatically mean a
high potency.
[0238] The term "selectively binds" in reference to an antibody
does not mean that the antibody only binds to a single substance,
but rather that the K.sub.D of the antibody to a first substance is
less than the K.sub.D of the antibody to a second substance. An
antibody that exclusively binds to an epitope only binds to that
single epitope.
[0239] When administered to humans, antibodies that contain rodent
(murine or rat) variable and/or constant regions are sometimes
associated with, for example, rapid clearance from the body or the
generation of an immune response by the body against the antibody.
In order to avoid the utilization of rodent-derived antibodies,
fully human antibodies can be generated through the introduction of
human antibody function into a rodent so that the rodent produces
fully human antibodies. Unless specifically identified herein,
"human" and "fully human" antibodies can be used interchangeably
herein. The term "fully human" can be useful when distinguishing
antibodies that are only partially human from those that are
completely, or fully human. The skilled artisan is aware of various
methods of generating fully human antibodies.
[0240] In order to address possible human anti-mouse antibody
responses, chimeric or otherwise humanized antibodies can be
utilized. Chimeric antibodies have a human constant region and a
murine variable region, and, as such, human anti-chimeric antibody
responses may be observed in some patients. Therefore, it is
advantageous to provide fully human antibodies against multimeric
enzymes in order to avoid possible human anti-mouse antibody or
human anti-chimeric antibody responses.
[0241] Fully human monoclonal antibodies can be prepared, for
example, by the generation of hybridoma cell lines by techniques
known to the skilled artisan. Other preparation methods involve the
use of sequences encoding particular antibodies for transformation
of a suitable mammalian host cell, such as a CHO cell.
Transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example, packaging
the polynucleotide in a virus (or into a viral vector) and
transducing a host cell with the virus (or vector) or by
transfection procedures known in the art. Methods for introducing
heterologous polynucleotides into mammalian cells are well known in
the art and include dextran-mediated transfection, calcium
phosphate precipitation, polybrene-mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei. Mammalian cell lines available as hosts for
expression are well known in the art and include, but are not
limited to CHO cells, HeLa cells, and human hepatocellular
carcinoma cells.
[0242] Antibodies can be used diagnostically and/or
therapeutically. For example, the antibodies can be used as a
diagnostic by detecting the level of one or more peptides provided
herein in a subject, and either comparing the detected level to
standard control level or to a baseline level in a subject
determined previously (e.g., prior to any illness). The antibodies
can be used as a therapeutic to modulate the activity of one or
more peptides provided herein and/or one or more additional agents
described herein, thereby having an effect on a condition or
disorder.
Kits
[0243] Also provided herein are kits including, but not limited to,
peptide sequences provided herein and/or one or more additional
agents for the treatment of a bile acid-related disease, disorder
or condition, or a composition comprising the foregoing, and one or
more pharmaceutically acceptable or physiologically acceptable
diluents, carriers or excipients, optionally in further combination
with one or more therapeutic agents distinct from those described
above, compositions and pharmaceutical compositions thereof,
packaged into suitable packaging material. A kit may include a
label or packaging insert including a description of the components
or instructions for use in vitro, in vivo, or ex vivo, of the
components therein. Exemplary instructions include instructions for
treatment and/or prevention of a bile acid related or associated
disorder, such as cholestasis, including, for example diseases of
intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal
cholestasis, and drug induced cholestasis (e.g., estrogen)), and
diseases of extrahepatic cholestasis (e.g., bile cut compression
from tumor, bile duct blockade by gall stones); bile acid
malabsorption and other disorders involving the distal small
intestine, including ileal resection, inflammatory bowel diseases
(e.g., Crohn's disease and ulcerative colitis), disorders impairing
absorption of bile acids not otherwise characterized (idiopathic))
leading to diarrhea (e.g., BAD) and GI symptoms, and GI, liver,
and/or biliary cancers (e.g., colon cancer and hepatocellular
cancer); and/or bile acid synthesis abnormalities, such as those
contributing to NASH, cirrhosis and portal hypertension, etc.
[0244] The term "packaging material" refers to a physical structure
housing the components of the kit. The packaging material can
maintain the components sterilely, and can be made of material
commonly used for such purposes (e.g., paper, corrugated fiber,
glass, plastic, foil, ampules, vials, tubes, etc.).
[0245] Kits provided herein can include labels or inserts. Labels
or inserts include "printed matter," e.g., paper or cardboard,
separate or affixed to a component, a kit or packing material
(e.g., a box), or attached to, for example, an ampule, tube or vial
containing a kit component. Labels or inserts can additionally
include a computer readable medium, such as a disk (e.g., hard
disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM,
DVD, MP3, magnetic tape, or an electrical storage media such as RAM
and ROM or hybrids of these such as magnetic/optical storage media,
FLASH media or memory type cards.
[0246] Labels or inserts can include, among other things,
identifying information of one or more components therein, dosing
parameters, and/or information on the clinical pharmacology of the
active ingredient(s), including mechanism of action,
pharmacokinetics and pharmacodynamics. Labels or inserts can
include information identifying manufacturer information, lot
numbers, manufacturer location and date.
[0247] Labels or inserts can include information on a condition,
disorder, disease or symptom for which a kit component may be used.
Labels or inserts can include instructions for the clinician or for
a subject for using one or more of the kit components in a method,
treatment protocol or therapeutic regimen. Instructions can include
dosage amounts, frequency or duration, and instructions for
practicing any of the methods, treatment protocols or therapeutic
regimens set forth herein. Exemplary instructions include
instructions for treatment or use of a peptide sequence as set
forth herein and/or the use of an additional agent or treatment
modality useful in treating a bile acid-related or associated
disorder or a disorder of bile acid homeostasis. Kits provided
herein therefore can additionally include labels or instructions
for practicing any of the methods and uses provided herein,
including treatment methods and uses.
[0248] Labels or inserts can include information on any benefit
that a component may provide, such as a prophylactic or therapeutic
benefit. Labels or inserts can include information on potential
adverse side effects, such as warnings to the subject or clinician
regarding situations where it would not be appropriate to use a
particular composition. Adverse effects could also occur when the
subject has, will be, or is currently taking one or more other
medications that may be incompatible with the composition, or the
subject has, will be, or is currently undergoing another treatment
protocol or therapeutic regimen which would be incompatible with
the composition and, therefore, instructions could include
information regarding such incompatibilities.
[0249] Kits provided herein can additionally include other
components. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package. In certain embodiments, kits are designed
for cold storage. Kits provided herein can further be designed to
contain peptide sequences provided herein, or that contain nucleic
acids encoding peptide sequences. Kits provided herein can also be
designed to contain, either separately or in combination with the
peptide sequences provided herein, one or more additional agents
useful in the treatment or prevention of a bile acid-related
disease or disorder. Any cells in the kit can be maintained under
appropriate storage conditions until ready to use.
[0250] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described herein.
[0251] All applications, publications, patents and other
references, GenBank citations and ATCC citations cited herein are
incorporated by reference in their entirety. In case of conflict,
the specification, including definitions, will control. As used
herein, the singular forms "a," "and," and "the" include plural
referents unless the context clearly indicates otherwise. Thus, for
example, reference to "a peptide sequence" or a "treatment,"
includes a plurality of such sequences, treatments, and so
forth.
[0252] As used herein, numerical values are often presented in a
range format throughout this document. The use of a range format is
merely for convenience and brevity and should not be construed as
an inflexible limitation on the scope of the invention unless the
context clearly indicates otherwise. Accordingly, the use of a
range expressly includes all possible subranges, all individual
numerical values within that range, and all numerical values or
numerical ranges including integers within such ranges and
fractions of the values or the integers within ranges, unless the
context clearly indicates otherwise. This construction applies
regardless of the breadth of the range and in all contexts
throughout this patent document. Thus, for example, reference to a
range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%,
91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range
of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc.,
as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%,
92.3%, 92.4%, 92.5%, etc., and so forth.
[0253] In addition, reference to a range of 1-3, 3-5, 5-10, 10-20,
20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,
110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180,
180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a further
example, reference to a range of 25-250, 250-500, 500-1000,
1000-2500, 2500-5000, 5000-25,000, or 5000-50,000 includes any
numerical value or range within or encompassing such values, e.g.,
25, 26, 27, 28, 29...250, 251, 252, 253, 254 . . . 500, 501, 502,
503, 504 . . . , etc.
[0254] A series of ranges are disclosed throughout this document.
The use of a series of ranges includes combinations of the upper
and lower ranges to provide another range. This construction
applies regardless of the breadth of the range and in all contexts
throughout this patent document. Thus, for example, reference to a
series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75,
75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50,
5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150,
and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth.
[0255] The invention is generally disclosed herein using
affirmative language to describe the numerous embodiments. The
invention also specifically includes embodiments in which
particular subject matter is excluded, in full or in part, such as
substances or materials, method steps and conditions, protocols,
procedures, assays or analysis. Thus, even though the invention is
generally not expressed herein in terms of what the invention does
not include, aspects that are not expressly included in the
invention are nevertheless disclosed herein.
[0256] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, the descriptions in the
Experimental section are intended to illustrate but not limit the
scope of invention described in the claims.
Experimental
[0257] The following descriptions are put forth so as to provide
those of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below were performed and are all of the experiments
that may be performed. It is to be understood that exemplary
descriptions written in the present tense were not necessarily
performed, but rather that the descriptions can be performed to
generate the data and the like associated with the teachings of the
present invention. Efforts have been made to ensure accuracy with
respect to numbers used (e.g., amounts, temperature, etc.), but
some experimental errors and deviations should be accounted
for.
[0258] Unless indicated otherwise, parts are parts by weight,
molecular weight is weight average molecular weight, temperature is
in degrees Celsius (.degree. C.), and pressure is at or near
atmospheric. Standard abbreviations are used, including the
following: bp=base pair(s); kb=kilobase(s); s or sec=second(s);
min=minute(s); h or hr=hour(s); aa=amino acid(s); kb=kilobase(s);
nt=nucleotide(s); pg=picogram; ng=nanogram; .mu.g=microgram;
mg=milligram; g=gram; kg=kilogram; pl or pL=picoliter(s); dl or
dL=deciliter; .mu.l or .mu.L=microliter; ml or mL=milliliter; 1 or
L=liter; .mu.M=micromolar; mM=millimolar; M=molar; kDa=kilodalton;
i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC or
SQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly;
TIW=three times a week; QM=monthly; HPLC=high performance liquid
chromatography; BW=body weight; U=unit; ns=not statistically
significant; PBS=phosphate-buffered saline; PCR=polymerase chain
reaction; NETS=N-Hydroxysuccinimide; HSA=human serum albumin;
BSA=bovine serum albumin; DMEM=Dulbeco's Modification of Eagle's
Medium; GC=genome copy; EDTA=ethylenediaminetetraacetic acid.
Materials and Methods
[0259] The following general materials and methods can be used.
[0260] Standard Molecular Biology Techniques. Standard methods in
molecular biology are described in the scientific literature (see,
e.g., Sambrook and Russell (2001) Molecular Cloning, 3.sup.rd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and
Ausubel, et al. (2001) Current Protocols in Molecular Biology,
Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which
describes cloning in bacterial cells and DNA mutagenesis (Vol. 1),
cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and
protein expression (Vol. 3), and bioinformatics (Vol. 4)).
[0261] The scientific literature describes methods for protein
purification, including immunoprecipitation, chromatography,
electrophoresis, centrifugation, and crystallization, as well as
chemical analysis, chemical modification, post-translational
modification, production of fusion proteins, and glycosylation of
proteins (see, e.g., Coligan, et al. (2000) Current Protocols in
Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).
[0262] Production, purification, and fragmentation of polyclonal
and monoclonal antibodies are described (e.g., Harlow and Lane
(1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY); standard techniques for characterizing
ligand/receptor interactions are available (see, e.g., Coligan et
al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley,
Inc., NY); methods for flow cytometry, including
fluorescence-activated cell sorting (FACS), are available (see,
e.g., Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons,
Hoboken, N.J.); and fluorescent reagents suitable for modifying
nucleic acids, including nucleic acid primers and probes,
polypeptides, and antibodies, for use, for example, as diagnostic
reagents, are available (Molecular Probes (2003) Catalogue,
Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003)
Catalogue, St. Louis, Mo.).
[0263] Software. Software packages and databases for determining,
e.g., antigenic fragments, leader sequences, protein folding,
functional domains, glycosylation sites, and sequence alignments,
are available (see, e.g., GCG Wisconsin Package.TM. (Accelrys,
Inc., San Diego, Calif); and DeCypher.TM. (TimeLogic Corp., Crystal
Bay, Nev.).
[0264] Animals. Mice can be purchased from The Jackson Laboratory
(Bar Harbor, Me.) and used in various models, assays and the like
familiar to the skilled artisan. By way of example, db/db mice (The
Jackson Laboratory) can be kept in accordance with welfare
guidelines under controlled light (12 hr light and 12 hr dark
cycle, dark 6:30 pm-6:30 am), temperature (22.+-.4.degree. C.) and
humidity (50%.+-.20%) conditions. Mice can have free access to
water (autoclaved distilled water) and can be fed ad libitum on a
commercial diet (Harlan Laboratories, Indianapolis, Ind.,
Irradiated 2018 Teklad Global 18% Protein Rodent Diet) containing
17 kcal % fat, 23 kcal % protein and 60 kcal % carbohydrate. All
animal studies can be approved by the NGM Institutional Animal Care
and Use Committee.
[0265] DNA and Amino Acid Sequences. cDNA of ORF encoding human
FGF19 (Homo sapiens FGF19, GenBank Accession No. NM_005117.2) and
protein sequence encoded by the cDNA (GenBank Accession No.
NP_005108.1) can be used herein.
[0266] PCR. FGF19 ORF can be amplified with polymerase chain
reaction (PCR) using recombinant DNA (cDNA) prepared from human
small intestinal tissue. PCR reagent kits with Phusion.RTM.
high-fidelity DNA polymerase can be purchased from New England
BioLabs (F-530L, Ipswich, Mass.). The following primers can be
used: forward PCR primer:
[0267] 5' CCGACTAGTCACCatgcggagcgggtgtgtgg (SEQ ID NO:136) and
reverse PCR primer:
[0268] 5' ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC (SEQ ID
NO:137). Amplified DNA fragment can be digested with restriction
enzymes Spe I and Not I (the restriction sites are frequently not
included in the 5' or 3' PCR primers, respectively) and then
ligated with AAV transgene vectors that have been digested with the
same restriction enzymes. The vector that can be used for
expression can contain a selectable marker and an expression
cassette comprising a strong eukaryotic promoter 5' of a site for
insertion of the cloned coding sequence, followed by a 3'
untranslated region and a bovine growth hormone polyadenylation
tail. The expression construct can also be flanked by internal
terminal repeats at the 5' and 3' ends.
[0269] CYP7A1 Repression Assay in Primary Human Hepatocytes.
Primary human hepatocytes can be plated on collagen-coated plates
(Becton Dickinson Biosciences) in Williams E media (Invitrogen)
supplemented with 100 nM dexamethasone (Sigma) and 0.25 mg/ml
MatriGel.TM. (Becton Dickinson Biosciences). Cells can be treated
with FGF19 or variants at 37.degree. C. for 6 hours. CYP7A1
expression can be evaluated in triplicate by quantitative RT-PCR
(TaqMan.RTM. ABI PRISM 7700, Applied Biosystems) and normalized to
GAPDH expression.
[0270] CYP7A1 In vivo Repression Assay. Nine-week-old male db/db
mice (Jackson Laboratories) can be injected intraperitoneally with
recombinant proteins FGF19 or FGF21 at 0.1 mg/kg, 1 mg/kg, and 10
mg/kg. Animals can be euthanized 5 hours post-injection. Livers can
be harvested and homogenized in TRIzol.RTM. reagent (Invitrogen).
Total RNA can be extracted and treated with DNase (Ambion) followed
by quantitative RT-PCR analysis and normalized to GAPDH
expression.
[0271] Production and Purification of AAV. AAV293 cells (which can
be obtained from Agilent Technologies, Santa Clara, Calif.) can be
cultured in Dulbeco's Modification of Eagle's Medium (DMEM,
Mediatech, Inc. Manassas, Va.) supplemented with 10% fetal bovine
serum and 1.times. antibiotic-antimycotic solution (Mediatech, Inc.
Manassas, Va.). The cells can be plated at 50% density on day 1 in
150 mm cell culture plates and can be transfected on day 2, using
calcium phosphate precipitation method with the following 3
plasmids (20 .mu.g/plate of each): AAV transgene plasmid,
pHelper.TM. plasmids (Agilent Technologies) and AAV2/9 plasmid (Gao
et al., J. Virol. 78:6381 (2004)). Forty-eight (48) hours after
transfection, the cells can be scraped off the plates, pelleted by
centrifugation at 3000.times.g and resuspended in buffer containing
20 mM Tris pH 8.5, 100 mM NaCl and 1 mM MgCl.sub.2. The suspension
can be frozen in an alcohol dry ice bath and then thawed in a
37.degree. C. water bath. The freeze and thaw cycles can be
repeated three times; Benzonase.RTM. (Sigma-aldrich, St. Louis,
Mo.) can be added to 50 units/ml; deoxycholate can be added to a
final concentration of 0.25%. After incubation at 37.degree. C. for
30 min, cell debris can be pelleted by centrifugation at
5000.times.g for 20 min. Viral particles in the supernatant can be
purified using a gradient comparable to discontinued iodixanal
(Sigma-aldrich, St. Louis, Mo.) gradient as previously described
(Zolotukhin S. et al (1999) Gene Ther. 6:973). The viral stock can
be concentrated using Vivaspin.RTM. 20 (MW cutoff 100,000 Dalton,
Sartorius Stedim Biotech, Aubagne, France) and re-suspended in
phosphate-buffered saline (PBS) with 10% glycerol and stored at
-80.degree. C. To determine the viral genome copy number, 2 .mu.l
of viral stock can be incubated in 6 .mu.l of solution containing
50 units/ml Benzonase.RTM., 50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2
and 10 mM CaCl.sub.2 at 37.degree. C. for 30 minutes.
[0272] Afterwards, 15 .mu.l of the solution containing 2 mg/ml of
Proteinase K, 0.5% SDS and 25 mM EDTA can be added and the mixture
can be incubated for an additional 20 min at 55.degree. C. to
release viral DNA. Viral DNA can be cleaned with mini DNeasy.RTM.
Kit (Qiagen, Valencia, Calif.) and eluted with 40 .mu.l of water.
Viral genome copy (GC) can be determined by using quantitative PCR.
Viral stock can be diluted with PBS to desirable GC/ml, and viral
working solution (200 .mu.l) can be delivered into mice via tail
vein injection.
[0273] HCC Assay. Liver specimens can be harvested from db/db mice
24 weeks after AAV injection. HCC scores can be recorded as the
number of HCC nodules on the surface of the entire liver from
variants-injected mice divided by the number of HCC nodules from
wild-type FGF19-injected mice.
[0274] Serum FGF19/FGF21/Variants Exposure Level Assay. Whole blood
(about 50 .mu.l/mouse) from mouse tail snips can be collected into
plain capillary tubes (BD Clay Adams SurePrep.TM., Becton Dickenson
and Co. Sparks, Md.). Serum and blood cells can be separated by
spinning the tubes in an Autocrit.TM. Ultra 3 (Becton Dickinson and
Co. Sparks, Md.). FGF19, FGF21, and variant exposure levels in
serum can be determined using EIA kits (Biovendor) by following the
manufacturer's instructions.
[0275] FGFR4 Binding and Activity Assays. Solid phase ELISA
(binding) and ERK phosphorylation assay can be performed using
purified recombinant proteins. FGFR binding assay can be conducted
using solid phase ELISA. Briefly, a 96-well plate can be coated
with 2 .mu.g/ml anti-hFc antibody and can be incubated with 1
.mu.g/ml FGFR1-hFc or FGFR4-hFc. Binding to FGF19 variants in the
presence of 1 .mu.g/ml soluble .beta.-klotho and 20 .mu.g/ml
heparin can be detected by biotinylated anti-FGF19 antibodies (0.2
.mu.g/mL), followed by streptavidin-HRP incubation (100 ng/mL). For
FGFR4 activation assay, Hep3B cells can be stimulated with FGF19
variants for 10 minutes at 37.degree. C., then can be immediately
lysed and assayed for ERK phosphorylation using a commercially
available kit from Cis-Bio.
[0276] In the examples that follow, wild-type FGF19, wild-type
FGF21, variants of FGF19 peptide sequences, fusions of FGF19 and/or
FGF21 peptide sequences, and variants of fusions (chimeras) of
FGF19 (for purposes of the Experimental section, collectively
"Invention Peptides") can be used to illustrate the procedures,
methodologies and the like useful in evaluating other Invention
Peptides, as well as in evaluating the one or more additional
agents or therapeutic modalities having a desired effect on one or
more bile acid-related or associated diseases, disorders or
conditions (for purposes of the Experimental section, "Additional
Agents") useful in combination with the Invention Peptides.
[0277] Example 1: Through assessment of wild-type FGF19 and FGF21,
this example illustrates how the peptides (e.g., M70) provided
herein can be evaluated for inhibition of CYP7A1 expression.
[0278] Briefly, at time0 db/db mice can be dosed intraperitoneally
with either recombinant FGF19 (0.1 mg/kg; 1 mg/kg; 10 mg/kg) or
recombinant FGF21 (0.1 mg/kg; 1 mg/kg; 10 mg/kg). Five hours after
dosing, livers can be harvested, RNA extracted, and CYP7A1
expression determined by real-time PCR (QPCR) using GADPH as a
normalization control. In each group of mice containing the desired
number of animals (e.g., n=3), CYP7A1 expression values for the
various FGF19 and FGF21 concentrations can be compared to mice
dosed with PBS vehicle control.
[0279] Example 2: Using the assays described above (e.g., in vitro
cell-based assay (primary human hepatocyte) and in vivo assay
(protein dosing in db/db mice)), repression of CYP7A1 in primary
human hepatocytes can be determined for Invention Peptides.
Invention Peptides that retain Cyp7a1 repression activity can be
further evaluated in the HCC assay (or other relevant assay or
model) described above to identify variants that can be useful for
modulating bile acid metabolism and/or for treating bile
acid-related diseases (e.g., PBC, NASH, and bile acid diarrhea)
without causing induction of HCC.
[0280] Example 3: Invention Peptides can be analyzed for lipid
elevating activity and tumorigenesis using the above-described
methods or any methods familiar to the skilled artisan. If a
positive correlation is observed between lipid elevation and
tumorigenesis, as determined by HCC formation in db/db mice, lipid
elevating activity can be used as an indicator and/or predictor of
HCC formation in animals.
[0281] Example 4: The teachings of this example can be used to
determine whether administration of Invention Peptides to human
patients can result in suppression of 7a-hydroxy-4-cholsten-3-one
(C4), a marker of bile acid synthesis.
[0282] Study Subjects: Healthy adults in the age range 18-65 years
and with normal body weight (body mass index, BMI 20-35) can be
enrolled in the study. The study protocol can be approved by the
Human Research Ethics Committee in Australia, and written informed
consent can be obtained from each subject. For inclusion in the
study, each subject can be required to be in good health as
determined by no clinically significant findings from medical
history, physical exam, 12-lead ECG, clinical laboratory findings,
and vital signs at screening. Subjects with a history or clinical
manifestation of any significant metabolic, allergic,
dermatological, hepatic, renal, hematological, pulmonary,
cardiovascular, GI, neurological, or psychiatric disorder can be
excluded from enrollment.
[0283] Study Design: The study can be a randomized, double-blind,
placebo-controlled design. Prescreening of subjects can be
performed 7-30 days prior to entry, and baseline evaluations can be
performed before treatment. Each subject can be given a SC
injection of 3 mg/day in a single bolus dose daily for 7 days of
one or more Invention Peptides. Blood samples can be collected into
heparinized tubes through an indwelling catheter. Blood samples
taken on Day 1 and Day 7 at 4.5 hrs or 24 hrs after administration
of one or more Invention Peptides or placebo can be analyzed. Serum
levels of 7a-hydroxy-4-cholesten-3-one (C4) can be used to monitor
CYP7A1 enzymatic activity (bile acid synthesis). They can be
analyzed from individual serum samples after sample extraction
followed by high-pressure liquid chromatography (HPLC) as described
previously (Galman et al. (2003) J Lipid Res. 2003;
44(4):859-66).
[0284] Example 5: The previously described assays for FGFR4 binding
and activity can be used to assess whether Invention Peptides show
activation of mouse FGFR4-.beta.-klotho signaling in a rat myoblast
cell line
[0285] Methods: An ELK luciferase assay can be performed in L6
cells transiently transfected with mouse FGFR4, b-klotho, and
reporter constructs containing 5xUAS luciferase and
GAL4-DNA-binding domain (DBD) fused to ELK1. In this system,
luciferase activity is regulated by the endogenous phosphorylated
extracellular signal-regulated kinase (ERK). Cells can be incubated
with ligands for 6 hours before being lysed for luciferase activity
measurements.
[0286] Thereafter, a cell-based receptor activation assay can be
used to evaluate the ability of mouse FGFR4 to mediate
ligand-dependent signaling in the presence of .beta.-klotho. To
this end, a rat L6 myoblast cell line, which lacks endogenous
expression of these proteins, can be transfected with DNAs encoding
FGFR4 and .beta.-klotho from mouse, as well as plasmids containing
an Elk1-dependent chimeric transcription factor-based reporter
system. Following transfection, concentration response of
ligand-dependent luciferase expression can be analyzed in
whole-cell lysates in the presence of luciferin substrate.
[0287] The resulting data can suggest that the formation of a
ternary complex between the FGFR4-.beta.-klotho co-receptors and
cognate ligands is important for potent activation of intracellular
signaling.
Sequence Listing
[0288] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing. The CRF entitled
13370-145-999_SEQLIST.txt was created on Dec. 20, 2021 and is
256,843 bytes in size. The CRF also serves as the paper copy of the
Sequence Listing, and are thus identical, and is incorporated
herein by reference in its entirety.
Sequence CWU 1
1
2121194PRTHomo sapiens 1Arg Pro Leu Ala Phe Ser Asp Ala Ser Pro His
Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120
125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys2194PRTHomo sapiens
2Arg Pro Leu Ala Phe Ser Asp Ser Ser Pro Leu Val His Tyr Gly Trp1 5
10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155
160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp
165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro
Ser Phe 180 185 190Glu Lys3194PRTHomo sapiens 3Arg Pro Leu Ala Phe
Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu
Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75
80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val
Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu
Lys4194PRTHomo sapiens 4Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His
Val His Tyr Ala Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120
125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys5191PRTHomo sapiens
5Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln1 5
10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155
160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly
165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
Lys 180 185 1906187PRTHomo sapiens 6Arg Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser Gly Pro
His Gly Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp Gly Val
Val Asp Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu Glu Ile
Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val His Ser
Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75 80Gln Gly
Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu 85 90 95Ile
Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 100 105
110Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg
115 120 125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met
Val Pro 130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser
Asp Met Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp Ser Met Asp
Pro Phe Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val Arg Ser Pro
Ser Phe Glu Lys 180 1857192PRTHomo sapiens 7Arg Pro Leu Ala Phe Ser
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly1 5 10 15Gln Val Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
1908191PRTHomo sapiens 8Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu
Gln Trp Gly Asp Pro1 5 10 15Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120
125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu
130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu
Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 185 1909193PRTHomo sapiens 9Arg His Pro
Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Trp Gly1 5 10 15Asp Pro
Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser
Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala 35 40
45Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
50 55 60Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met65 70 75 80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val Pro
Glu Glu Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170 175Phe
Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185
190Lys10193PRTHomo sapiens 10Arg His Pro Ile Pro Asp Ser Ser Pro
His Val His Tyr Gly Trp Gly1 5 10 15Asp Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala 35 40 45Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu 50 55 60Arg Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met65 70 75 80Gly Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp 85 90 95Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg 100 105
110Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro 130 135 140Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185 190Lys11192PRTHomo
sapiens 11Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro Leu Leu Gln Trp
Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150
155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro
Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
Phe Glu Lys 180 185 19012194PRTHomo sapiens 12Arg Pro Leu Ala Phe
Ser Asp Ala Gly Pro Leu Leu Gln Phe Gly Trp1 5 10 15Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu
Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75
80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu
Lys13192PRTHomo sapiens 13Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
Leu Leu Gln Phe Gly Gly1 5 10 15Gln Val Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu
Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120
125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met
130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185 19014191PRTHomo sapiens 14Arg
His Pro Ile Pro Asp Ser Ser Pro His Val His Tyr Gly Gly Gln1 5 10
15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser
20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg
Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro
Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155 160Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170
175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185 19015192PRTHomo sapiens 15Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro His Val His Tyr Gly Gly1 5 10 15Gln Val Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19016192PRTHomo sapiens
16Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Trp Gly Asp1
5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155
160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19017192PRTHomo sapiens 17Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro His Val Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19018192PRTHomo sapiens 18Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
His Tyr Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu
Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120
125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185 19019192PRTHomo sapiens 19Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro Val Tyr Gly Trp Gly Asp1 5 10
15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155 160Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170
175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys
180 185 19020192PRTHomo sapiens 20Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro Val His Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19021192PRTHomo sapiens
21Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro Val His Tyr Trp Gly Asp1
5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155
160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19022193PRTHomo sapiens 22Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro His Val His Gly Trp Gly1 5 10 15Asp Pro Ile Arg Leu
Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser Ser Cys Phe
Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala 35 40 45Arg Gly Gln
Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu 50 55 60Arg Thr
Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met65 70 75
80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp
85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr
Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala
Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu
Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val Pro Glu Glu Pro
Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe Ser
Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu Val
Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185
190Lys23192PRTHomo sapiens 23Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro His His Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19024192PRTHomo sapiens
24Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His His Tyr Trp Gly Asp1
5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155
160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19025192PRTHomo sapiens 25Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro His Val Tyr Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19026192PRTHomo sapiens 26Arg Pro Leu Ala Phe Ser Asp Ser Ser Pro
Leu Val His Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19027192PRTHomo sapiens 27Arg Pro Leu Ala Phe Ser Asp Ser Ser Pro
His Val His Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu
Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120
125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185 19028191PRTHomo sapiens 28Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val Trp Gly Asp Pro1 5 10
15Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser
20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg
Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro
Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155 160Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170
175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185 19029193PRTHomo sapiens 29Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro His Val His Tyr Trp Gly1 5 10 15Asp Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala 35 40 45Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu 50 55 60Arg Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met65 70 75 80Gly Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp 85 90 95Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg 100 105
110Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro 130 135 140Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185 190Lys30194PRTHomo
sapiens 30Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Ala Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys31191PRTHomo sapiens 31Arg His Pro
Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Ala Gln1 5 10 15Val Arg
Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys
Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40
45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr
50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly
Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19032191PRTHomo sapiens 32Arg His Pro Ile Pro Asp Ser Ser Pro Leu
Leu Gln Phe Gly Asp Gln1 5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met
Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu
Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys
His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120
125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu
130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu
Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 185 19033191PRTHomo sapiens 33Arg His
Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Pro Gln1 5 10 15Val
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25
30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly
35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg
Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met
Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser
Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu
Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met
Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170
175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185 19034191PRTHomo sapiens 34Arg His Pro Ile Pro Asp Ser Ser Pro
Leu Leu Gln Phe Gly Gly Ala1 5 10 15Val Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105
110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu
115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr
Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala
Val Arg Ser Pro Ser Phe Glu Lys 180 185 19035191PRTHomo sapiens
35Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Glu1
5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155
160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly
165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
Lys 180 185 19036191PRTHomo sapiens 36Arg His Pro Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Asn1 5 10 15Val Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90
95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu
100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19037191PRTHomo
sapiens 37Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln1 5 10 15Ala Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19038191PRTHomo sapiens 38Arg His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln1 5 10 15Ile Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75
80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala
85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser
Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19039191PRTHomo
sapiens 39Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln1 5 10 15Thr Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90
95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu
100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19040193PRTHomo
sapiens 40Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Trp Gly1 5 10 15Gln Pro Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro
His Gly Leu 20 25 30Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val
Val Asp Cys Ala 35 40 45Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile
Lys Ala Val Ala Leu 50 55 60Arg Thr Val Ala Ile Lys Gly Val His Ser
Val Arg Tyr Leu Cys Met65 70 75 80Gly Ala Asp Gly Lys Met Gln Gly
Leu Leu Gln Tyr Ser Glu Glu Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile
Arg Pro Asp Gly Tyr Asn Val Tyr Arg 100 105 110Ser Glu Lys His Arg
Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg 115 120 125Gln Leu Tyr
Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro 130 135 140Met
Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu145 150
155 160Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp
Pro 165 170 175Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro
Ser Phe Glu 180 185 190Lys41182PRTHomo sapiens 41Arg Pro Leu Ala
Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro
Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser
Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala
Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55
60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65
70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser
Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu
Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Glu Pro Pro Gly
Ile Leu Ala Pro Gln Pro Pro Asp145 150 155 160Val Gly Ser Ser Asp
Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg 165 170 175Ser Pro Ser
Tyr Ala Ser 18042178PRTHomo sapiens 42His Pro Ile Pro Asp Ser Ser
Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25 30Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp65 70 75 80Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe 85 90
95Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys
100 105 110His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met Leu Pro 130 135 140Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val Gly Ser Ser145 150 155 160Asp Pro Leu Ser Met Val Gly
Pro Ser Gln Gly Arg Ser Pro Ser Tyr 165 170 175Ala Ser43192PRTHomo
sapiens 43Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Gly Gly1 5 10 15Asp Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150
155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro
Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
Phe Glu Lys 180 185 19044185PRTHomo sapiens 44Arg Pro Leu Ala Phe
Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln 20 25 30Thr Glu Ala
His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala 35 40 45Ala Asp
Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro 50 55 60Gly
Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln65 70 75
80Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala
85 90 95Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr
Gln 100 105 110Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn
Lys Ser Pro 115 120 125His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg
Phe Leu Pro Leu Pro 130 135 140Gly Leu Pro Pro Ala Leu Pro Glu Pro
Pro Gly Ile Leu Ala Pro Gln145 150 155 160Pro Pro Asp Val Gly Ser
Ser Asp Pro Leu Ser Met Val Gly Pro Ser 165 170 175Gln Gly Arg Ser
Pro Ser Tyr Ala Ser 180 18545193PRTHomo sapiens 45His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55
60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65
70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu
Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185
190Lys46232PRTHomo sapiens 46Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln 20 25 30Thr Glu Ala His Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala 35 40 45Ala Asp Gln Ser Pro Glu
Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro 50 55 60Gly Val Ile Gln Ile
Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln65 70 75 80Arg Pro Asp
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala 85 90 95Cys Ser
Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln 100 105
110Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro
115 120 125His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro
Leu Pro 130 135 140Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln145 150 155 160Pro Pro Asp Val Gly Ser Ser Asp Pro
Leu Ser Met Val Gly Pro Ser 165 170 175Gln Gly Arg Ser Pro Ser Tyr
Ala Ser Pro Met Val Pro Glu Glu Pro 180 185 190Glu Asp Leu Arg Gly
His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu 195 200 205Glu Thr Asp
Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala 210 215 220Val
Arg Ser Pro Ser Phe Glu Lys225 23047190PRTHomo sapiens 47His Pro
Ile Pro Asp Ser Ser Pro Leu Leu Gln Trp Gly Asp Pro Ile1 5 10 15Arg
Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25
30Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln
35 40 45Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr
Val 50 55 60Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly
Ala Asp65 70 75 80Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp Cys Ala Phe 85 90 95Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys 100 105 110His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg Gln Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro Met Leu Pro 130 135 140Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp145 150 155 160Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170
175Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19048187PRTHomo sapiens 48Arg Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln Val Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75 80Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu 85 90 95Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 100 105 110Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg 115 120
125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro
130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met
Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 18549192PRTHomo sapiens 49Arg Pro Leu Ala Phe Ser Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly1 5 10 15Gln Val Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala
Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90
95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser
100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19050191PRTHomo
sapiens 50Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Asp Gln1 5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Leu Glu
Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19051191PRTHomo sapiens 51Arg His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Asn1 5 10 15Val Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75
80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala
85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser
Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19052187PRTHomo sapiens 52Arg Asp Ser Ser Pro Leu
Leu Gln Trp Gly Asp Pro Ile Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75
80Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu
85 90 95Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg
Leu 100 105 110Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr
Lys Asn Arg 115 120 125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
Leu Pro Met Val Pro 130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu Ser Asp Met Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 18553189PRTHomo sapiens 53Met Asp
Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg1 5 10 15Leu
Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25
30Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser
35 40 45Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val
Ala 50 55 60Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala
Asp Gly65 70 75 80Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp
Cys Ala Phe Glu 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr
Arg Ser Glu Lys His 100 105 110Arg Leu Pro Val Ser Leu Ser Ser Ala
Lys Gln Arg Gln Leu Tyr Lys 115 120 125Asn Arg Gly Phe Leu Pro Leu
Ser His Phe Leu Pro Met Leu Pro Met 130 135 140Val Pro Glu Glu Pro
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met145 150 155 160Phe Ser
Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170
175Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
18554192PRTHomo sapiens 54Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
Leu Leu Gln Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu
Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120
125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185 19055192PRTHomo sapiens 55Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Tyr Gly Trp Gly Asp1 5 10
15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155 160Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170
175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys
180 185 19056192PRTHomo sapiens 56Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro Val Tyr Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19057192PRTHomo sapiens
57Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro Val His Gly Trp Gly Asp1
5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155
160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19058192PRTHomo sapiens 58Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro Val His Tyr Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19059192PRTHomo sapiens 59Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
His His Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu
Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120
125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185 19060192PRTHomo sapiens 60Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His His Tyr Trp Gly Asp1 5 10
15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155 160Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170
175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys
180 185 19061192PRTHomo sapiens 61Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val Gly Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19062192PRTHomo sapiens
62Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val Tyr Trp Gly Asp1
5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155
160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 19063192PRTHomo sapiens 63Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro His Val His Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19064192PRTHomo sapiens 64Arg Pro Leu Ala Phe Ser Asp Ser Ser Pro
Leu Val His Trp Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100
105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 19065192PRTHomo
sapiens 65Arg Pro Leu Ala Phe Ser Asp Ser Ser Pro His Val His Trp
Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150
155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro
Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
Phe Glu Lys 180 185 19066192PRTHomo sapiens 66Arg Pro Leu Ala Phe
Ser Asp Ala Gly Pro His Leu Gln Trp Gly Asp1 5 10 15Pro Ile Arg Leu
Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe
Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln
Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr
Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75
80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr
Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19067191PRTHomo sapiens 67Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
His Val Trp Gly Asp Pro1 5 10 15Ile Arg Leu Arg His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met
Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu
Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys
His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120
125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu
130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu
Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 185 19068193PRTHomo sapiens 68Arg Pro
Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Trp Gly1 5 10 15Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25
30Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
35 40 45Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu 50 55 60Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met65 70 75 80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170
175Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
180 185 190Lys69189PRTHomo sapiens 69Arg Asp Ser Ser Pro Leu Val
His Tyr Gly Trp Gly Asp Pro Ile Arg1 5 10 15Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly65 70 75 80Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90
95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His
100 105 110Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu
Tyr Lys 115 120 125Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met Leu Pro Met 130 135 140Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
His Leu Glu Ser Asp Met145 150 155 160Phe Ser Ser Pro Leu Glu Thr
Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175Thr Gly Leu Glu Ala
Val Arg Ser Pro Ser Phe Glu Lys 180 18570190PRTHomo sapiens 70Met
Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile1 5 10
15Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys
20 25 30Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly
Gln 35 40 45Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg
Thr Val 50 55 60Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met
Gly Ala Asp65 70 75 80Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu Asp Cys Ala Phe 85 90 95Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Arg Ser Glu Lys 100 105 110His Arg Leu Pro Val Ser Leu Ser
Ser Ala Lys Gln Arg Gln Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu
Pro Leu Ser His Phe Leu Pro Met Leu Pro 130 135 140Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp145 150 155 160Met
Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170
175Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19071181PRTHomo sapiens 71His Pro Ile Pro Asp Ser Ser Pro Leu Leu
Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp
Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr
Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu
Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Ser
Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120
125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro
Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala Ser 18072181PRTHomo
sapiens 72His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly
Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr
Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala
Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys
Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu
Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe
Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg
Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala
Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150
155 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg
Ser 165 170 175Pro Ser Tyr Ala Ser 18073212PRTHomo sapiens 73His
Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10
15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln
Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val
Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg
Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val
Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu His Leu Pro Gly
Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro Arg Gly Pro Ala
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140Ala Leu Pro Glu
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145 150 155 160Gly
Ser Ser Asp Pro Leu Ser Met Val Val Gln Asp Glu Leu Gln Gly 165 170
175Val Gly Gly Glu Gly Cys His Met His Pro Glu Asn Cys Lys Thr Leu
180 185 190Leu Thr Asp Ile Asp Arg Thr His Thr Glu Lys Pro Val Trp
Asp Gly 195 200 205Ile Thr Gly Glu 21074189PRTHomo sapiens 74Arg
Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Asp Pro Ile Arg1 5 10
15Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe
20 25 30Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln
Ser 35 40 45Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr
Val Ala 50 55 60Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly
Ala Asp Gly65 70 75 80Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp Cys Ala Phe Glu 85 90 95Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys His 100 105 110Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met145 150 155 160Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170
175Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
18575184PRTHomo sapiens 75Arg Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr1 5 10 15Thr Ser Gly Pro His Gly Leu Ser Ser
Cys Phe Leu Arg Ile Arg Ala 20 25 30Asp Gly Val Val Asp Cys Ala Arg
Gly Gln Ser Ala His Ser Leu Leu 35 40 45Glu Ile Lys Ala Val Ala Leu
Arg Thr Val Ala Ile Lys Gly Val His 50 55 60Ser Val Arg Tyr Leu Cys
Met Gly Ala Asp Gly Lys Met Gln Gly Leu65 70 75 80Leu Gln Tyr Ser
Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 85 90 95Asp Gly Tyr
Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 100 105 110Leu
Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 115 120
125Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro
130 135 140Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser
Pro Leu145 150 155 160Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val
Thr Gly Leu Glu Ala 165 170 175Val Arg Ser Pro Ser Phe Glu Lys
18076179PRTHomo sapiens 76Arg Gly Asp Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His1 5 10 15Gly Leu Ser Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp 20 25 30Cys Ala Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val 35 40 45Ala Leu Arg Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu 50 55 60Cys Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu65 70 75 80Glu Asp Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val 85 90 95Tyr Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys 100 105 110Gln
Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe 115 120
125Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
130 135 140His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
Ser Met145 150 155 160Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala
Val Arg Ser Pro Ser 165 170 175Phe Glu Lys77175PRTHomo sapiens
77Arg Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser1
5 10 15Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg
Gly 20 25 30Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
Arg Thr 35 40 45Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met Gly Ala 50 55 60Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu Asp Cys Ala65 70 75 80Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg Ser Glu 85
90 95Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
Leu 100 105 110Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met Leu 115 120 125Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu Ser 130 135 140Asp Met Phe Ser Ser Pro Leu Glu Thr
Asp Ser Met Asp Pro Phe Gly145 150 155 160Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170 17578188PRTHomo sapiens
78Arg Ala Gly Pro His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu1
5 10 15Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe
Leu 20 25 30Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln
Ser Ala 35 40 45His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr
Val Ala Ile 50 55 60Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly
Ala Asp Gly Lys65 70 75 80Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp Cys Ala Phe Glu Glu 85 90 95Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys His Arg 100 105 110Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg Gln Leu Tyr Lys Asn 115 120 125Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro Met Leu Pro Met Val 130 135 140Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe145 150 155
160Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr
165 170 175Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
18579187PRTHomo sapiens 79Arg Gly Pro His Val His Tyr Gly Trp Gly
Asp Pro Ile Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75 80Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu 85 90 95Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 100 105 110Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg 115 120
125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro
130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met
Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 18580186PRTHomo sapiens 80Arg Pro His Val His Tyr Gly
Trp Gly Asp Pro Ile Arg Leu Arg His1 5 10 15Leu Tyr Thr Ser Gly Pro
His Gly Leu Ser Ser Cys Phe Leu Arg Ile 20 25 30Arg Ala Asp Gly Val
Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser 35 40 45Leu Leu Glu Ile
Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly 50 55 60Val His Ser
Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln65 70 75 80Gly
Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile 85 90
95Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro
100 105 110Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn
Arg Gly 115 120 125Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro
Met Val Pro Glu 130 135 140Glu Pro Glu Asp Leu Arg Gly His Leu Glu
Ser Asp Met Phe Ser Ser145 150 155 160Pro Leu Glu Thr Asp Ser Met
Asp Pro Phe Gly Leu Val Thr Gly Leu 165 170 175Glu Ala Val Arg Ser
Pro Ser Phe Glu Lys 180 18581185PRTHomo sapiens 81Arg His Val His
Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu1 5 10 15Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg 20 25 30Ala Asp
Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 35 40 45Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 50 55
60His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly65
70 75 80Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile
Arg 85 90 95Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu
Pro Val 100 105 110Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys
Asn Arg Gly Phe 115 120 125Leu Pro Leu Ser His Phe Leu Pro Met Leu
Pro Met Val Pro Glu Glu 130 135 140Pro Glu Asp Leu Arg Gly His Leu
Glu Ser Asp Met Phe Ser Ser Pro145 150 155 160Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu 165 170 175Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 18582194PRTHomo sapiens 82Arg Pro Leu
Ala Phe Ser Ala Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys83194PRTHomo sapiens 83Arg Pro Leu Ala Phe Ser Asp Ala
Ala Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly
Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105
110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys84194PRTHomo
sapiens 84Arg Pro Leu Ala Phe Ser Asp Ala Gly Ala His Val His Tyr
Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys85194PRTHomo sapiens 85Arg Pro Leu
Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Ala1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys86194PRTHomo sapiens 86Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Ala Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly
Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105
110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys87167PRTHomo
sapiens 87Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Gly Trp1 5 10 15Gly Asp Ala Ile Cys Ala Arg Gly Gln Ser Ala His Ser
Leu Leu Glu 20 25 30Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser 35 40 45Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys
Met Gln Gly Leu Leu 50 55 60Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu
Glu Glu Ile Arg Pro Asp65 70 75 80Gly Tyr Asn Val Tyr Arg Ser Glu
Lys His Arg Leu Pro Val Ser Leu 85 90 95Ser Ser Ala Lys Gln Arg Gln
Leu Tyr Lys Asn Arg Gly Phe Leu Pro 100 105 110Leu Ser His Phe Leu
Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu 115 120 125Asp Leu Arg
Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu 130 135 140Thr
Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val145 150
155 160Arg Ser Pro Ser Phe Glu Lys 16588194PRTHomo sapiens 88Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10
15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro Ala Gly
20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro
Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ala His Phe Leu 130 135 140Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190Glu Lys89194PRTHomo sapiens 89Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg
Leu Arg His Leu Tyr Thr Ser Gly Pro Ala Gly 20 25 30Leu Ser Ser Cys
Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly
Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg
Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75
80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser Ala Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu
Lys90194PRTHomo sapiens 90Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu
Ser Ser Ala Ala Gln 115 120 125Ala Gln Leu Tyr Lys Asn Arg Gly Phe
Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro
Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe
Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys91194PRTHomo sapiens 91Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly
Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105
110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Ala Gln
115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ala His
Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys92194PRTHomo
sapiens 92Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Ala Gln 115 120 125Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser Ala Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys93194PRTHomo sapiens 93Arg Pro Leu
Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125Ala Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ala His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys94194PRTHomo sapiens 94Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly
Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105
110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ala Ala
Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys95194PRTHomo
sapiens 95Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Ala Gln 115 120 125Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser Ala Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys96194PRTHomo sapiens 96Arg Pro Leu
Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Ala Gln 115 120 125Ala Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ala His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys97194PRTHomo sapiens 97Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly
Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105
110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Ala Gln
115 120 125Ala Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser Ala
Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu Lys98194PRTHomo
sapiens 98Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr
Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Ala Gln 115 120 125Ala Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ala Ala Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys99194PRTHomo sapiens 99Arg Pro Leu
Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys100181PRTHomo sapiens 100His Pro Ile Pro Asp Ser Ser Pro
Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr
Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu
Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu
Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105
110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu
Pro Pro 130 135 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val
Gly Pro Ser Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala Ser
1801014PRTHomo sapiens 101Val His Tyr Gly11029PRTHomo sapiens
102Asp Ala Ser Pro His Val His Tyr Gly1 51039PRTHomo sapiens 103Asp
Ser Ser Pro Leu Val His Tyr Gly1 51047PRTHomo sapiens 104Asp Ser
Ser Pro Leu Leu Gln1 510512PRTHomo sapiens 105Asp Ser Ser Pro Leu
Leu Gln Phe Gly Gly Gln Val1 5 101065PRTHomo sapiens 106Arg His Pro
Ile Pro1 51074PRTHomo sapiens 107His Pro Ile Pro11085PRTHomo
sapiens 108Arg Pro Leu Ala Phe1 51094PRTHomo sapiens 109Pro Leu Ala
Phe11106PRTHomo sapiens 110Met Asp Ser Ser Pro Leu1 51117PRTHomo
sapiens 111Met Ser Asp Ser Ser Pro Leu1 51126PRTHomo sapiens 112Ser
Asp Ser Ser Pro Leu1 51135PRTHomo sapiens 113Met Ser Ser Pro Leu1
51144PRTHomo sapiens 114Ser Ser Pro Leu11154PRTHomo sapiens 115Arg
Asp Ser Ser11164PRTHomo sapiens 116Met Asp Ser Ser11175PRTHomo
sapiens 117Met Arg Asp Ser Ser1 51185PRTHomo sapiens 118Met Ser Ser
Pro Leu1 51196PRTHomo sapiens 119Met Asp Ser Ser Pro Leu1
51207PRTHomo sapiens 120Met Ser Asp Ser Ser Pro Leu1 51215PRTHomo
sapiens 121Asp Ser Ser Pro Leu1 51225PRTHomo sapiens 122Asp Ala Ser
Pro His1 51234PRTHomo sapiens 123Arg Asp Ser Ser11244PRTHomo
sapiens 124Met Asp Ser Ser11255PRTHomo sapiens 125Met Arg Asp Ser
Ser1 51266PRTHomo sapiens 126Met Asp Ser Ser Pro Leu1 51277PRTHomo
sapiens 127Met Ser Asp Ser Ser Pro Leu1 51285PRTHomo sapiens 128Met
Ser Ser Pro Leu1 51295PRTArtificial SequenceDescription of
Artificial Sequence Linker sequence 129Gly Ser Gly Gly Ser1
51304PRTArtificial SequenceDescription of Artificial Sequence
Linker sequence 130Gly Gly Gly Ser11314PRTArtificial
SequenceDescription of Artificial Sequence Linker sequence 131Gly
Gly Ser Gly11325PRTArtificial SequenceDescription of Artificial
Sequence Linker sequence 132Gly Gly Ser Gly Gly1 51335PRTArtificial
SequenceDescription of Artificial Sequence Linker sequence 133Gly
Ser Gly Ser Gly1 51345PRTArtificial SequenceDescription of
Artificial Sequence Linker sequence 134Gly Ser Gly Gly Gly1
51355PRTArtificial SequenceDescription of Artificial Sequence
Linker sequence 135Gly Ser Ser Ser Gly1 513632DNAArtificial
SequenceDescription of Artificial Sequence Forward primer
136ccgactagtc accatgcgga gcgggtgtgt gg 3213741DNAArtificial
SequenceDescription of Artificial Sequence Reverse primer
137ataagaatgc ggccgcttac ttctcaaagc tgggactcct c 41138186PRTHomo
sapiens 138Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Leu
Arg His1 5 10
15Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile
20 25 30Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His
Ser 35 40 45Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile
Lys Gly 50 55 60Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly
Lys Met Gln65 70 75 80Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala
Phe Glu Glu Glu Ile 85 90 95Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser
Glu Lys His Arg Leu Pro 100 105 110Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln Leu Tyr Lys Asn Arg Gly 115 120 125Phe Leu Pro Leu Ser His
Phe Leu Pro Met Leu Pro Met Val Pro Glu 130 135 140Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser145 150 155 160Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu 165 170
175Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185139194PRTHomo
sapiens 139Arg Pro Leu Ala Phe Ser Asp Ala Ser Pro His Val His Tyr
Gly Trp1 5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150
155 160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp 165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe 180 185 190Glu Lys140194PRTHomo sapiens 140Arg Pro Leu
Ala Phe Ser Asp Ser Ser Pro Leu Val His Tyr Gly Trp1 5 10 15Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190Glu Lys141188PRTHomo sapiens 141Asp Ser Ser Pro Leu Val His Tyr
Gly Trp Gly Asp Pro Ile Arg Leu1 5 10 15Arg His Leu Tyr Thr Ser Gly
Pro His Gly Leu Ser Ser Cys Phe Leu 20 25 30Arg Ile Arg Ala Asp Gly
Val Val Asp Cys Ala Arg Gly Gln Ser Ala 35 40 45His Ser Leu Leu Glu
Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile 50 55 60Lys Gly Val His
Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys65 70 75 80Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu 85 90 95Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg 100 105
110Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn
115 120 125Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro
Met Val 130 135 140Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu
Ser Asp Met Phe145 150 155 160Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp Pro Phe Gly Leu Val Thr 165 170 175Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe Glu Lys 180 185142193PRTHomo sapiens 142Arg His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Trp Gly1 5 10 15Asp Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala 35 40 45Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu 50 55
60Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met65
70 75 80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185
190Lys143191PRTHomo sapiens 143Arg His Pro Ile Pro Asp Ser Ser Pro
Leu Leu Gln Trp Gly Asp Pro1 5 10 15Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105
110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu
115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr
Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala
Val Arg Ser Pro Ser Phe Glu Lys 180 185 190144194PRTHomo sapiens
144Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro Leu Leu Gln Phe Gly Trp1
5 10 15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly 20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155
160Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp
165 170 175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro
Ser Phe 180 185 190Glu Lys145193PRTHomo sapiens 145Arg His Pro Ile
Pro Asp Ser Ser Pro His Val His Tyr Gly Trp Gly1 5 10 15Asp Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25 30Ser Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala 35 40 45Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu 50 55
60Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met65
70 75 80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
Tyr Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170 175Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu 180 185
190Lys146192PRTHomo sapiens 146Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro Leu Leu Gln Phe Gly Gly1 5 10 15Gln Val Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190147191PRTHomo
sapiens 147Arg His Pro Ile Pro Asp Ser Ser Pro His Val His Tyr Gly
Gly Gln1 5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 190148187PRTHomo sapiens 148Arg Asp Ser Ser Pro Leu
Leu Gln Phe Gly Gly Gln Val Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75
80Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu
85 90 95Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg
Leu 100 105 110Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr
Lys Asn Arg 115 120 125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
Leu Pro Met Val Pro 130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu Ser Asp Met Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185149192PRTHomo sapiens 149Arg Pro
Leu Ala Phe Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly1 5 10 15Gln
Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25
30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg
35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys Asn Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu Pro Met Val Pro
Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150 155 160Ser Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 165 170
175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys
180 185 190150191PRTHomo sapiens 150Arg His Pro Ile Pro Asp Ser Ser
Pro Leu Leu Gln Phe Gly Ala Gln1 5 10 15Val Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105
110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu
115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
190151191PRTHomo sapiens 151Arg His Pro Ile Pro Asp Ser Ser Pro Leu
Leu Gln Phe Gly Asp Gln1 5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met
Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu
Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys
His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120
125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu
130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu
Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 185 190152191PRTHomo sapiens 152Arg His
Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Pro Gln1 5 10 15Val
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25
30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly
35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg
Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met
Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser
Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu
Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met
Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170
175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185 190153191PRTHomo sapiens 153Arg His Pro Ile Pro Asp Ser Ser Pro
Leu Leu Gln Phe Gly Gly Ala1 5 10 15Val Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu
Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105
110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu
115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro
Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr
Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala
Val Arg Ser Pro Ser Phe Glu Lys 180 185 190154191PRTHomo sapiens
154Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Glu1
5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150 155
160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly
165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
Lys 180 185 190155191PRTHomo sapiens 155Arg His Pro Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Asn1 5 10 15Val Arg Leu Arg His Leu
Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile
Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile
Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp
Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90
95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu
100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190156191PRTHomo
sapiens 156Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln1 5 10 15Ala Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Arg Pro
Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 190157191PRTHomo sapiens 157Arg His Pro Ile Pro Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln1 5 10 15Ile Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75
80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala
85 90 95Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser
Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
190158191PRTHomo sapiens 158Arg His Pro Ile Pro Asp Ser Ser Pro Leu
Leu Gln Phe Gly Gly Gln1 5 10 15Thr Arg Leu Arg His Leu Tyr Thr Ser
Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp
Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu
Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val
His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met
Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu
Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys
His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120
125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu
130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu
Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro Ser Phe Glu Lys 180 185 190159193PRTHomo sapiens 159Arg His
Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Trp Gly1 5 10 15Gln
Pro Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu 20 25
30Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
35 40 45Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu 50 55 60Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met65 70 75 80Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp 85 90 95Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr Arg 100 105 110Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg 115 120 125Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro 130 135 140Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu145 150 155 160Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 165 170
175Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
180 185 190Lys160190PRTHomo sapiens 160His Pro Ile Pro Asp Ser Ser
Pro Leu Leu Gln Phe Gly Gly Gln Val1 5 10 15Arg Leu Arg His Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25 30Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45Ser Ala His Ser
Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60Ala Ile Lys
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp65 70 75 80Gly
Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe 85 90
95Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys
100 105 110His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met Leu Pro 130 135 140Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu Ser Asp145 150 155 160Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170 175Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190161186PRTHomo
sapiens 161Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Leu
Arg His1 5 10 15Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe
Leu Arg Ile 20 25 30Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln
Ser Ala His Ser 35 40 45Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr
Val Ala Ile Lys Gly 50 55 60Val His Ser Val Arg Tyr Leu Cys Met Gly
Ala Asp Gly Lys Met Gln65 70 75 80Gly Leu Leu Gln Tyr Ser Glu Glu
Asp Cys Ala Phe Glu Glu Glu Ile 85 90 95Arg Pro Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys His Arg Leu Pro 100 105 110Val Ser Leu Ser Ser
Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly 115 120 125Phe Leu Pro
Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu 130 135 140Glu
Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser145 150
155 160Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly
Leu 165 170 175Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185162190PRTHomo sapiens 162His Pro Ile Pro Asp Ser Ser Pro Leu Leu
Gln Trp Gly Asp Pro Ile1 5 10 15Arg Leu Arg His Leu Tyr Thr Ser Gly
Pro His Gly Leu Ser Ser Cys 20 25 30Phe Leu Arg Ile Arg Ala Asp Gly
Val Val Asp Cys Ala Arg Gly Gln 35 40 45Ser Ala His Ser Leu Leu Glu
Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60Ala Ile Lys Gly Val His
Ser Val Arg Tyr Leu Cys Met Gly Ala Asp65 70 75 80Gly Lys Met Gln
Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe 85 90 95Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys 100 105 110His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr 115 120
125Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro
130 135 140Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu
Ser Asp145 150 155 160Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
Asp Pro Phe Gly Leu 165 170 175Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser Phe Glu Lys 180 185 190163192PRTHomo sapiens 163His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Trp Gly Asp1 5 10 15Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 20 25 30Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 35 40
45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg
50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met
Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn
Val Tyr Arg Ser 100 105
110Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln
115 120 125Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu
Pro Met 130 135 140Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
Gly His Leu Glu145 150 155 160Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met Asp Pro Phe 165 170 175Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190164192PRTHomo
sapiens 164His Pro Ile Pro Asp Ser Ser Pro His Val His Tyr Gly Trp
Gly Asp1 5 10 15Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
Gly Leu Ser 20 25 30Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val
Asp Cys Ala Arg 35 40 45Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val Ala Leu Arg 50 55 60Thr Val Ala Ile Lys Gly Val His Ser Val
Arg Tyr Leu Cys Met Gly65 70 75 80Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu Asp Cys 85 90 95Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr Asn Val Tyr Arg Ser 100 105 110Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 115 120 125Leu Tyr Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 130 135 140Leu
Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu145 150
155 160Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro
Phe 165 170 175Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
Phe Glu Lys 180 185 190165190PRTHomo sapiens 165His Pro Ile Pro Asp
Ser Ser Pro His Val His Tyr Gly Gly Gln Val1 5 10 15Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25 30Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp65 70 75
80Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe
85 90 95Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu
Lys 100 105 110His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met Leu Pro 130 135 140Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu Ser Asp145 150 155 160Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170 175Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190166188PRTHomo
sapiens 166Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu1 5 10 15Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser
Cys Phe Leu 20 25 30Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg
Gly Gln Ser Ala 35 40 45His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu
Arg Thr Val Ala Ile 50 55 60Lys Gly Val His Ser Val Arg Tyr Leu Cys
Met Gly Ala Asp Gly Lys65 70 75 80Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu Asp Cys Ala Phe Glu Glu 85 90 95Glu Ile Arg Pro Asp Gly Tyr
Asn Val Tyr Arg Ser Glu Lys His Arg 100 105 110Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn 115 120 125Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val 130 135 140Pro
Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe145 150
155 160Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val
Thr 165 170 175Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185167183PRTHomo sapiens 167Val His Tyr Gly Trp Gly Asp Pro Ile Arg
Leu Arg His Leu Tyr Thr1 5 10 15Ser Gly Pro His Gly Leu Ser Ser Cys
Phe Leu Arg Ile Arg Ala Asp 20 25 30Gly Val Val Asp Cys Ala Arg Gly
Gln Ser Ala His Ser Leu Leu Glu 35 40 45Ile Lys Ala Val Ala Leu Arg
Thr Val Ala Ile Lys Gly Val His Ser 50 55 60Val Arg Tyr Leu Cys Met
Gly Ala Asp Gly Lys Met Gln Gly Leu Leu65 70 75 80Gln Tyr Ser Glu
Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp 85 90 95Gly Tyr Asn
Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu 100 105 110Ser
Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro 115 120
125Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu
130 135 140Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu145 150 155 160Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr
Gly Leu Glu Ala Val 165 170 175Arg Ser Pro Ser Phe Glu Lys
180168174PRTHomo sapiens 168Arg Leu Arg His Leu Tyr Thr Ser Gly Pro
His Gly Leu Ser Ser Cys1 5 10 15Phe Leu Arg Ile Arg Ala Asp Gly Val
Val Asp Cys Ala Arg Gly Gln 20 25 30Ser Ala His Ser Leu Leu Glu Ile
Lys Ala Val Ala Leu Arg Thr Val 35 40 45Ala Ile Lys Gly Val His Ser
Val Arg Tyr Leu Cys Met Gly Ala Asp 50 55 60Gly Lys Met Gln Gly Leu
Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe65 70 75 80Glu Glu Glu Ile
Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys 85 90 95His Arg Leu
Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr 100 105 110Lys
Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro 115 120
125Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp
130 135 140Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly Leu145 150 155 160Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
Phe Glu Lys 165 17016914PRTArtificial SequenceSynthetic peptide
169Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly1 5
101705PRTArtificial SequenceSynthetic peptide 170Trp Gly Asp Pro
Ile1 51714PRTArtificial SequenceSynthetic peptide 171Trp Gly Pro
Ile11725PRTArtificial SequenceSynthetic peptide 172Trp Gly Asp Pro
Val1 51734PRTArtificial SequenceSynthetic peptide 173Trp Gly Asp
Ile11744PRTArtificial SequenceSynthetic peptide 174Gly Asp Pro
Ile11755PRTArtificial SequenceSynthetic peptide 175Trp Gly Gln Pro
Ile1 51765PRTArtificial SequenceSynthetic peptide 176Trp Gly Ala
Pro Ile1 51775PRTArtificial SequenceSynthetic peptide 177Ala Gly
Asp Pro Ile1 51785PRTArtificial SequenceSynthetic peptide 178Trp
Ala Asp Pro Ile1 51795PRTArtificial SequenceSynthetic peptide
179Trp Gly Asp Ala Ile1 51805PRTArtificial SequenceSynthetic
peptide 180Trp Gly Asp Pro Ala1 51814PRTArtificial
SequenceSynthetic peptide 181Trp Asp Pro Ile11824PRTArtificial
SequenceSynthetic peptide 182Trp Gly Asp Ile11834PRTArtificial
SequenceSynthetic peptide 183Trp Gly Asp Pro11845PRTArtificial
SequenceSynthetic peptide 184Phe Gly Asp Pro Ile1
51859PRTArtificial SequenceSynthetic peptide 185Arg Leu Arg His Leu
Tyr Thr Ser Gly1 51869PRTArtificial Sequencecore sequence 186Arg
Gln Arg Tyr Leu Tyr Thr Asp Asp1 518713PRTArtificial
SequenceSynthetic peptide 187Ala Gly Pro His Val His Tyr Gly Trp
Gly Asp Pro Ile1 5 10188165PRTHomo sapiensFGF19 C-terminal sequence
188Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val1
5 10 15Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile
Lys 20 25 30Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser
Val Arg 35 40 45Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu
Leu Gln Tyr 50 55 60Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg
Pro Asp Gly Tyr65 70 75 80Asn Val Tyr Arg Ser Glu Lys His Arg Leu
Pro Val Ser Leu Ser Ser 85 90 95Ala Lys Gln Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser 100 105 110His Phe Leu Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu 115 120 125Arg Gly His Leu Glu
Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp 130 135 140Ser Met Asp
Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser145 150 155
160Pro Ser Phe Glu Lys 1651895PRTArtificial SequenceLinker sequence
189Gly Gly Gly Ser Gly1 519011PRTHomo sapiensSheet-8/Loop-8/Sheet-9
region of FGF19 190Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr1 5
1019111PRTHomo sapiensSheet-8/Loop-8/Sheet-9 region of FGF21 191Glu
Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr1 5 10192187PRTArtificial
SequenceM53 sequence 192Met Asp Ser Ser Pro Leu Leu Gln Trp Gly Asp
Pro Ile Arg Leu Arg1 5 10 15His Leu Tyr Thr Ser Gly Pro His Gly Leu
Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala Asp Gly Val Val Asp Cys
Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu Leu Glu Ile Lys Ala Val
Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly Val His Ser Val Arg Tyr
Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75 80Gln Gly Leu Leu Gln
Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu 85 90 95Ile Arg Pro Asp
Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 100 105 110Pro Val
Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg 115 120
125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro
130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met
Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185193194PRTArtificial SequenceM139 sequence 193Arg Pro
Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10 15Gly
Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25
30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys
35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Pro Asp
Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val
Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg
Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu
Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190Glu Lys194194PRTArtificial SequenceM140 sequence 194Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10
15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Arg Glu
Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190Glu Lys195194PRTArtificial SequenceM141 sequence 195Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10
15Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Cys
Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190Glu Lys196194PRTArtificial SequenceM160 sequence 196Arg
Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp1 5 10
15Gly Asp Pro Ile Arg Gln Arg His Leu Tyr Thr Ser Gly Pro His Gly
20 25 30Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys 35 40 45Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala 50 55 60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys65 70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Glu
Asp Gly Tyr Asn Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190Glu Lys197189PRTArtificial SequenceM200
Sequence 197Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro
Ile Arg1 5 10 15Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser
Ser Cys Phe 20 25 30Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala
Arg Gly Gln Ser 35 40 45Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
Leu Arg Thr Val Ala 50 55 60Ile Lys Gly Val His Ser Val Arg Tyr Leu
Cys Met Gly Ala Asp Gly65 70 75 80Lys Met Gln Gly Leu Leu Gln Tyr
Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95Glu Glu Ile Leu Glu Asp Gly
Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met145 150
155 160Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu
Val 165 170 175Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys
180 185198194PRTArtificial SequenceM201 Sequence 198Arg Pro Leu Ala
Phe Ser Asp Ser Ser Pro Leu Val His Tyr Gly Trp1 5 10 15Gly Asp Pro
Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser
Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala
Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55
60Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65
70 75 80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
Glu 85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn
Val Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser
Ser Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu
Pro Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met
Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly
Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu
Lys199194PRTArtificial SequenceM202 Sequence 199Arg Pro Leu Ala Phe
Ser Asp Ala Ser Pro His Val His Tyr Gly Trp1 5 10 15Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30Leu Ser Ser
Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45Ala Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60Leu
Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys65 70 75
80Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu
85 90 95Asp Cys Ala Phe Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val
Tyr 100 105 110Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser
Ala Lys Gln 115 120 125Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro
Leu Ser His Phe Leu 130 135 140Pro Met Leu Pro Met Val Pro Glu Glu
Pro Glu Asp Leu Arg Gly His145 150 155 160Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185 190Glu
Lys200187PRTArtificial SequenceM203 Sequence 200Arg Asp Ser Ser Pro
Leu Leu Gln Trp Gly Asp Pro Ile Arg Leu Arg1 5 10 15His Leu Tyr Thr
Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg 20 25 30Ile Arg Ala
Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His 35 40 45Ser Leu
Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys 50 55 60Gly
Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met65 70 75
80Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu
85 90 95Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg
Leu 100 105 110Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr
Lys Asn Arg 115 120 125Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met
Leu Pro Met Val Pro 130 135 140Glu Glu Pro Glu Asp Leu Arg Gly His
Leu Glu Ser Asp Met Phe Ser145 150 155 160Ser Pro Leu Glu Thr Asp
Ser Met Asp Pro Phe Gly Leu Val Thr Gly 165 170 175Leu Glu Ala Val
Arg Ser Pro Ser Phe Glu Lys 180 185201191PRTArtificial SequenceM204
Sequence 201Arg His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Asp Gln1 5 10 15Val Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly
Leu Ser Ser 20 25 30Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
Cys Ala Arg Gly 35 40 45Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala
Val Ala Leu Arg Thr 50 55 60Val Ala Ile Lys Gly Val His Ser Val Arg
Tyr Leu Cys Met Gly Ala65 70 75 80Asp Gly Lys Met Gln Gly Leu Leu
Gln Tyr Ser Glu Glu Asp Cys Ala 85 90 95Phe Glu Glu Glu Ile Leu Glu
Asp Gly Tyr Asn Val Tyr Arg Ser Glu 100 105 110Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 115 120 125Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 130 135 140Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser145 150
155 160Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe
Gly 165 170 175Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
Glu Lys 180 185 190202187PRTArtificial SequenceM205 Sequence 202Arg
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Leu Arg1 5 10
15His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg
20 25 30Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala
His 35 40 45Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala
Ile Lys 50 55 60Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp
Gly Lys Met65 70 75 80Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
Ala Phe Glu Glu Glu 85 90 95Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg
Ser Glu Lys His Arg Leu 100 105 110Pro Val Ser Leu Ser Ser Ala Lys
Gln Arg Gln Leu Tyr Lys Asn Arg 115 120 125Gly Phe Leu Pro Leu Ser
His Phe Leu Pro Met Leu Pro Met Val Pro 130 135 140Glu Glu Pro Glu
Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser145 150 155 160Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly 165 170
175Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180
185203191PRTArtificial SequenceM206 Sequence 203Arg His Pro Ile Pro
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln1 5 10 15Val Arg Leu Arg
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 20 25 30Cys Phe Leu
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 35 40 45Gln Ser
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 50 55 60Val
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala65 70 75
80Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala
85 90 95Phe Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg Ser
Glu 100 105 110Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
Arg Gln Leu 115 120 125Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe Leu Pro Met Leu 130 135 140Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser145 150 155 160Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 165 170 175Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
190204190PRTArtificial SequenceM207 Sequence 204Met Arg Asp Ser Ser
Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile1 5 10 15Arg Leu Arg His
Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25 30Phe Leu Arg
Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45Ser Ala
His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp65 70 75
80Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe
85 90 95Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg Ser Glu
Lys 100 105 110His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg
Gln Leu Tyr 115 120 125Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
Leu Pro Met Leu Pro 130 135 140Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly His Leu Glu Ser Asp145 150 155 160Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170 175Val Thr Gly Leu
Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185
19020511PRTArtificial SequenceModified Sheet-8/Loop-8/Sheet-9
region of FGF19 205Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val Tyr1 5
102065PRTArtificial SequenceLinker sequence 206Gly Ser Gly Gly Ser1
52074PRTArtificial SequenceLinker sequence 207Gly Gly Gly
Ser12084PRTArtificial SequenceLinker sequence 208Gly Gly Ser
Gly12095PRTArtificial SequenceLinker sequence 209Gly Gly Ser Gly
Gly1 52105PRTArtificial SequenceLinker sequence 210Gly Ser Gly Ser
Gly1 52115PRTArtificial SequenceLinker sequence 211Gly Ser Gly Gly
Gly1 52125PRTArtificial SequenceLinker sequence 212Gly Ser Ser Ser
Gly1 5
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