U.S. patent application number 16/533224 was filed with the patent office on 2020-02-20 for methods and compositions for the treatment of metabolic disorders and diseases.
The applicant listed for this patent is NGM Biopharmaceuticals, Inc.. Invention is credited to Darrin Anthony Lindhout, Lei Ling.
Application Number | 20200054714 16/533224 |
Document ID | / |
Family ID | 54700035 |
Filed Date | 2020-02-20 |
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United States Patent
Application |
20200054714 |
Kind Code |
A1 |
Ling; Lei ; et al. |
February 20, 2020 |
Methods and Compositions for the Treatment of Metabolic Disorders
and Diseases
Abstract
Provided herein are 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), having one or more activities, such as glucose
lowering activity, and methods for and uses in treatment of
hyperglycemia and other disorders.
Inventors: |
Ling; Lei; (Foster City,
CA) ; Lindhout; Darrin Anthony; (Mountain View,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NGM Biopharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
54700035 |
Appl. No.: |
16/533224 |
Filed: |
August 6, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15313528 |
Nov 22, 2016 |
10398758 |
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PCT/US15/32581 |
May 27, 2015 |
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16533224 |
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62004043 |
May 28, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61K 38/00 20130101; C07K 14/50 20130101; G01N 2400/00 20130101;
C12N 15/62 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/50 20060101 C07K014/50 |
Claims
1.-81. (canceled)
82. A peptide having an amino acid sequence comprising or
consisting of SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:200, SEQ ID
NO: 201, SEQ ID NO:202, or SEQ ID NO:203.
83. A pharmaceutical composition, comprising the peptide of claim
82 and a pharmaceutically acceptable carrier.
84. A pharmaceutical composition, comprising the peptide of claim
82, a glucose lowering agent, and a pharmaceutically acceptable
carrier.
85. A method of treating a subject having a hyperglycemic
condition, comprising administering an effective amount of the
peptide of claim 82 to the subject.
86. The method of claim 85, wherein the method further comprises
supplementary therapy.
87. The method of claim 86, wherein the supplementary therapy is a
weight loss surgery.
88. The method of claim 86, wherein the supplementary therapy is a
gastric bypass, gastrectomy, gastric banding, gastric balloon, or
gastric sleeve.
89. The method of claim 86, wherein the supplementary therapy is
administration of a glucose lowering agent, insulin, GLP1 analogue,
biguanide, sulphonylurea, thiazolidinedione, a dipeptidyl
peptidase-4 (DPP-4) inhibitor, a bromocriptine formulation, a bile
acid sequestrant, metformin, a thiazolidinedione (TZD), a SGLT-2
inhibitor, or any combination thereof.
90. The method of claim 86, wherein the supplementary therapy is
provided prior to said method.
91. The method of claim 86, wherein the supplementary therapy is
provided contemporaneously with said method.
92. The method of claim 86, wherein the supplementary therapy is
provided following said method.
93. The method of claim 85, wherein the hyperglycemic condition
comprises diabetes.
94. The method of claim 85, wherein the hyperglycemic condition
comprises insulin-dependent (type I) diabetes, type II diabetes, or
gestational diabetes.
95. The method of claim 85, wherein the hyperglycemic condition
comprises obesity.
96. A method of reducing glucose levels in a subject in need
thereof, comprising administering an effective amount of the
peptide of claim 82 to the subject.
97. The method of claim 96, wherein the subject has a fasting
plasma glucose level greater than 100 mg/dl or has a hemoglobin A1c
(HbA1c) level above 6%.
98. The method of claim 96, wherein the subject has a hyperglycemic
condition, insulin resistance, hyperinsulinemia, glucose
intolerance or metabolic syndrome.
99. The method of claim 96, wherein the subject has diabetes or
obesity.
100. The method of claim 96, wherein the subject has
insulin-dependent (type I) diabetes, type II diabetes, or
gestational diabetes.
101. A nucleic acid encoding the peptide of claim 82.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
15/313,528, filed Nov. 22, 2016, which is a U.S. National Stage
Application under 35 U.S.C. .sctn. 371 of International Patent
Application No. PCT/US2015/032581, filed May 27, 2015, which claims
the benefit of priority to U.S. Ser. No. 62/004,043 filed May 28,
2014, each of which is incorporated herein by reference in its
entirety.
FIELD
[0002] Provided herein are 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) having glucose lowering activity, and methods
for and uses of the variants and fusions in treatment of
hyperglycemia and other disorders.
INTRODUCTION
[0003] Diabetes mellitus is a debilitating metabolic disease caused
by absent insulin production (type 1) or insulin resistance or
insufficient insulin production (type 2) from pancreatic
.beta.-cells. .beta.-cells are specialized endocrine cells that
manufacture and store insulin for release following a meal. Insulin
is a hormone that facilitates the transfer of glucose from the
blood into tissues where it is needed. Patients with diabetes must
frequently monitor blood glucose levels and many require multiple
daily insulin injections to survive. However, such patients rarely
attain ideal glucose levels by insulin injection (Turner, R. C. et
al. JAMA 281:2005(1999)). Furthermore, prolonged elevation of
insulin levels can result in detrimental side effects such as
hypoglycemic shock and desensitization of the body's response to
insulin. Consequently, diabetic patients still develop long-term
complications, such as cardiovascular diseases, kidney disease,
blindness, nerve damage and wound healing disorders (UK Prospective
Diabetes Study (UKPDS) Group, Lancet 352:837 (1998)).
[0004] Bariatric surgery has been proposed as a potential treatment
for diabetes. It has been postulated that changes in gut hormone
secretion after the surgery are responsible for the resolution of
diabetic conditions. The underlying molecular mechanism has yet to
be elucidated, although glucagon-like peptide 1 (GLP-1) has been
speculated as a possible candidate (Rubino, F. Diabetes Care 32
Suppl 2:S368 (2009)). FGF19 is highly expressed in the distal small
intestine and transgenic over-expression of FGF19 improves glucose
homeostasis (Tomlinson, E. Endocrinology 143(5):1741-7(2002)).
Serum levels of FGF19 in humans are elevated following gastric
bypass surgery. Augmented expression and secretion of FGF19 could
at least partially explain the diabetes remission experienced
following surgery.
[0005] Accordingly, there is a need for alternative treatments of
hyperglycemic conditions such as diabetes, prediabetes, insulin
resistance, hyperinsulinemia, glucose intolerance or metabolic
syndrome, and other disorders and diseases associated with elevated
glucose levels, in humans. The compositions and methods provided
herein satisfy this need and provide related advantages.
SUMMARY
[0006] The invention is based, in part, on 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, such as glucose lowering
activity. Such variants and fusions (chimeras) of FGF19 and/or
FGF21 peptide sequences include sequences that do not substantially
or significantly increase or induce hepatocellular carcinoma (HCC)
formation or HCC tumorigenesis. Such variants and fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences further include
sequences that do not induce a substantial elevation or increase in
lipid profile.
[0007] In one embodiment, a chimeric peptide sequence comprises or
consists of: a) 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 b) 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.
[0008] In another embodiment, a chimeric peptide sequence comprises
or consists of: a) 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 b) 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.
[0009] In a further embodiment, a chimeric peptide sequence
comprises or consists of any of: a) 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 b) 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.
[0010] In an additional embodiment, a peptide sequence comprises or
consists of any of: a) a FGF19 sequence variant having one or more
amino acid substitutions, insertions or deletions compared to a
reference or wild type FGF19; b) a FGF21 sequence variant having
one or more amino acid substitutions, insertions or deletions
compared to a reference or wild type FGF21; c) a portion of an
FGF19 sequence fused to a portion of an FGF21 sequence; or d) 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.
[0011] In particular aspects, the N-terminal region comprises at
least 6 contiguous amino acids (or more, e.g., 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-40, 40-50, 50-75,
75-100 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 (or more, e.g., 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-40, 40-50, 50-75,
75-100 contiguous amino acids) of SEQ ID NO:100 (FGF21), including
the amino acid residue GQV.
[0012] In still further embodiments, a peptide sequence or a
chimeric peptide sequence comprises or consists of 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).
[0013] 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), 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.
[0014] Methods and uses provided herein can 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 205 a peptide sequence that
comprises or consists of any sequence set forth in Tables 1-9, FIG.
1 or 9, or a peptide sequence that comprises or consists of any
sequence set forth in the Sequence Listing herein.
[0015] 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.
[0016] In some embodiments, the N-terminal R residue is deleted. In
other embodiments, the peptide comprises at least one (e.g., from 1
to 20, from 1 to 15, from 1 to 10 or from 1 to 5) amino acid
substitution(s). In another embodiment, the peptide comprises at
least one (e.g., from 1 to 20, from 1 to 15, from 1 to 10 or from 1
to 5) amino acid deletion(s). In other embodiments, the peptide
comprises at least one (e.g., from 1 to 20, from 1 to 15, from 1 to
10 or from 1 to 5) amino acid insertion(s).
[0017] 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 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.
[0018] 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 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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).
[0025] In some embodiments, a peptide sequence or a chimeric
peptide 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.
[0026] 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 205 a
peptide sequence that comprises or consists of any sequence set
forth in Tables 1-9, FIG. 1 or 9, or a peptide sequence that
comprises or consists of any sequence set forth in the Sequence
Listing herein.
[0027] 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)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M3); (SEQ ID NO: 194)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M140); (SEQ ID NO: 196)
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M160); (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M69); (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M52); (SEQ ID NO: 5)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M5); (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M5-R); (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71);
(SEQ ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72);
(SEQ ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPE
NCKTLLTDIDRTHTEKPVWDGITGE (M73); (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M1); (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK (M2); (SEQ ID NO:48 or 6 or 148)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M48); (SEQ ID NO: 49 or 7 or 149)
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL
RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA
KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR
SPSFEK (M49); (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M50); (SEQ ID NO: 51 or 36 or 155)
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M51); (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVA
IKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M53); (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M70); (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M139); or (SEQ ID NO: 195)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M141);
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.
[0028] In other embodiments, the peptide comprises or consists of:
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M200) (SEQ ID NO: 197); or a subsequence or fragment thereof. In
one embodiment, the N-terminal R residue is deleted.
[0029] In some embodiments, the peptide comprises or consists of:
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK (M201) (SEQ ID NO: 198); or a subsequence or fragment
thereof. In one embodiment, the N-terminal R residue is
deleted.
[0030] In certain embodiments, the peptide comprises or consists
of: RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M202) (SEQ ID NO: 199); or a subsequence or fragment
thereof. In one embodiment, the N-terminal R residue is
deleted.
[0031] In other embodiments, the peptide comprises or consists of:
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M203) (SEQ ID NO:200); 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:
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M204) (SEQ ID NO:201); 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: RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M205) (SEQ ID NO:202); or a subsequence or fragment thereof. In
one embodiment, the N-terminal R residue is deleted.
[0034] In some embodiments, the peptide comprises or consists of:
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M206) (SEQ ID NO:203); or a subsequence or fragment thereof.
In one embodiment, the N-terminal R residue is deleted.
[0035] In other embodiments, the peptide comprises or consists of:
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M207) (SEQ ID NO:204); or a subsequence or fragment
thereof.
[0036] 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.
[0037] In various additional particular aspects, the N-terminus of
the peptide sequence includes or consists of any of:
HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO:
160); DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID
NO:6); RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID
NO:7); HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID
NO: 8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ
ID NO:9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of
SEQ ID NO: 10); RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27
of SEQ ID NO: 11); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids
1-29 of SEQ ID NO: 12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino
acids 1-27 of SEQ ID NO: 13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R)
(amino acids 2-26 of SEQ ID NO: 14); RPLAFSDAGPHVHYGGQVRLRHLYTSG
(M15) (amino acids 1-27 of SEQ ID NO: 15);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO:
16); RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID
NO: 17); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ
ID NO: 18); RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of
SEQ ID NO: 19); RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27
of SEQ ID NO:20); RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids
1-27 of SEQ ID NO:21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino
acids 1-27 of SEQ ID NO:22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23)
(amino acids 1-27 of SEQ ID NO:23); RPLAFSDAGPHHYWGDPIRLRHLYTSG
(M24) (amino acids 1-27 of SEQ ID NO:24);
RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID
NO:25); RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ
ID NO:26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of
SEQ ID NO:27); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26
of SEQ ID NO:28); RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids
1-28 of SEQ ID NO:29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino
acids 1-29 of SEQ ID NO:30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M31)
(amino acids 1-26 of SEQ ID NO:31); RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32) (amino acids 1-26 of SEQ ID NO:32);
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID
NO:33); RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ
ID NO:34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of
SEQ ID NO:35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26
of SEQ ID NO:36); RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids
1-26 of SEQ ID NO:37); RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino
acids 1-26 of SEQ ID NO:38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39)
(amino acids 1-26 of SEQ ID NO:39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG
(M40) (amino acids 1-28 of SEQ ID NO:40); DAGPHVHYGWGDPIRLRHLYTSG
(M74-R) (amino acids 2-24 of SEQ ID NO: 74); VHYGWGDPIRLRHLYTSG
(M75-R) (amino acids 2-19 of SEQ ID NO:75); RLRHLYTSG (M77-R)
(amino acids 2-10 of SEQ ID NO:77); RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9) (amino acids 1-28 of SEQ ID NO:9); RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8) (amino acids 1-26 of SEQ ID NO: 8);
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO:
12); RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID
NO:10); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ
ID NO: 13); RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of
SEQ ID NO: 14); RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27
of SEQ ID NO:43); or RDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22
of SEQ ID NO:6). In certain embodiments, the peptide comprises or
consists of any of:
[0038] HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID
NO: 160); DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID
NO:6); RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID
NO:7); HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID
NO: 8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ
ID NO:9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of
SEQ ID NO: 10); RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27
of SEQ ID NO: 11); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids
1-29 of SEQ ID NO: 12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino
acids 1-27 of SEQ ID NO: 13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R)
(amino acids 2-26 of SEQ ID NO: 14); RPLAFSDAGPHVHYGGQVRLRHLYTSG
(M15) (amino acids 1-27 of SEQ ID NO: 15);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO:
16); RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID
NO: 17); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ
ID NO: 18); RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of
SEQ ID NO: 19); RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27
of SEQ ID NO:20); RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids
1-27 of SEQ ID NO:21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino
acids 1-27 of SEQ ID NO:22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23)
(amino acids 1-27 of SEQ ID NO:23); RPLAFSDAGPHHYWGDPIRLRHLYTSG
(M24) (amino acids 1-27 of SEQ ID NO:24);
RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID
NO:25); RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ
ID NO:26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of
SEQ ID NO:27); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26
of SEQ ID NO:28); RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids
1-28 of SEQ ID NO:29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino
acids 1-29 of SEQ ID NO:30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M31)
(amino acids 1-26 of SEQ ID NO:31); RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32) (amino acids 1-26 of SEQ ID NO:32);
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID
NO:33); RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ
ID NO:34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of
SEQ ID NO:35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26
of SEQ ID NO:36); RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids
1-26 of SEQ ID NO:37); RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino
acids 1-26 of SEQ ID NO:38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39)
(amino acids 1-26 of SEQ ID NO:39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG
(M40) (amino acids 1-28 of SEQ ID NO:40); DAGPHVHYGWGDPIRLRHLYTSG
(M74-R) (amino acids 2-24 of SEQ ID NO: 74); VHYGWGDPIRLRHLYTSG
(M75-R) (amino acids 2-19 of SEQ ID NO:75); RLRHLYTSG (M77-R)
(amino acids 2-10 of SEQ ID NO:77); RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9) (amino acids 1-28 of SEQ ID NO:9); RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8) (amino acids 1-26 of SEQ ID NO: 8);
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO:
12); RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID
NO:10); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ
ID NO: 13); RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of
SEQ ID NO: 14); RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27
of SEQ ID NO:43); or RDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22
of SEQ ID NO:6). 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, 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. In various further particular
aspects, a peptide sequence includes or consists of:
TABLE-US-00002 (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP
SFEK; (SEQ ID NO: 138 or 161)
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;
(SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK; (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK; or (SEQ ID NO: 141)
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTV
AIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSF
EK;
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.
[0039] 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. 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.
[0040] 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 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.
[0041] 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).
[0042] In various still more particular aspects, a peptide or
chimeric sequence includes all or a portion of an FGF19 sequence
set forth as:
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGL
LQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188)
positioned at the C-terminus of the peptide, or the amino terminal
"R" residue is deleted from the sequence.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] In still additional embodiments, chimeric peptide sequences
and peptide sequences isolated or purified, and/or chimeric peptide
sequences and peptide sequences can be included in compositions. In
one embodiment, a chimeric peptide sequence or peptide sequence is
included in a pharmaceutical composition. Such compositions include
combinations of inactive or other active ingredients. In one
embodiment, a compositions, such as a pharmaceutical composition
includes chimeric peptide sequence or peptide sequence and a
glucose lowering agent.
[0049] In still additional embodiments, a chimeric peptide or
peptide sequence is included in a pharmaceutical composition, which
in turn can be used for practicing the methods and uses provided
herein. Such compositions include combinations of inactive or other
active ingredients. In one embodiment, a composition, such as a
pharmaceutical composition includes chimeric peptide sequence or
peptide sequence and a glucose lowering agent.
[0050] In yet further embodiments, nucleic acid molecules encoding
the chimeric peptide sequence or peptide sequence are provided.
Such molecules can further include an expression control element in
operable linkage that confers expression of the nucleic acid
molecule encoding the peptide in vitro, in a cell or in vivo, or a
vector comprising the nucleic acid molecule (e.g., a viral vector).
Transformed and host cells that express the chimeric peptide
sequences and peptide sequences are also provided.
[0051] Uses and methods of treatment that include administration or
delivery of any chimeric peptide sequence or peptide sequence are
also provided. 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, such as a subject
having, or at risk of having, a disease or disorder treatable by a
peptide sequence provided herein, in an amount effective for
treating the disease or disorder. In a further embodiment, a method
includes administering a chimeric peptide or peptide sequence
provided herein to a subject, such as a subject having a
hyperglycemic condition (e.g., diabetes, such as insulin-dependent
(type I) diabetes, type II diabetes, or gestational diabetes),
insulin resistance, hyperinsulinemia, glucose intolerance or
metabolic syndrome, or is obese or has an undesirable body
mass.
[0052] In particular aspects of the methods and uses, a chimeric
peptide sequence or peptide sequence is administered to a subject
in an amount effective to improve glucose metabolism in the
subject. In more particular aspects, a subject has a fasting plasma
glucose level greater than 100 mg/dl or has a hemoglobin A1c
(HbA1c) level above 6%, prior to administration.
[0053] In further embodiments, a use or method of treatment of a
subject is intended to or results in reduced glucose levels,
increased insulin sensitivity, reduced insulin resistance, reduced
glucagon, an improvement in glucose tolerance, or glucose
metabolism or homeostasis, improved pancreatic function, or reduced
triglyceride, cholesterol, IDL, LDL or VLDL levels, or a decrease
in blood pressure, a decrease in intimal thickening of the blood
vessel, or a decrease in body mass or weight gain.
[0054] Methods of analyzing and/or identifying a chimeric peptide
sequence or peptide sequence are also provided, such as chimeric
peptide sequences and peptide sequences that have glucose lowering
activity without substantial HCC activity. In one embodiment, a
method includes: a) providing a candidate chimeric peptide sequence
or peptide sequence; b) administering the candidate peptide
sequence to a test animal (e.g., a db/db mouse); c) measuring
glucose levels of the animal after administration of the candidate
peptide sequence, to determine if the candidate peptide sequence
reduces glucose levels. In a particular aspect, the chimeric
peptide sequence or peptide sequence is also analyzed for induction
of HCC in the animal (e.g., assessing a hepatic tissue sample from
the test animal), or expression of a marker correlating with HCC
activity, wherein a candidate peptide having glucose lowering
activity and not substantial HCC activity. Such methods identify
the candidate as having glucose lowering activity, optionally also
without substantial HCC activity.
DESCRIPTION OF DRAWINGS
[0055] FIGS. 1A-1C show FGF19 and FGF21 protein sequences (SEQ ID
NOs:99 and 100, respectively), and representative variant
sequences, namely variant M5 (SEQ ID NO: 5), variant M1 (SEQ ID NO:
1), variant M2 (SEQ ID NO:2), variant M69 (SEQ ID NO:69), variant
M3 (SEQ ID NO:3), variant M48 (SEQ ID NO:48), variant M49 (SEQ ID
NO:49), variant M50 (SEQ ID NO:50), variant M51 (SEQ ID NO:51),
variant M52 (SEQ ID NO:52), variant M53 (SEQ ID NO:192) and variant
M70 (SEQ ID NO:70) peptide sequences. Three additional allelic
(polymorphic) forms of FGF21, namely M71 (SEQ ID NO:71), M72 (SEQ
ID NO:72) and M73 (SEQ ID NO:73), are also shown.
[0056] FIG. 2 shows representative domain exchanges between FGF21
(no shading) and FGF19 (grey shading) protein sequences, and the
resultant fusion (chimeric) sequences. The amino acid regions from
each of FGF21 and FGF19 present in the fusion (chimera) are
indicated by the numbers. Glucose lowering and lipid elevation are
shown for each of the chimeric sequences.
[0057] FIGS. 3A-3I show glucose lowering and body weight data. A)
variant M5; B) variant M1; C) variant M2 and variant M69; D)
variant M3; E) variant M48 and variant M49; F) variant M51 and
variant M50; G) variant M52 peptide; H) variant M53 peptide; and I)
variant M70 peptide sequences all have glucose lowering (i.e.,
anti-diabetic) activity in db/db mice. Mice were injected with AAV
vector expressing FGF19, FGF21, the selected variants, and saline
and GFP are negative controls.
[0058] FIGS. 4A-4I show serum lipid profile (triglyceride, total
cholesterol, HDL and non-HDL) of db/db mice injected with AAV
vector expressing FGF19, FGF21 or A) variant M5; B) variant M1; C)
variant M2 and variant M69; D) variant M3; E) variant M48 and
variant M49; F) variant M51 and variant M50; G) variant M52
peptide; H) variant M53 peptide; and I) variant M70 peptide
sequences. Variant M5 peptide sequence did not increase or elevate
lipids, in contrast to FGF19, M1, M2 and M69 which increases and
elevates lipids. Serum levels of all variants were comparable.
Saline and GFP are negative controls.
[0059] FIGS. 5A-5I show HCC-related data for A) variant M5; B)
variant M1; C) variant M2 and variant M69; D) variant M3; E)
variant M48 and variant M49; F) variant M51 and variant M50; G)
variant M52; H) variant M53 peptide; and I) variant M70 peptide
sequences. None of the variants significantly increased or induced
HCC formation or HCC tumorigenesis, in contrast to FGF19. HCC score
is 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.
[0060] FIGS. 6A-6I show lean mass or fat mass data for A) variant
M5; B) variant M1; C) variant M2 and variant M69; D) variant M3; E)
variant M48 and variant M49; F) variant M51 and variant M50; G)
variant M52; H) variant M53 peptide; and I) variant M70 peptide
sequences. Except for M2, M5 and M69, the variant peptide sequences
reduce lean mass or fat mass, in contrast to FGF21.
[0061] FIGS. 7A-7B show graphical data demonstrating that injection
of the recombinant A) variant M5; and B) variant M69 polypeptides
reduce blood glucose in ob/ob mice.
[0062] FIG. 8 depicts that the expression of FGFR4/.beta.-klotho
complex in L6 cells potentiates activation of intracellular
signaling pathways by FGF19, M3 and M70.
[0063] FIG. 9 shows representative variant sequences, namely
variant M200 (SEQ ID NO: 197), variant M201 (SEQ ID NO:198),
variant M202 (SEQ ID NO:199), variant M203 (SEQ ID NO:200), variant
M204 (SEQ ID NO:201), variant M205 (SEQ ID NO:202), variant M206
(SEQ ID NO:203), and variant M207 (SEQ ID NO:204) peptide
sequences.
[0064] FIGS. 10A-10D show that M200 activates intracellular
signaling pathways in 293T cells expressing FGFR1c/.beta.-klotho
(KLB) receptor complexes or FGFR4/.beta.-klotho receptor complexes
as effectively as FGF19, and M200 also induced phosphorylation and
activation of ERK with a similar potency and efficacy as wild
type.
[0065] FIGS. 11A-11C show glucose lowering and body weight data. A)
Variant M200 and M202; B) variant M201; C) variant M203, M204, M205
and M206 peptide sequences all have glucose lowering (i.e.,
anti-diabetic) activity in db/db mice. Mice were injected with AAV
vector expressing FGF19, FGF21, the selected variants, and saline
and GFP are negative controls.
[0066] FIGS. 12A-12C show serum lipid profile (triglyceride, total
cholesterol, HDL and non-HDL) of db/db mice injected with AAV
vector expressing FGF19, FGF21 or A) variant M200 or M202; B)
variant M201; or C) variant M203, M204, M205 or M206. Saline and
GFP are negative controls.
[0067] FIGS. 13A-13C show HCC-related data for A) variant M200 and
M202; B) variant M201; and C) variant M203, M204, M205 and M206
peptide sequences. None of the variants significantly increased or
induced HCC formation or HCC tumorigenesis, in contrast to FGF19.
HCC score is 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.
[0068] FIGS. 14A-14C show lean mass or fat mass data for A) variant
M200 and M202; B) variant M201; and C) variant M203, M204, M205 and
M206 peptide sequences.
DETAILED DESCRIPTION
[0069] Provided herein are chimeric and peptide sequences that are
able to lower or reduce levels of glucose. In one embodiment, a
chimeric peptide sequence comprises 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.
[0070] In another embodiment, a chimeric peptide sequence comprises
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.
[0071] In further embodiments, a peptide sequence comprises or
consists of a FGF19 sequence 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
comprises 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 comprises or consists of a portion of an FGF19
sequence fused to a portion of an FGF21 sequence. In still
additional embodiments, a peptide sequence comprises 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.
[0072] Also provided herein are methods and uses of treating a
subject having or at risk of having a metabolic disorder treatable
using variants and fusions of FGF19 and/or FGF21 peptide sequences.
In one embodiment, a method includes contacting or administering to
a subject one or more variant or fusion FGF19 and/or FGF21 peptide
sequences in an amount effective for treating the disorder. In
another embodiment, a method includes contacting or administering
to a subject one or more nucleic acid molecules encoding a variant
or fusion FGF19 and/or FGF21 peptide sequence (for example, an
expression control element in operable linkage with the nucleic
acid encoding the peptide sequence, optionally including a vector),
in an amount effective for treating the disorder.
[0073] Although an understanding of the underlying mechanism of
action of the peptides provided herein is not required in order to
practice the invention, without being bound to any particular
theory or hypothesis, it is believed that peptides provided herein
mimic, at least in part, the effect that bariatric surgery has on,
for example, glucose homeostasis and weight loss. Changes in
gastrointestinal hormone secretion (e.g., glucagon-like peptide 1
(GLP-1)) after bariatric surgery are believed responsible for the
resolution of, for example, diabetic conditions. FGF19 is highly
expressed in the distal small intestine, and transgenic
over-expression of FGF19 improves glucose homeostasis. Because
levels of FGF19 in humans are also elevated following gastric
bypass surgery, the elevated FGF19 might be involved with the
remission of diabetes observed following bariatric surgery.
[0074] A representative reference or wild type FGF19 sequence is
set forth as:
TABLE-US-00003 (SEQ ID NO: 99)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.
[0075] A representative reference or wild type FGF21 sequence is
set forth as:
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID
NO: 100). FGF21 allelic variants are illustrated in FIG. 1 (e.g.,
M70, M71 and M72).
[0076] 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 abbreviation or 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.
[0077] Exemplified herein are peptide sequences, distinct from
reference FGF19 and FGF21 polypeptides set forth herein, that
reduce or lower glucose, in vivo (e.g., Tables 1-9, FIGS. 1 and 9,
and the Sequence Listing). Non-limiting particular examples are a
peptide sequence with amino-terminal amino acids 1-16 of FGF21
fused to carboxy-terminal amino acids 21-194 of FGF19; a peptide
sequence with amino-terminal amino acids 1-147 of FGF19 fused to
carboxy-terminal amino acids 147-181 of FGF21; a peptide sequence
with amino-terminal amino acids 1-20 of FGF19 fused to
carboxy-terminal amino acids 17-181 of FGF21; a peptide sequence
with amino-terminal amino acids 1-146 of FGF21 fused to
carboxy-terminal amino acids 148-194 of FGF19; and a peptide
sequence with amino-terminal amino acids 1-20 of FGF19 fused to
internal amino acids 17-146 of FGF21 fused to carboxy-terminal
amino acids 148-194 of FGF19.
[0078] Additional particular peptides sequences have a WGDPI (SEQ
ID NO: 170) sequence motif corresponding to the WGDPI sequence of
amino acids 16-20 of FGF19 (SEQ ID NO:99), lack a WGDPI SEQ ID NO:
170) sequence motif corresponding to the WGDPI sequence of amino
acids 16-20 of FGF19 (SEQ ID NO:99), or have a substituted (i.e.,
mutated) WGDPI SEQ ID NO: 170) sequence motif corresponding to the
WGDPI sequence of amino acids 16-20 of FGF19 (SEQ ID NO:99).
[0079] Particular peptide sequences provided herein also include
sequences distinct from FGF19 and FGF21 (e.g., as set forth
herein), and FGF19 variant sequences having any 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 FGF19 WGDPI (SEQ ID NO: 170) sequence at amino
acids 16-20. Accordingly, the wild-type FGF19 and FGF21 (e.g., as
set forth herein as SEQ ID NOS:99 and 100, respectively) may be
excluded sequences, and FGF19 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 may
also be excluded. This exclusion, however, does not apply to where
a sequence has, for example, 3 FGF21 residues fused to FGF19
having, for example, any of GQV, GQV, GDI, or GPI, or 2 FGF21
residues fused to any of WGPI (SEQ ID NO:171), WGDI (SEQ ID
NO:173), GDPI (SEQ ID NO:174), WDPI (SEQ ID NO:181), WGDI (SEQ ID
NO: 182), or WGDP (SEQ ID NO: 183).
[0080] Particular non-limiting examples of peptide sequences
include or consist of all or a part of a sequence variant specified
herein as M1-M98 (SEQ ID NOs:1-52, 192, and 54-98, respectively).
More particular non-limiting examples of peptide sequences include
or consist of all or a part of a sequence set forth as:
TABLE-US-00004 (SEQ ID NO: 160)
EIPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M5-R)(FGF21 sequences can also include an "R" residue at the amino
terminus); (SEQ ID NO: 138 and 161)
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;
(SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M1); (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK (M2); (SEQ ID NO: 141)
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTV
AIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSF EK;
(SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M69); (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M52); (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M5-R); (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71);
(SEQ ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72);
(SEQ ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPE
NCKTLLTDIDRTHTEKPVWDGITGE (M73); (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M3); (SEQ ID NO: 48, 6 or 148)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M48); (SEQ ID NO: 49, 7 or 149)
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL
RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA
KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR
SPSFEK (M49); (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M50); (SEQ ID NO: 51, 36 or 155)
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M51); (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVA
IKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M53); (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M70); (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M139); (SEQ ID NO: 194)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M140); (SEQ ID NO: 195)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M141); or (SEQ ID NO: 196)
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (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 is deleted.
[0081] In other embodiments, the peptide comprises or consists of:
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
(M200) (SEQ ID NO: 197); or a subsequence or fragment thereof. In
one embodiment, the N-terminal R residue is deleted.
[0082] In some embodiments, the peptide comprises or consists of:
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK (M201) (SEQ ID NO: 198); or a subsequence or fragment
thereof. In one embodiment, the N-terminal R residue is
deleted.
[0083] In certain embodiments, the peptide comprises or consists
of: RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (M202) (SEQ ID NO: 199); or a subsequence or fragment
thereof. In one embodiment, the N-terminal R residue is
deleted.
[0084] In other embodiments, the peptide comprises or consists of:
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M203) (SEQ ID NO:200); or a subsequence or fragment thereof. In
one embodiment, the N-terminal R residue is deleted.
[0085] In some embodiments, the peptide comprises or consists of:
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M204) (SEQ ID NO:201); or a subsequence or fragment thereof.
In one embodiment, the N-terminal R residue is deleted.
[0086] In certain embodiments, the peptide comprises or consists
of: RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
(M205) (SEQ ID NO:202); or a subsequence or fragment thereof. In
one embodiment, the N-terminal R residue is deleted.
[0087] In some embodiments, the peptide comprises or consists of:
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M206) (SEQ ID NO:203); or a subsequence or fragment thereof.
In one embodiment, the N-terminal R residue is deleted.
[0088] In other embodiments, the peptide comprises or consists of:
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK (M207) (SEQ ID NO:204); or a subsequence or fragment
thereof.
[0089] Additional particular non-limiting examples of peptide
sequences, having at the N-terminus, a peptide sequence including
or consisting of all or a part of any of: HPIPDSSPLLQFGGQVRLRHLYTSG
(M5-R) (amino acids 1-25 of SEQ ID NO: 160); DSSPLLQFGGQVRLRHLYTSG
(M6) (M6-R) (amino acids 2-22 of SEQ ID NO:6);
RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO:7);
HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO:
8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID
NO:9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ
ID NO: 10); RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of
SEQ ID NO: 11); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids
1-29 of SEQ ID NO: 12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino
acids 1-27 of SEQ ID NO: 13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R)
(amino acids 2-26 of SEQ ID NO: 14); RPLAFSDAGPHVHYGGQVRLRHLYTSG
(M15) (amino acids 1-27 of SEQ ID NO: 15);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO:
16); RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID
NO: 17); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ
ID NO: 18); RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of
SEQ ID NO: 19); RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27
of SEQ ID NO:20); RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids
1-27 of SEQ ID NO:21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino
acids 1-27 of SEQ ID NO:22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23)
(amino acids 1-27 of SEQ ID NO:23); RPLAFSDAGPHHYWGDPIRLRHLYTSG
(M24) (amino acids 1-27 of SEQ ID NO:24);
RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID
NO:25); RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ
ID NO:26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of
SEQ ID NO:27); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26
of SEQ ID NO:28); RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids
1-28 of SEQ ID NO:29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino
acids 1-29 of SEQ ID NO:30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M31)
(amino acids 1-26 of SEQ ID NO:31); RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32) (amino acids 1-26 of SEQ ID NO:32);
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID
NO:33); RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ
ID NO:34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of
SEQ ID NO:35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26
of SEQ ID NO:36); RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids
1-26 of SEQ ID NO:37); RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino
acids 1-26 of SEQ ID NO:38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39)
(amino acids 1-26 of SEQ ID NO:39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG
(M40) (amino acids 1-28 of SEQ ID NO:40); DAGPHVHYGWGDPIRLRHLYTSG
(M74-R) (amino acids 2-24 of SEQ ID NO: 74); VHYGWGDPIRLRHLYTSG
(M75-R) (amino acids 2-19 of SEQ ID NO:75); RLRHLYTSG (M77-R)
(amino acids 2-10 of SEQ ID NO:77); RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9) (amino acids 1-28 of SEQ ID NO:9); RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8) (amino acids 1-26 of SEQ ID NO: 8);
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO:
12); RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID
NO:10); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ
ID NO: 13); RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of
SEQ ID NO: 14); RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27
of SEQ ID NO:43); or RDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22
of SEQ ID NO:6); and for any of the foregoing peptide sequences the
amino terminal R residue may be deleted. In certain embodiments,
the peptide comprises or consists of any of:
HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO:
160); DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID
NO:6); RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID
NO:7); HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID
NO: 8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ
ID NO:9); HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of
SEQ ID NO: 10); RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27
of SEQ ID NO: 11); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids
1-29 of SEQ ID NO: 12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino
acids 1-27 of SEQ ID NO: 13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R)
(amino acids 2-26 of SEQ ID NO: 14); RPLAFSDAGPHVHYGGQVRLRHLYTSG
(M15) (amino acids 1-27 of SEQ ID NO: 15);
RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO:
16); RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID
NO: 17); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ
ID NO: 18); RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of
SEQ ID NO: 19); RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27
of SEQ ID NO:20); RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids
1-27 of SEQ ID NO:21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino
acids 1-27 of SEQ ID NO:22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23)
(amino acids 1-27 of SEQ ID NO:23); RPLAFSDAGPHHYWGDPIRLRHLYTSG
(M24) (amino acids 1-27 of SEQ ID NO:24);
RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID
NO:25); RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ
ID NO:26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of
SEQ ID NO:27); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26
of SEQ ID NO:28); RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids
1-28 of SEQ ID NO:29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino
acids 1-29 of SEQ ID NO:30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M31)
(amino acids 1-26 of SEQ ID NO:31); RHPIPDSSPLLQFGDQVRLRHLYTSG
(M32) (amino acids 1-26 of SEQ ID NO:32);
RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID
NO:33); RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ
ID NO:34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of
SEQ ID NO:35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26
of SEQ ID NO:36); RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids
1-26 of SEQ ID NO:37); RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino
acids 1-26 of SEQ ID NO: 38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39)
(amino acids 1-26 of SEQ ID NO:39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG
(M40) (amino acids 1-28 of SEQ ID NO:40); DAGPHVHYGWGDPIRLRHLYTSG
(M74-R) (amino acids 2-24 of SEQ ID NO: 74); VHYGWGDPIRLRHLYTSG
(M75-R) (amino acids 2-19 of SEQ ID NO:75); RLRHLYTSG (M77-R)
(amino acids 2-10 of SEQ ID NO:77); RHPIPDSSPLLQFGWGDPIRLRHLYTSG
(M9) (amino acids 1-28 of SEQ ID NO:9); RHPIPDSSPLLQWGDPIRLRHLYTSG
(M8) (amino acids 1-26 of SEQ ID NO: 8);
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO:
12); RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID
NO:10); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ
ID NO: 13); RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of
SEQ ID NO: 14); RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27
of SEQ ID NO:43); or RDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22
of SEQ ID NO:6). 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, 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
[0090] Peptide sequences provided herein additionally include those
with reduced or absent induction or formation of HCC 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. Peptide sequences provided herein also include those with
greater glucose lowering activity compared to FGF19, or an FGF19
variant sequence having any of GQV, GDI, WGPI, 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.
Peptide sequences provided herein moreover include those with less
lipid (e.g., 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.
[0091] 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.
[0092] 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.
[0093] Also provided herein are subsequences, variants and modified
forms of the exemplified peptide sequences (including the FGF19 and
FGF21 variants and subsequences listed in Tables 1-9, FIGS. 1 and
9, and the Sequence Listing, and the FGF19/FGF21 fusions and
chimeras listed in Tables 1-9, FIGS. 1 and 9, and the Sequence
Listing), so long as the foregoing retains at least a detectable or
measureable activity or function. For example, certain exemplified
variant peptides have FGF19 C-terminal sequence,
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGL
LQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188) at the
C-terminal portion, e.g., following the "TSG" amino acid residues
of the variant.
[0094] 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, in cells 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 "VM" 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)).
[0095] Accordingly, the "peptide," "polypeptide," and "protein"
sequences provided herein include subsequences, variants and
modified forms of the FGF19 and FGF21 variants and subsequences
listed in Tables 1-9, FIGS. 1 and 9, and the Sequence Listing, and
the FGF19/FGF21 fusions and chimeras listed in Tables 1-9, FIGS. 1
and 9, and the Sequence Listing, so long as the subsequence,
variant or modified form (e.g., fusion or chimera) retains at least
a detectable activity or function.
[0096] 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.
[0097] Particular examples of such subsequences, variants and
modified forms of the peptide sequences exemplified herein (e.g., a
peptide sequence listed in Tables 1-9, FIGS. 1 and 9, and the
Sequence Listing) 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.
[0098] 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.
[0099] 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.
[0100] 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.,
glucose lowering activity. 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., glucose lowering activity.
[0101] Particular examples of subsequences, variants and modified
forms of the peptide sequences exemplified herein (e.g., a peptide
sequence listed in Tables 1-9, FIGS. 1 and 9, and the Sequence
Listing) 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 (for example, a peptide sequence in any of Table 1-9,
FIGS. 1 and 9, and the Sequence Listing). 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 these regions.
[0102] 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)).
[0103] In the peptide sequences, including subsequences, variants
and modified forms of the peptide sequences exemplified herein
(e.g., sequences listed in Table 1-9, FIGS. 1 and 9, and the
Sequence Listing) 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)).
[0104] 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.
[0105] 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, preferably 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 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., glucose lowering, improved glucose or lipid
metabolism, anti-diabetic activity, absence of substantial HCC
formation or tumorigenesis, absence of substantial modulation of
lean or fat mass, etc.).
[0106] 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.
[0107] 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 otherwise 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 C1 to C6 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.
[0108] 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 substituents
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.
[0109] Covalent modifications of the peptide sequences provided
herein, including subsequences, variants and modified forms of the
peptide sequences exemplified herein (e.g., sequences listed in
Table 1-9, FIGS. 1 and 9, and the Sequence Listing), are also
provided herein. One 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.
[0110] 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.
[0111] Exemplified peptide sequences, and subsequences, variants
and modified forms of the peptide sequences exemplified herein
(e.g., sequences listed in Table 1-9, FIG. 1, and the Sequence
Listing), 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.
[0112] 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.
[0113] Another type of modification of the peptide sequences
provided herein, including subsequences, sequence variants and
modified forms of the exemplified peptide sequences (including the
peptides listed in Table 1-9, FIGS. 1 and 9, and the Sequence
Listing), is glycosylation. As used herein, "glycosylation" broadly
refers to the presence, addition or attachment of one or more sugar
(e.g., carbohydrate) moieties to proteins, lipids or other organic
molecules. The use of the term "deglycosylation" herein is
generally intended to mean the removal or deletion, of one or more
sugar (e.g., carbohydrate) moieties. In addition, the phrase
includes qualitative changes in the glycosylation of the native
proteins involving a change in the type and proportions (amount) of
the various sugar (e.g., carbohydrate) moieties present.
[0114] Glycosylation can be achieved by modification of an amino
acid residue, or by adding one or more glycosylation sites that may
or may not be present in the native sequence. For example, a
typically non-glycosylated residue can be substituted for a residue
that may be glycosylated. Addition of glycosylation sites can be
accomplished by altering the amino acid sequence. The alteration to
the peptide sequence 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.
[0115] Peptide sequences provided herein may optionally be altered
through changes at the nucleotide (e.g., DNA) level, particularly
by mutating the DNA encoding the peptide at preselected bases such
that codons are generated that will translate into the desired
amino acids. Another means of increasing the number of carbohydrate
moieties on the peptide is by chemical or enzymatic coupling of
glycosides to the polypeptide (see, for example, in WO 87/05330).
De-glycosylation can be accomplished by removing the underlying
glycosylation site, by deleting the glycosylation by chemical
and/or enzymatic means, or by substitution of codons encoding amino
acid residues that are glycosylated. Chemical deglycosylation
techniques are known, and enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo- and exo-glycosidases.
[0116] Various cell lines can be used to produce proteins that are
glycosylated. One non-limiting example is Dihydrofolate reductase
(DHFR)--deficient Chinese Hamster Ovary (CHO) cells, which are a
commonly used host cell for the production of recombinant
glycoproteins. These cells do not express the enzyme
beta-galactoside alpha-2,6-sialyltransferase and therefore do not
add sialic acid in the alpha-2,6 linkage to N-linked
oligosaccharides of glycoproteins produced in these cells.
[0117] Another type of modification is to conjugate (e.g., link)
one or more additional components or molecules at the N- and/or
C-terminus of a peptide sequence provided herein, such as another
protein (e.g., a protein having an amino acid sequence heterologous
to the subject protein), or a carrier molecule. Thus, an exemplary
peptide sequence can be a conjugate with another component or
molecule.
[0118] In certain embodiments, the amino- or carboxy-terminus of a
peptide 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 can increase
the systemic half-life of biopharmaceuticals, and thus the
biopharmaceutical product may have prolonged activity or require
less frequent administration. 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.
Well-known and validated Fc-fusion drugs consist of two copies of a
biopharmaceutical linked to the Fc region of an antibody to improve
pharmacokinetics, solubility, and production efficiency. More
recent Fc-fusion technology links a single copy of a
biopharmaceutical to Fc region of an antibody to optimize the
pharmacokinetic and pharmacodynamic properties of the
biopharmaceutical as compared to traditional Fc-fusion
conjugates.
[0119] A conjugate modification can be used to produce a peptide
sequence that retains activity with an additional or complementary
function or activity of the second molecule. For example, a peptide
sequence may be conjugated to a molecule, e.g., to facilitate
solubility, storage, in vivo or shelf half-life or stability,
reduction in immunogenicity, delayed or controlled release in vivo,
etc. Other functions or activities include a conjugate that reduces
toxicity relative to an unconjugated peptide sequence, a conjugate
that targets a type of cell or organ more efficiently than an
unconjugated peptide sequence, or a drug to further counter the
causes or effects associated with a disorder or disease as set
forth herein (e.g., diabetes).
[0120] Clinical effectiveness of protein therapeutics may be
limited by short plasma half-life and susceptibility to
degradation. Studies of various therapeutic proteins have shown
that various modifications, including conjugating or linking the
peptide sequence to any of a variety of nonproteinaceous polymers,
e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes (see, for example, typically via a linking moiety
covalently bound to both the protein and the nonproteinaceous
polymer (e.g., a PEG) can prolong half-life. 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.
[0121] PEGs suitable for conjugation to a peptide sequence provided
herein is 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 peptide sequence can be linear or branched.
Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are also
provided herein. A molecular weight of the PEG used is not
restricted to any particular range, but certain embodiments have a
molecular weight between 500 and 20,000 while other embodiments
have a molecular weight between 4,000 and 10,000.
[0122] Also 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.
[0123] PEG may directly or indirectly (e.g., through an
intermediate) bind to the peptide sequences provided herein. For
example, in one embodiment, PEG binds via a terminal reactive group
(a "spacer"). The spacer, is, for example, a terminal reactive
group which mediates a bond between the free amino or carboxyl
groups of one or more of the peptide 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 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.
[0124] Conjugation of one or more of peptide 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.gtoreq.7), and longer
reaction time tend to increase the number of PEGs attached. Various
methods 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.
[0125] Peptide sequences provided herein, including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (including the peptides listed in Table 1-9, FIGS. 1 and
9, and the Sequence Listing), further include conjugation to large,
slowly metabolized macromolecules such as proteins;
polysaccharides, such as sepharose, agarose, cellulose, cellulose
beads; polymeric amino acids such as polyglutamic acid, polylysine;
amino acid copolymers; inactivated virus particles; inactivated
bacterial toxins such as toxoid from diphtheria, tetanus, cholera,
leukotoxin molecules; inactivated bacteria; and dendritic cells.
Such conjugated forms, if desired, can be used to produce
antibodies against peptide sequences provided herein.
[0126] Additional suitable components and molecules for conjugation
include, for example, thyroglobulin; albumins such as human serum
albumin (HSA); 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.
[0127] Fusion of albumin to a peptide sequence provided herein can,
for example, be achieved by genetic manipulation, such that the DNA
coding for HSA (human serum albumin), or a fragment thereof, is
joined to the DNA coding for a peptide sequence. Thereafter, a
suitable host can be transformed or transfected with the fused
nucleotide sequence in the form of, for example, a suitable
plasmid, so as to express a fusion polypeptide. The expression may
be effected in vitro from, for example, prokaryotic or eukaryotic
cells, or in vivo from, for example, a transgenic organism. In some
embodiments, the expression of the fusion protein is performed in
mammalian cell lines, for example, CHO cell lines.
[0128] Further means for genetically fusing target proteins or
peptides to albumin include a technology known as Albufuse.RTM.
(Novozymes Biopharma A/S; Denmark), and the conjugated therapeutic
peptide sequences frequently become much more effective with better
uptake in the body. The technology has been utilized commercially
to produce Albuferon.RTM. (Human Genome Sciences), a combination of
albumin and interferon .alpha.-2B used to treat hepatitis C
infection.
[0129] Another embodiment entails the use of one or more human
domain antibodies (dAb). dAbs are the smallest functional binding
units of human antibodies (IgGs) and have favorable stability and
solubility characteristics. The technology entails a dAb(s)
conjugated to HSA (thereby forming a "AlbudAb"; see, e.g.,
EP1517921B, WO2005/118642 and WO2006/051288) and a molecule of
interest (e.g., a peptide sequence provided herein). AlbudAbs are
often smaller and easier to manufacture in microbial expression
systems, such as bacteria or yeast, than current technologies used
for extending the serum half-life of peptides. As HSA has a
half-life of about three weeks, the resulting conjugated molecule
improves the half-life. Use of the dAb technology may also enhance
the efficacy of the molecule of interest.
[0130] 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.
[0131] 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 -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, preferably 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 accordingly 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] Other means and methods for prolonging the circulation
half-life, increasing stability, reducing clearance, or altering
immunogenicity or allergenicity of a peptide sequence provided
herein involves modification of the peptide sequence by hesylation,
which utilizes hydroxyethyl starch derivatives linked to other
molecules in order to modify the molecule's characteristics.
Various aspects of hesylation are described in, for example, U.S.
Patent Appln. Nos. 2007/0134197 and 2006/0258607.
[0136] Any of the foregoing components and molecules used to modify
peptide 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 peptide 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, 2,
3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 amino acids (e.g.,
Gly).
[0137] Exemplary flexible linkers include glycine polymers
(G).sub.n, glycine-serine polymers (for example, (GS).sub.n,
GSGGS.sub.n (SEQ ID NO:129) and GGGS.sub.n(SEQ ID NO:130), 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 a neutral tether between components. Exemplary
flexible linkers include, but are not limited to GGSG (SEQ ID NO:
131), GGSGG (SEQ ID NO: 132), GSGSG (SEQ ID NO: 133), GSGGG (SEQ ID
NO: 134), GGGSG (SEQ ID NO: 189), and GSSSG (SEQ ID NO: 135).
[0138] Peptide sequences provided herein, including the FGF19 and
FGF21 variants and subsequences and the FGF19/FGF21 fusions and
chimeras listed in Table 1-9, FIG. 1, and the Sequence Listing, as
well as subsequences, sequence variants and modified forms of the
sequences listed in Table 1-9, FIGS. 1 and 9, and the Sequence
Listing have one or more activities as set forth herein. One
example of an activity is glucose lowering activity. Another
example of an activity is reduced stimulation or formation of HCC,
for example, as compared to FGF19. An additional example of an
activity is lower or reduced lipid (e.g., triglyceride,
cholesterol, non-HDL) or HDL increasing activity, for example, as
compared to FGF21. A further example of an activity is a lower or
reduced lean muscle mass reducing activity, for example, as
compared to FGF21. Yet another example of an activity is binding to
FGFR4, or activating FGFR4, for example, peptide sequences that
bind to FGFR4 with an affinity comparable to or greater than FGF19
binding affinity for FGFR4; and peptide sequences that activate
FGFR4 to an extent or amount comparable to or greater than FGF19
activates FGFR4. Still further examples of activities include
down-regulation or reduction of aldo-keto reductase gene
expression, for example, compared to FGF19; up-regulation or
increased Slc1a2 gene expression compared to FGF21.
[0139] More particularly, peptide sequences provided herein,
including the FGF19 and FGF21 variants and subsequences and the
FGF19/FGF21 fusions and chimeras listed in Table 1-9, FIGS. 1 and
9, and the Sequence Listing, as well as subsequences, variants and
modified forms of the sequences listed in Table 1-9, FIGS. 1 and 9,
and the Sequence Listing include those with the following
activities: peptide sequences having 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; peptide sequences having greater glucose lowering
activity compared to FGF19, or 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; peptide sequences having less lipid
increasing activity (e.g., less triglyceride, cholesterol, non-HDL)
or more 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; and peptide
sequences having less lean mass reducing activity as compared to
FGF21.
[0140] More particularly, peptide sequences provided herein,
including the FGF19 and FGF21 variants and subsequences and the
FGF19/FGF21 fusions and chimeras listed in Table 1-9, FIGS. 1 and
9, and the Sequence Listing, as well as subsequences, variants and
modified forms of the sequences listed in Table 1-9, FIGS. 1 and 9,
and the Sequence Listing include those with the following
activities: peptide sequences that bind to FGFR4, or activate
FGFR4, such as peptide sequences that bind to FGFR4 with an
affinity comparable to or greater than FGF19 binding affinity for
FGFR4; peptide sequences that activate FGFR4 to an extent or amount
comparable to or greater than FGF19 activates FGFR4; peptide
sequences that down-regulate or reduce aldo-keto reductase gene
expression, for example, compared to FGF19; and peptide sequences
that up-regulate or increase solute carrier family 1, member 2
(Slc1a2) gene expression as compared to FGF21.
[0141] As disclosed herein, variants include various N-terminal
modifications and/or truncations of FGF19, including variants in
which there has been a substitution of one or several N-terminal
FGF19 amino acids with amino acids from FGF21. Such variants
include variants having glucose lowering activity, as well as a
favorable lipid profile and are not measurably or detectably
tumorigenic.
[0142] In various particular aspects, modifications to the Loop-8
region of FGF19 (residues 127-129 are defined as constituting the
Loop-8 region) are disclosed herein that have glucose lowering
activity and also 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). As set
forth in Example 9-14 and Tables 8 and 9, certain combinations of
R127L and P128E substitutions to the FGF19 framework had an
unexpectedly positive effect on HCC formation. A combination of
R127L and P128E substitutions and a substitution of Gln (Q) for Leu
(L) in the FGF19 core region (see, e.g., core region sequence
denoted in Tables 1-4, 8 and 9) 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).
[0143] Activities such as, for example, HCC formation or
tumorigenesis, glucose lowering activity, lipid increasing
activity, or lean mass reducing activity can be ascertained in an
animal, such as a db/db mouse. Measurement of binding to FGFR4 or
activation of FGFR4 can be ascertained by assays disclosed herein
(see, for example, Example 1) or known to the skilled artisan.
[0144] The term "bind," or "binding," when used in reference to a
peptide sequence, means that the peptide sequence interacts at the
molecular level. Thus, a peptide sequence that binds to FGFR4 binds
to all or a part of the FGFR4 sequence. 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).
[0145] 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.
[0146] 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.
[0147] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in Table 1-9, FIGS. 1 and 9, and
the Sequence Listing), can also be modified to form a chimeric
molecule. Also 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.
[0148] The term "peptide" also includes dimers or multimers
(oligomers) of peptides. Also provided herein are dimers or
multimers (oligomers) of the exemplified peptide sequences as well
as subsequences, variants and modified forms of the exemplified
peptide sequences (e.g., sequences listed in Table 1-9, FIGS. 1 and
9, and the Sequence Listing).
[0149] Also provided herein are nucleic acid molecules encoding
peptide sequences provided herein, including subsequences, sequence
variants and modified forms of the sequences listed in Table 1-9,
FIGS. 1 and 9, and the Sequence Listing, and vectors that include
nucleic acid that encodes the peptide. 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. For example,
a subsequence, a variant or modified form of an exemplified peptide
sequence disclosed herein (e.g., a sequence listed in Table 1-9,
FIGS. 1 and 9, and the Sequence Listing) that retains some ability
to lower or reduce glucose, provide normal glucose homeostasis, or
reduce the histopathological conditions associated with chronic or
acute hyperglycemia in vivo, etc.
[0150] 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 analogues and derivatives.
[0151] As a result of the degeneracy of the genetic code, nucleic
acid molecules include sequences degenerate with respect to nucleic
acid molecules encoding the peptide sequences provided herein.
Thus, degenerate nucleic acid sequences encoding peptide sequences,
including subsequences, variants and modified forms of the peptide
sequences exemplified herein (e.g., sequences listed in Table 1-9,
FIGS. 1 and 9, and the Sequence Listing), 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.
[0152] 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 sequencing, gel
electrophoresis, UV spectrometry.
[0153] 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.
[0154] 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.
[0155] 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' and 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.
[0156] 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. As used herein, 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
(e.g., a subject in need of treatment), or producing peptide
sequences, for example.
[0157] As used herein, the term "transgene" means a polynucleotide
that has been introduced into a cell or organism by artifice. For
example, 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.
[0158] Bacterial system promoters include T7 and inducible
promoters such as pL of bacteriophage .lamda., 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.
[0159] 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.
[0160] 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).
[0161] 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
Biolovgy, Cold Spring Harbor Laboratory).
[0162] Also provided herein are 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.
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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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 lowered glucose and is
anti-diabetic.
[0170] 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, also provided herein are 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.
[0171] 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.
[0172] 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.
[0173] 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. Peptide sequences provided herein are generally not known
or believed to exist in nature. However, for a composition that
does exist in nature, an isolated composition will generally be
free of some, a substantial number of, or most or all other
materials with which it typically associates with in nature. Thus,
an isolated peptide sequence that also occurs in nature does not
include polypeptides or polynucleotides present among millions of
other sequences, such as proteins of a protein library or nucleic
acids in a genomic or cDNA library, for example. A "purified"
composition includes combinations with one or more other inactive
or active molecules. For example, a peptide sequence provided
herein combined with another drug or agent, such as a glucose
lowering drug or therapeutic agent, for example.
[0174] 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 where 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.
[0175] Also provided are compositions and mixtures of peptide
sequences provided herein, including subsequences, variants and
modified forms of the exemplified peptide sequences (including the
FGF19 and FGF21 variants and subsequences listed in Table 1-9,
FIGS. 1 and 9, and the Sequence Listing, and the FGF19/FGF21
fusions and chimeras listed in Table 1-9, FIGS. 1 and 9, and the
Sequence Listing). In one embodiment, a mixture includes one or
more peptide sequences and a pharmaceutically acceptable carrier or
excipient. In another embodiment, a mixture includes one or more
peptide sequences and an adjunct drug or therapeutic agent, such as
an anti-diabetic, or glucose lowering, drug or therapeutic agent.
Examples of drugs and therapeutic agents are set forth hereafter.
Combinations, such as one or more peptide sequences in a
pharmaceutically acceptable carrier or excipient, with one or more
of an anti-diabetic, or glucose lowering drug or therapeutic agent
are also provided. Such combinations of peptide sequence provided
herein with another drug or agent, such as a glucose lowering drug
or therapeutic agent, for example are useful in accordance with the
methods and uses provided herein, for example, for treatment of a
subject.
[0176] Combinations also include incorporation of peptide sequences
or nucleic acids provided herein into particles or a polymeric
substances, such as polyesters, carbohydrates, polyamine acids,
hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate,
methylcellulose, carboxymethylcellulose, protamine sulfate, or
lactide/glycolide copolymers, polylactide/glycolide copolymers, or
ethylenevinylacetate copolymers; entrapment in microcapsules
prepared by coacervation techniques or by interfacial
polymerization, for example, by the use of hydroxymethylcellulose
or gelatin-microcapsules, or poly (methylmethacrolate)
microcapsules, respectively; incorporation in colloid drug delivery
and dispersion systems such as macromolecule complexes,
nano-capsules, microspheres, beads, and lipid-based systems (e.g.,
N-fatty acyl groups such as N-lauroyl, N-oleoyl, fatty amines such
as dodecyl amine, oleoyl amine, etc., see U.S. Pat. No. 6,638,513),
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes, for example.
[0177] In some embodiments, provided herein is a peptide sequence,
comprising or consisting of a fibroblast growth factor 19 (FGF19)
sequence variant having one or more amino acid substitutions,
insertions or deletions compared to a reference or wild type FGF19.
In other embodiments, provided herein is 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. In some embodiments, the peptide
sequence comprises at least one amino acid substitution to amino
acid residues 125-129 of SEQ ID NO:99 (FGF19), EIRPD, or a
corresponding region thereof in a variant polypeptide provided
herein. In one embodiment, the peptide sequence comprises at least
one amino acid substitution to amino acid residues 126-128 of SEQ
ID NO:99 (FGF19), IRP, or a corresponding region thereof in a
variant polypeptide provided herein. In another embodiments, the
peptide sequence comprises at least one amino acid substitution to
amino acid residues 127-128 of SEQ ID NO:99 (FGF19), RP, or a
corresponding region thereof in a variant polypeptide 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.
[0178] In some embodiments, the 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
wherein 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). In one embodiment, the
peptide sequence comprises at least one amino acid 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. In
one embodiment, the at least one amino acid substitution is R127L
and P128E.
[0179] In certain embodiments, the peptide sequence further
comprises 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). In some embodiments, the
peptide sequence is
TABLE-US-00005 (SEQ ID NO: 196)
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160).
[0180] In some embodiments, the peptide sequence (a) has a WGDPI
sequence motif (SEQ. ID NO: 170) corresponding to the WGDPI
sequence of amino acids 16-20 of SEQ ID NO:99 (FGF19); (b) the
peptide sequence has a substituted, mutated or absent WGDPI
sequence motif corresponding to FGF19 WGDPI sequence of amino acids
16-20 of FGF19; or (c) 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 sequence at amino acids 16-20.
[0181] In some embodiments, the peptide has an N-terminal region
and a C-terminal region, wherein the N-terminal region comprises
amino acid residues DSSPLLQ (SEQ ID NO: 104), and wherein the Q
residue is the last amino acid position of the N-terminal region.
In certain embodiments, the N-terminal region further comprises:
RHPIP (SEQ ID NO: 106), wherein R is the first amino acid position
of the N-terminal region; or HPIP (SEQ ID NO: 107), wherein H is
the first amino acid position of the N-terminal region; or RPLAF
(SEQ ID NO: 108), wherein R is the first amino acid position of the
N-terminal region; or PLAF (SEQ ID NO: 109), wherein P is the first
amino acid position of the N-terminal region; or R, wherein R is
the first amino acid position of the N-terminal region.
[0182] In some embodiments, the 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. In an embodiment, amino acid residues HPIP (SEQ ID NO:
107) are the first 4 amino acid residues of the N-terminal region.
In some embodiments, the first position of the N-terminal region is
an R residue; the first position of the N-terminal region is an M
residue; the first and second positions of the N-terminal region is
an MR sequence; the first and second positions of the N-terminal
region is an RM sequence; the first and second positions of the
N-terminal region is an RD sequence; the first and second positions
of the N-terminal region is an DS sequence; the first and second
positions of the N-terminal region is an MD sequence; the first and
second positions of the N-terminal region is an MS sequence; the
first through third positions of the N-terminal region is an MDS
sequence; the first through third positions of the N-terminal
region is an RDS sequence; the first through third positions of the
N-terminal region is an MSD sequence; the first through third
positions of the N-terminal region is an MSS sequence; the first
through third positions of the N-terminal region is an DSS
sequence; the first through fourth positions of the N-terminal
region is an RDSS (SEQ ID NO: 115) sequence; the first through
fourth positions of the N-terminal region is an MDSS (SEQ ID NO:
116) sequence; the first through fifth positions of the N-terminal
region is an MRDSS (SEQ ID NO: 117) sequence; the first through
fifth positions of the N-terminal region is an MSSPL (SEQ ID NO:
118) sequence; the first through sixth positions of the N-terminal
region is an MDSSPL (SEQ ID NO: 119) sequence; the first through
seventh positions of the N-terminal region is an MSDSSPL (SEQ ID
NO: 120) sequence. In some embodiments, the last position of the
C-terminal region corresponds to about residue 194 of SEQ ID NO:99
(FGF19). In some embodiments, the N-terminal region 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 some embodiments, the C-terminal region
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 one embodiment, the FGF19
sequence portion, 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. In one embodiment, the FGF21 sequence portion, 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 FGF21. In certain embodiments, the
N-terminal region, the C-terminal region, the FGF19 sequence
portion, and/or the FGF21 sequence portion, are joined by a linker
or spacer.
[0183] In some embodiments, the reference or wild type FGF19
sequence is set forth as:
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK (SEQ ID NO:99). In other embodiments, the reference or
wild type FGF21 sequence is set forth as:
TABLE-US-00006 (SEQ ID NO: 100)
RHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSP
ESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELL
LEDGYNVYQSEAHGLPLHLPGNKSPEIRDPAPRGPARFLPLPGLPPALPE
PPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS.
[0184] In certain embodiments, the N-terminal region first amino
acid position is a "M" residue, "R" residue, "S" residue, "H"
residue, "P" residue, "L" residue or "D" residue. In other
embodiments, the peptide sequence does not have a "M" residue or an
"R" residue at the first amino acid position of the N-terminal
region.
[0185] In some embodiments, the N-terminal region comprises any one
of the following sequences: MDSSPL (SEQ ID NO:119), MSDSSPL (SEQ ID
NO:120), SDSSPL (SEQ ID NO:112), MSSPL (SEQ ID NO: 113) or SSPL
(SEQ ID NO: 114). In yet other embodiments, the peptide sequence
comprises one or more L-amino acids, D-amino acids, non-naturally
occurring amino acids, amino acid mimetics, derivatives or
analogues.
[0186] In certain embodiments, the peptide sequence comprises any
one of SEQ ID NOS: 193-204. In other embodiments, the peptide
sequence consists of any one of SEQ ID NOS: 193-204. In other
embodiments, the peptide sequence comprises a subsequence or
fragment of any of the foregoing peptide sequences, or any of the
foregoing peptide sequences wherein the R terminal residue is
deleted. In other embodiments, the peptide sequence consists of a
subsequence or fragment of any of the foregoing peptide sequences,
or any of the foregoing peptide sequences wherein the R terminal
residue is deleted. In certain embodiments, the subsequence or
fragment thereof 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.
[0187] In certain embodiments, the peptide sequence has reduced
hepatocellular carcinoma (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 sequence at amino acids 16-20 of FGF19. In some embodiments,
the peptide sequence 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 sequence at amino acids 16-20 of FGF19.
In other embodiments, the peptide sequence 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
sequence at amino acids 16-20 of FGF19. In certain embodiments, the
peptide sequence 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) sequence at amino acids 16-20 of FGF19. In
some embodiments, the peptide sequence has less lean mass reducing
activity compared to FGF21. Any combination of two or more of the
foregoing activities is also contemplated. In other embodiments,
the HCC formation, glucose lowering activity, lipid increasing
activity, or lean mass reducing activity is ascertained in a db/db
mouse.
[0188] In some embodiments, the peptide sequence: (a) binds to
fibroblast growth factor receptor 4 (FGFR4) or activates FGFR4, or
does not detectably bind to fibroblast growth factor receptor 4
(FGFR4) or activate FGFR4; (b) binds to FGFR4 with an affinity less
than, comparable to or greater than FGF19 binding affinity for
FGFR4; and/or (c) activates FGFR4 to an extent or amount less than,
comparable to or greater than FGF19 the extent or amount that
activates FGFR4.
[0189] In some embodiments, the peptide sequence has 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 amino acid substitutions, deletions or insertions.
In certain embodiments, the amino acid deletions are at the N- or
C-terminus, or internal; or wherein the amino acid substitution, or
deletion is at any of amino acid positions 8-20 of FGF19
(AGPHVHYGWGDPI).
[0190] Also provided are compositions comprising any of the peptide
sequences provided herein.
[0191] Also provided are nucleic acid molecules encoding a peptide
sequence provide herein. In some embodiments, the nucleic acid
molecule further comprises an expression control element in
operable linkage that confers expression of the nucleic acid
molecule encoding the peptide in vitro, in a cell or in vivo. Also
provided is a vector comprising the nucleic acid molecule. In some
embodiments, the vector comprises a viral vector. Also provided is
a transformed or host cell that expresses the peptide sequence.
[0192] Peptides, including subsequences, variants and modified
forms of the exemplified peptide sequences (including the FGF19 and
FGF21 variants and subsequences listed in Table 1-9, FIGS. 1 and 9,
and the Sequence Listing, and the FGF19/FGF21 fusions and chimeras
listed in Table 1-9, FIGS. 1 and 9, and the Sequence Listing) as
set forth herein, can be used to modulate glucose metabolism and
facilitate transport of glucose from the blood to key metabolic
organs such as muscle, liver and fat. Such peptide sequences can be
produced in amounts sufficient or effective to restore glucose
tolerance and/or to improve or provide normal glucose
homeostasis.
[0193] As disclosed herein, administration of various FGF19
and/FGF21 variants and fusion peptide sequences to mice
successfully reduced glucose levels. Furthermore, in contrast to
FGF19, certain peptide sequences did not stimulate or induce HCC
formation or tumorigenesis in mice. Thus, administration of
peptides provided herein, including subsequences, variants and
modified forms of the exemplified peptide sequences (including the
FGF19 and FGF21 variants and subsequences listed in Table 1-9,
FIGS. 1 and 9, and the Sequence Listing, and the FGF19/FGF21
fusions and chimeras listed in Table 1-9, FIGS. 1 and 9, and the
Sequence Listing), into an animal, either by direct or indirect in
vivo or by ex vivo methods (e.g., administering the variant or
fusion peptide, a nucleic acid encoding the variant or fusion
peptide, or a transformed cell or gene therapy vector expressing
the variant or fusion peptide), can be used to treat various
disorders.
[0194] Accordingly, 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.
[0195] Also provided herein are methods of treating a subject
having, or at risk of having, a disorder. In various embodiments, a
method includes administering a peptide sequence, such as an FGF19
or FGF21 variant, fusion or chimera listed in Table 1-9, FIGS. 1
and 9, and the Sequence Listing, or a subsequence, a variant or
modified form of an FGF19 or FGF21 variant, fusion or chimera
listed in Table 1-9, FIGS. 1 and 9, and the Sequence Listing, to a
subject in an amount effective for treating the disorder.
[0196] Exemplary disorders treatable, preventable, and the like
with peptides provided herein, and methods and uses, include
metabolic diseases and disorders. Non limiting examples of diseases
and disorders include: 1. Glucose utilization disorders and the
sequelae associated therewith, including diabetes mellitus (Type I
and Type-2), gestational diabetes, hyperglycemia, insulin
resistance, abnormal glucose metabolism, "pre-diabetes" (Impaired
Fasting Glucose (IFG) or Impaired Glucose Tolerance (IGT)), and
other physiological disorders associated with, or that result from,
the hyperglycemic condition, including, for example,
histopathological changes such as pancreatic .beta.-cell
destruction. For treatment, peptide sequences provided herein can
be administered to subjects having a fasting plasma glucose (FPG)
level greater than about 100 mg/dl. Peptide sequences provided
herein may also be useful in 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; 2. Dyslipidemias and their sequelae
such as, for example, atherosclerosis, coronary artery disease,
cerebrovascular disorders and the like; 3. Other conditions which
may be associated with the 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,
cardiovascular disease, stroke and heart failure; 4. 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; 5. Disorders
of cell cycle or cell differentiation processes such as adipose
cell tumors, lipomatous carcinomas including, for example,
liposarcomas, solid tumors, and neoplasms; 6. 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; 7. Skin and dermatological disorders
and/or disorders of wound healing processes, including
erythemato-squamous dermatoses; and 8. Other disorders such as
syndrome X, osteoarthritis, and acute respiratory distress
syndrome.
[0197] Also provided herein is a method of treating a subject
having, or at risk of having, a disease or disorder treatable by a
peptide sequence provided herein, comprising administering the
chimeric peptide sequence or peptide sequence provided herein to a
subject in an amount effective for treating the disorder. In some
embodiments, the disease or disorder comprises a hyperglycemic
condition. In some embodiment, the hyperglycemic condition
comprises diabetes. In some embodiments, the diabetes is
insulin-dependent (type I) diabetes, type II diabetes, or
gestational diabetes. In other embodiments, the disease or disorder
comprises insulin resistance. In certain embodiments, the disease
or disorder comprises hyperinsulinemia. In some embodiments, the
disease or disorder comprises glucose intolerance. In other
embodiments, the disease or disorder comprises obesity. In yet
other embodiments, the disease or disorder comprises an undesirable
body mass. In some embodiments, the disease or disorder comprises
metabolic syndrome. Also provided herein is a method of improving
glucose metabolism in a subject in need thereof, comprising
administering a peptide sequence provided herein to a subject in an
amount effective to improve glucose metabolism in the subject. In
some embodiments, the subject has a fasting plasma glucose level
greater than 100 mg/dl and/or has a hemoglobin A1c (HbA1c) level
above 6%. In some embodiments of the methods provided herein, the
method results in reduced glucose levels, increased insulin
sensitivity, reduced insulin resistance, reduced glucagon, an
improvement in glucose tolerance, or glucose metabolism or
homeostasis, improved pancreatic function, a reduced triglyceride,
cholesterol, IDL, LDL or VLDL levels, a decrease in blood pressure,
a decrease in intimal thickening of the blood vessel, or a decrease
in body mass or weight gain, or any combination thereof.
[0198] As used herein, the term "hyperglycemic" or "hyperglycemia,"
when used in reference to a condition of a subject means a
transient or chronic abnormally high level of glucose present in
the blood of a subject. The condition can be caused by a delay in
glucose metabolism or absorption such that the subject exhibits
glucose intolerance or a state of elevated glucose not typically
found in normal subjects (e.g., in glucose-intolerant pre-diabetic
subjects at risk of developing diabetes, or in diabetic subjects).
Fasting plasma glucose (FPG) levels for normoglycemia are less than
about 100 mg/dl, for impaired glucose metabolism, between about 100
and 126 mg/dl, and for diabetics greater than about 126 mg/dl.
[0199] As disclosed herein, also provided 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) obesity or an undesirable body mass
(e.g., a greater than normal body mass index, or "BMI" relative to
an appropriate matched subject of comparable age, gender, race,
etc.). Thus, in various embodiments, a method provided herein for,
for example, treating obesity or an undesirable body mass
(including the co-morbid conditions of obesity, e.g., obstructive
sleep apnea, arthritis, cancer (e.g., breast, endometrial, and
colon), gallstones or hyperglycemia, includes contacting or
administering a peptide as set forth herein (e.g., a variant or
fusion of FGF19 and/or FGF21 as set forth in Table 1-9 or FIG. 1,
for example) in an amount effective to treat obesity or an
undesirable body mass. In particular aspects, a subject has a body
mass index greater than 25, for example, 25-30, 30-35, 35-40, or
greater than 40.
[0200] Moreover, also provided herein are methods of preventing
(e.g., in subjects predisposed to having a particular disorder(s)),
slowing or inhibiting the progression of, delaying the onset of, or
treating undesirable levels or abnormally elevated serum/plasma
LDL, VLDL, triglycerides or cholesterol, all of which, alone or in
combination, can lead to, for example, plaque formation, narrowing
or blockage of blood vessels, and increased risk of hypertension,
stroke and coronary artery disease. Such disorders can be due to,
for example, genetic predisposition or diet, for example.
[0201] 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.
[0202] Subjects include those having a disorder, e.g., a
hyperglycemic disorder, such as diabetes, or subjects that do not
have a disorder but may be at risk of developing the disorder,
e.g., pre-diabetic subjects having FPG levels greater than 100
mg/dl, for example, between about 100 and 126 mg/dl. Subjects at
risk of developing a disorder include, for example, those whose
diet may contribute to development of acute or chronic
hyperglycemia (e.g., diabetes), undesirable body mass or obesity,
as well as those which may have a family history or genetic
predisposition towards development of acute or chronic
hyperglycemia, or undesirable body mass or obesity.
[0203] 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 Table 1-9 or FIG. 1,
for example) 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 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 hyperglycemic disorder, for example,
treatment can lower or reduce blood glucose, improve glucose
tolerance, improve glucose metabolism, provide normal glucose
homeostasis, lower or reduce insulin resistance, lower or reduce
insulin levels, or decrease, prevent, improve, or reverse metabolic
syndrome, or a histopathological change associated with or that
results from the hyperglycemic disorder, such as diabetes.
[0204] For example, a peptide sequence, method or use can lower or
reduce glucose in one or more subjects having FPG levels greater
than 100 mg/dl, for example, between about 100 and 125 mg/dl, or
greater than 125 mg/dl, by 5-10%, 10-20%, 20-30%, or 30-50%, or
more, or for example from greater than 200 mg/dl to less than 200
mg/dl, for greater than 150 mg/dl to less than 150 mg/dl, from
greater than 125 mg/dl to less than 125 mg/dl, etc. In addition, a
peptide sequence, method or use can lower or reduce glucose, for
example, for pre-diabetes or for diabetes (e.g., Type 2) subjects
with baseline HbAIc levels greater than about 5%, 6%, 7%, 8%, 9% or
10%, in particular 5%, 6%, or 7%.
[0205] Non-limiting examples of an improvement of a
histopathological change associated with a hyperglycemic condition
include, for example, decreasing, inhibiting, reducing or
arresting: the destruction or degeneration of pancreas cells (e.g.,
.beta.-cells), kidney damage such as tubule calcification or
nephropathy, degeneration of liver, eye damage (e.g., diabetic
retinopathy, cataracts), diabetic foot, ulcerations in mucosa such
as mouth and gums, periodontitis, excess bleeding, slow or delayed
healing of injuries or wounds (e.g., that lead to diabetic
carbuncles), skin infections and other cutaneous disorders,
cardiovascular and coronary heart disease, peripheral vascular
disease, stroke, dyslipidemia, hypertension, obesity, or the risk
of developing any of the foregoing. Improvement in undesirable body
mass or obesity can include, for example, a reduction of body mass
(as reflected by BMI or the like) or an improvement in an
associated disorder, such as a decrease in triglyceride,
cholesterol, LDL or VLDL levels, a decrease in blood pressure, a
decrease in intimal thickening of the blood vessel, a decreased or
reduced risk of cardiovascular disease, or stroke, decrease in
resting heart rate, etc.
[0206] An "effective amount" or a "sufficient amount" for use
and/or for treating a subject refer to an amount that provides, in
single or multiple doses, alone, or in combination with one or more
other compositions (therapeutic agents such as a drug or treatment
for hyperglycemia), treatments, protocols, or therapeutic regimens
agents, 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, 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.
[0207] 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
hyperglycemic disorder (e.g., diabetes, insulin resistance, glucose
intolerance, metabolic syndrome, etc.), for example, an improvement
can be a lowering or a reduction in blood glucose, a reduction in
insulin resistance, a reduction in glucagon, an improvement in
glucose tolerance, or glucose metabolism or homeostasis. An
improvement in a hyperglycemic disorder also can include improved
pancreatic function (e.g., inhibit or prevent .beta.-cell/islet
destruction or enhance .beta.-cell number and/or function), a
decrease in a pathology associated with or resulting from the
disorder, such as an improvement in histopathology of an affected
tissue or organ, as set forth herein. In the case of undesirable
body mass or obesity, for example, an improvement can be a decrease
in weight gain, a reduction of body mass (as reflected in reduced
BMI, for example) or an improvement in a condition associated with
undesirable body mass obesity, for example, as set forth herein
(e.g., a lowering or a reduction of blood glucose, triglyceride,
cholesterol, LDL or VLDL levels, a decrease in blood pressure, a
decrease in intimal thickening of the blood vessel, etc.).
[0208] 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.).
[0209] Thus, in the case of a disorder treatable by a peptide
sequence provided herein, the amount of peptide 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 glucose
homeostasis in a subject can reduce the dosage amount or frequency
of insulin injection, even though complete freedom from insulin has
not resulted.
[0210] An effective amount can be ascertained, for example, by
measuring one or more relevant physiological effects. In a
particular non-limiting example in the case of a hyperglycemic
condition, a lowering or reduction of blood glucose or an
improvement in glucose tolerance test can be used to determine
whether the amount of a peptide sequence, including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in Table 1-9, FIGS. 1 and 9, and
the Sequence Listing) provided herein is effective to treat a
hyperglycemic condition. In another particular non-limiting
example, an effective amount is an amount sufficient to reduce or
decrease any level (e.g., a baseline level) of FPG, wherein, for
example, an amount sufficient to reduce a FPG level greater than
200 mg/dl to less than 200 mg/dl, an amount sufficient to reduce a
FPG level between 175 mg/dl and 200 mg/dl to less than the
pre-administration level, an amount sufficient to reduce a FPG
level between 150 mg/dl and 175 mg/dl to less than the
pre-administration level, an amount sufficient to reduce a FPG
level between 125 mg/dl and 150 mg/dl to less than the
pre-administration level, and so on (e.g., reducing FPG levels to
less than 125 mg/dl, to less than 120 mg/dl, to less than 115
mg/dl, to less than 110 mg/dl, etc.). In the case of HbAIc levels,
an effective amount includes an amount sufficient to reduce or
decrease levels by more than about 10% to 9%, by more than about 9%
to 8%, by more than about 8% to 7%, by more than about 7% to 6%, by
more than about 6% to 5%, and so on. More particularly, a reduction
or decrease of HbAIc levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%,
0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%,
35%, 40%, 45%, 50%, or more is an effective amount in certain
embodiments. In yet another particular non-limiting example in the
case of undesirable body mass or obesity, an effective amount is an
amount sufficient to decrease or reduce the body mass index (BMI)
of a subject, a decrease or reduction of glucose, a decrease or
reduction in serum/plasma levels of triglyceride, lipid,
cholesterol, fatty acids, LDL and/or VLDL. In yet further
particular non-limiting examples, an amount is an amount sufficient
to decrease or reduce any of the aforementioned parameters by, for
example, about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,
1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or
more.
[0211] Methods and uses provided herein for treating a subject are
applicable for prophylaxis to prevent a disorder in a subject, such
as a hyperglycemic disorder, or development of undesirable body
mass or obesity. Alternatively, methods and uses can be practiced
during or following treatment of a subject. For example, prior to,
during or following treatment of a subject to lower glucose using
insulin or another glucose lowering drug or therapeutic agent, for
example, a method or use provided herein can, for example, a
peptide sequence provided herein can be administered to the
subject. In addition, a composition such as a peptide sequence
provided herein can be combined with another drug or agent, such as
a glucose lowering drug or therapeutic agent, for example.
[0212] Accordingly, methods and uses provided herein for treating a
subject can be practiced prior to, substantially contemporaneously
with or following another treatment, and 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. Drugs for treating diabetes include, for example,
biguanides and sulphonylureas (e.g., tolbutamide, chlorpropamide,
acetohexamide, tolazamide, glibenclamide and glipizide),
thiazolidinediones (rosiglitazone, pioglitazone), GLP-1 analogues,
Dipeptidyl peptidase-4 (DPP-4) inhibitors, bromocriptine
formulations (e.g. and bile acid sequestrants (e.g., colesevelam),
and insulin (bolus and basal analogs), metformin (e.g., metformin
hydrochloride) with or without a thiazolidinedione (TZD), and
SGLT-2 inhibitors. Appetite suppression drugs are also well known
and can be used in combination with the methods provided herein.
Supplementary therapies can be administered prior to,
contemporaneously with or following methods and uses provided
herein.
[0213] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (sequences listed in Table 1-9, FIGS. 1 and 9, and the
Sequence Listing), 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 hyperglycemia. 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.
[0214] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (sequences listed in Table 1-9, FIGS. 1 and 9, and the
Sequence Listing) 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 .mu.g/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).
[0215] Peptide sequences provided herein including subsequences,
variants and modified forms of the exemplified peptide sequences
(sequences listed in Table 1-9, FIGS. 1 and 9, and the Sequence
Listing) 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
(sequences listed in Table 1-9, FIGS. 1 and 9, and the Sequence
Listing) and methods provided herein including pharmaceutical
compositions can be administered via a (micro)encapsulated delivery
system or packaged into an implant for administration.
[0216] 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. The subcutaneous system is being evaluated for the
continuous, subcutaneous delivery of exenatide (marketed as
Byetta.TM.) in a once-yearly, injection-free GLP-1 therapy for
treatment of type 2 diabetes.
[0217] 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 (sequences listed in Table 1-9, FIGS.
1 and 9, and the Sequence Listing), and one or more
pharmaceutically acceptable or physiologically acceptable diluent,
carrier or excipient. In particular embodiments, a peptide sequence
or sequences 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.
[0218] 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. In addition, the pharmaceutical compositions may further
comprise other therapeutically active agents or compounds disclosed
herein (e.g., glucose lowering agents) or known to the skilled
artisan which can be used in the treatment or prevention of various
diseases and disorders as set forth herein.
[0219] 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 (sequences
listed in Table 1-9, FIGS. 1 and 9, and the Sequence Listing) 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 provided 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.
[0220] 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.
[0221] Pharmaceutical compositions provided herein may contain
still other pharmaceutically-acceptable formulation agents for
modifying or maintaining the rate of release of a peptide provided
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.
[0222] 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 provided 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 provided 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.
[0223] 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).
[0224] 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).
[0225] 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.
[0226] 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 for preparation of such formulations are known to those
skilled in the art and are commercially available.
[0227] Other sustained-release preparations can also be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
peptides provided herein, which matrices are in the form of shaped
articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] Also provided herein are peptides in the form of
suppositories for rectal administration. The suppositories can be
prepared by mixing a peptide provided 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.
[0235] In certain embodiments, provided herein are nucleic acids
encoding a peptide (or a subsequence, variant or modified form
thereof) as set forth herein. In a specific embodiment, nucleic
acids comprising sequences encoding any of the peptide sequences
provided herein, are administered to a subject for use in a methods
and uses provided herein, for example, to prevent, manage or treat
a disease or disorder in a subject, such as a hyperglycemic
disorder, or development of undesirable body mass or obesity. Such
therapy encompasses that performed by the administration to a
subject of an expressed or expressible nucleic acid. In an
embodiment, the nucleic acids produce their encoded peptide
sequence, and the peptide sequence mediates a prophylactic or
therapeutic effect. For example, a nucleic acid can encode any
peptide sequence or chimeric peptide sequence provided herein,
including but not limited to those that comprise or consist 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 205 a
peptide sequence that comprises or consists of any sequence set
forth in Tables 1-9, FIG. 1 or 9, or a peptide sequence that
comprises or consists of any sequence set forth in the Sequence
Listing herein; or any fragment, subsequence or variant peptide
thereof.
[0236] Any of the methods for recombinant gene expression (or gene
therapy) available in the art can be used. Exemplary methods are
described below and are provided in the Examples section.
[0237] For general review of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu, 1991,
Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan, 1993,
Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215.
Methods commonly known in the art of recombinant DNA technology
which can be used are described in Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, N Y (1993);
and Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY (1990).
[0238] In a specific embodiment, a composition comprises a nucleic
acid encoding a peptide sequence provided herein, the nucleic acid
being part of an expression vector that expresses the peptide
thereof in a suitable host. In particular, such nucleic acids have
promoters, preferably heterologous promoters, operably linked to
the peptide coding region, the promoter being inducible or
constitutive, and, optionally, tissue-specific. In another
particular embodiment, nucleic acid molecules are used in which the
peptide coding sequences and any other desired sequences are
flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the peptide encoding nucleic acids (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0239] Delivery of the nucleic acids into a subject can be either
direct, in which case the subject is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the subject. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0240] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where the sequences are expressed to
produce the encoded product. This can be accomplished by any of
numerous methods known in the art, e.g., by constructing them as
part of an appropriate nucleic acid expression vector and
administering the vector so that the sequences become
intracellular, e.g., by infection using defective or attenuated
retroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or
by direct injection of naked DNA, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic.RTM., Dupont), or coating
with lipids or cell surface receptors or transfecting agents,
encapsulation in liposomes, microparticles, or microcapsules, or by
administering them in linkage to a peptide which is known to enter
the nucleus, by administering it in linkage to a ligand subject to
receptor-mediated endocytosis (see, e.g., Wu, 1987, J. Biol. Chem.
262:4429-4432) (which can be used to target cell types specifically
expressing the receptors), etc. In another embodiment, nucleic
acid-ligand complexes can be formed in which the ligand comprises a
fusogenic viral peptide to disrupt endosomes, allowing the nucleic
acid to avoid lysosomal degradation. In yet another embodiment, the
nucleic acid can be targeted in vivo for cell specific uptake and
expression, by targeting a specific receptor (see, e.g., PCT
Publications WO 92/06180; WO 92/22635; WO 92/20316; WO93/14188, WO
93/20221). Alternatively, the nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression, by homologous recombination (Koller and Smithies, 1989,
Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989,
Nature 342:435-438).
[0241] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an peptide sequence provided herein
are used. For example, a retroviral vector can be used (see Miller
et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the peptide to be used in gene therapy can
be cloned into one or more vectors, which facilitates delivery of
the gene into a subject. More detail about retroviral vectors can
be found in Boesen et al., 1994, Biotherapy 6:291-302, which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., 1994, J.
Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0242] Adenoviruses are other viral vectors that can be used in the
recombinant production of peptides provided herein. Adenoviruses
are especially attractive vehicles for delivering genes to
respiratory epithelia. Adenoviruses naturally infect respiratory
epithelia where they cause a mild disease. Other targets for
adenovirus-based delivery systems are liver, the central nervous
system, endothelial cells, and muscle. Adenoviruses have the
advantage of being capable of infecting non-dividing cells.
Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang et al., 1995, Gene Therapy
2:775-783. In a specific embodiment, adenovirus vectors are
used.
[0243] Adeno-associated virus (AAV) can also be utilized (Walsh et
al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; and U.S. Pat.
No. 5,436,146). In a specific embodiment, AAV vectors are used to
express a peptide provided herein. In some embodiments of the
methods provided herein, a subject is administered an AAV
comprising a nucleic acid encoding a peptide provided herein. In
certain embodiments, the peptides are over-expressed.
[0244] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a subject. In this embodiment, the nucleic acid is introduced into
a cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcellmediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993, Meth. Enzymol. 217:618-644; Clin. Pharma. Ther. 29:69-92
(1985)) and can be used in accordance with the methods provided
herein, provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0245] The resulting recombinant cells can be delivered to a
subject by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0246] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0247] In a specific embodiment, the cell used for gene therapy is
autologous to the subject.
[0248] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an peptide are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the methods provided herein (see
e.g., PCT Publication WO 94/08598; Stemple and Anderson, 1992, Cell
7 1:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and
Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
[0249] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0250] Also provided herein are methods of identifying a peptide
(or a subsequence, variant or modified form as set forth herein)
having glucose lowering activity without substantial HCC activity.
In one embodiment, a method includes: screening (e.g., assaying or
measuring) a peptide sequence (or a subsequence, variant or
modified form as set forth herein) for glucose lowering activity;
and screening (e.g., assaying or measuring) a peptide sequence (or
a subsequence, variant or modified form as set forth herein) for
HCC activity, or expression of a marker correlating with HCC
activity. A peptide having glucose lowering activity and reduced or
absent HCC activity thereby identifies the peptide. In particular
aspects, the marker correlating with HCC activity comprises lipid
profile--a peptide that has less lipid increasing activity compared
to FGF19 indicates the peptide has reduced or absent HCC activity;
or the marker correlating with HCC activity comprises aldo-keto
reductase gene expression--a peptide that down-regulates or
decreases aldo-keto reductase gene expression compared to FGF19
indicates that the peptide has reduced or absent HCC activity; or
the marker indicative of HCC activity comprises Slc1a2 gene
expression--a peptide that up-regulates or increases Slc1a2 gene
expression compared to FGF19 indicates that the peptide has reduced
or absent HCC activity.
[0251] 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, gene expression can be
assayed or measured by a Northern blot, Western blot,
immunoprecipitation assay, or by measuring activity, function or
amount of the expressed protein (e.g., aldo-keto reductase or
Slc1a2).
[0252] 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.
[0253] 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 MRI. For
example, 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.
[0254] 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).
[0255] 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 Table 1-9, FIGS. 1 and 9, and the Sequence
Listing).
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] The term "Fab" refers to a fragment of an antibody that
comprises the constant domain of the light chain and the CH.sub.1
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.
[0264] The term "complementarity determining regions" or "CDRs"
refers 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".
[0265] 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
NM, preferably .ltoreq.100 nM, and most preferably .ltoreq.10 nM.
An increased equilibrium constant ("K.sub.D") 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.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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, thereby having an effect on a
condition or disorder.
[0271] Also provided herein are kits, including, but not limited
to, peptide sequences provided herein, optionally in combination
with one or more therapeutic agents, compositions and
pharmaceutical compositions thereof, packaged into suitable
packaging material. A kit optionally includes 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 reducing or
lowering blood glucose, treatment of hyperglycemia, treatment of
diabetes, etc.
[0272] A kit can contain a collection of such components, e.g., two
or more peptide sequences alone, or a combination of a peptide
sequence with another therapeutically useful composition (e.g., an
anti-diabetic drug, such as a gastrin compound).
[0273] 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.).
[0274] 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.
[0275] Labels or inserts can include identifying information of one
or more components therein, dose amounts, 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.
[0276] 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
regimes set forth herein. Exemplary instructions include
instructions for treatment or use of a peptide sequence as set
forth herein. Kits provided herein therefore can additionally
include labels or instructions for practicing any of the methods
and uses provided herein described herein including treatment
methods and uses.
[0277] 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 side 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.
[0278] 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. Kits provided herein can be 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. The cells in the kit can be
maintained under appropriate storage conditions until ready to
use.
[0279] 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 provided herein,
suitable methods and materials are described herein.
[0280] 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.
[0281] 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%, 95%, 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.
[0282] 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 or 2500-5000, 5000-25,000, 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.
[0283] As also used herein a series of ranges are disclosed
throughout this document. The use of a series of ranges include
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.
[0284] For the sake of conciseness, certain abbreviations are used
herein. One example is the single letter abbreviation to represent
amino acid residues. The amino acids and their corresponding three
letter and single letter abbreviations are as follows:
TABLE-US-00007 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)
[0285] 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.
[0286] 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 following examples are
intended to illustrate but not limit the scope of invention
described in the claims.
EXAMPLES
Example 1
[0287] The following is a description of various methods and
materials used in the studies herein.
[0288] Animals.
[0289] db/db mice were purchased from The Jackson Laboratory (Bar
Harbor, Me.), Mice were 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. They had free access to water (autoclaved
distilled water) and were 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. For diet-induced obesity,
C57BL6/J mice (Jackson Laboratory) were maintained on a high-fat
diet (D12492, Research Diet, New Brunswick, N.J. USA) containing 60
kcal % fat, 20 kcal % protein and 20 kcal % carbohydrate for 16-20
weeks. All animal studies were approved by the NGM Institutional
Animal Care and Use Committee.
[0290] DNA and Amino Acid Sequences.
[0291] cDNA of ORF encoding human FGF19 (Homo sapiens FGF19,
GenBank Accession No. NM 005117.2) variants. Protein sequence
encoded by the cDNA (GenBank Accession No. NP_005108.1)
PCR.
[0292] FGF19 ORF was amplified with polymerase chain reaction (PCR)
using recombinant DNA (cDNA) prepared from human small intestinal
tissue. PCR reagents kits with Phusion high-fidelity DNA polymerase
were purchased from New England BioLabs (F-530L, Ipswich, Mass.).
The following primers were used: forward PCR primer:
TABLE-US-00008 (SEQ ID NO: 136) 5'
CCGACTAGTCACCatgcggagcgggtgtgtgg
and reverse PCR primer:
TABLE-US-00009 (SEQ ID NO: 137) 5.varies.
ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC.
Amplified DNA fragment was digested with restriction enzymes Spe I
and Not I (the restriction sites were included in the 5' or 3' PCR
primers, respectively) and was then ligated with AAV transgene
vectors that had been digested with the same restriction enzymes.
The vector used for expression contained a selectable marker and an
expression cassette composed of a strong eukaryotic promoter 5' of
a site for insertion of the cloned coding sequence, followed by a
3' untranslated region and bovine growth hormone polyadenylation
tail. The expression construct is also flanked by internal terminal
repeats at the 5' and 3' ends.
[0293] Production and Purification of AAV.
[0294] AAV293 cells (obtained from Agilent Technologies, Santa
Clara, Calif.) were 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 were plated at 50%
density on day 1 in 150 mm cell culture plates and transfected on
day 2, using calcium phosphate precipitation method with the
following 3 plasmids (20 .mu.g/plate of each): AAV transgene
plasmid, pHelper plasmids (Agilent Technologies) and AAV2/9 plasmid
(Gao et al., J. Virol. 78:6381 (2004)). 48 hours after
transfection, the cells were 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
was frozen in an alcohol dry ice bath and was then thawed in
37.degree. C. water bath. The freeze and thaw cycles were repeated
three times; Benzonase.RTM. (Sigma-aldrich, St. Louis, Mo.) was
added to 50 units/ml; deoxycholate was added to a final
concentration of 0.25%. After an incubation at 37.degree. C. for 30
min, cell debris was pelleted by centrifugation at 5000.times.g for
20 min. Viral particles in the supernatant were purified using a
discontinued iodixanal (Sigma-aldrich, St. Louis, Mo.) gradient as
previously described (Zolotukhin S. et al (1999) Gene Ther. 6:973).
The viral stock was concentrated using Vivaspin Benzonase.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 were 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.
[0295] Afterwards, 15 .mu.l of the solution containing 2 mg/ml of
Proteinase K, 0.5% SDS and 25 mM EDTA were added and the mixture
was incubated for additional 20 min at 55.degree. C. to release
viral DNA. Viral DNA was cleaned with mini DNeasy.RTM. Kit (Qiagen,
Valencia, Calif.) and eluted with 40 .mu.l of water. Viral genome
copy (GC) was determined by using quantitative PCR.
[0296] Viral stock was diluted with PBS to desirable GC/ml. Viral
working solution (200 .mu.l) was delivered into mice via tail vein
injection.
[0297] Blood Glucose Assay.
[0298] Blood glucose in mouse tail snip was measured using
ACCU-CHEK Active test strips read by ACCU-CHEK Active meter (Roche
Diagnostics, Indianapolis, Ind.) following manufacturer's
instruction.
[0299] Lipid Profile Assay.
[0300] Whole blood from mouse tail snips was collected into plain
capillary tubes (BD Clay Adams SurePrep.TM., Becton Dickenson and
Co. Sparks, Md.). Serum and blood cells were separated by spinning
the tubes in an Autocrit.TM. Ultra 3 (Becton Dickinson and Co.
Sparks, Md.). Serum samples were assayed for lipid profile
(triglyceride, total cholesterol, HDL, and non-HDL) using
Integra.TM. 400 Clinical Analyzer (Roche Diagnostics, Indianapolis,
Ind.) following the manufacturer's instructions.
[0301] Serum FGF19/FGF21/Variants Exposure Level Assay.
[0302] Whole blood (about 50 .mu.l/mouse) from mouse tail snips was
collected into plain capillary tubes (BD Clay Adams SurePrep,
Becton Dickenson and Co. Sparks, Md.). Serum and blood cells were
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 were determined using EIA kits (Biovendor) by
following the manufacturer's instructions.
[0303] Hepatocellular Carcinoma (HCC) Assay.
[0304] Liver specimen was harvested from db/db mice 6 months after
AAV injection. HCC score is 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.
[0305] Liver Gene Expression Assay.
[0306] Liver specimen was harvested and homogenized in TRIzol.RTM.
reagent (Invitrogen). Total RNA was extracted following
manufacturer's instruction. RNA was treated with DNase (Ambion)
followed by quantitative RT-PCR analysis using TaqMan.RTM. primers
and reagents from Applied Biosystems. Relative mRNA levels of
aldo-keto reductase and slc1a2 in the liver was calculated using
.DELTA..DELTA.Ct method.
[0307] FGFR4 Binding and Activity Assays.
[0308] 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.
Example 2
[0309] The following is a description of studies showing the
glucose lowering activity of various sequence variants of FGF19 and
FGF21, and FGF19/FGF21 fusion constructs.
[0310] FIG. 2 illustrates exemplary FGF19/FGF21 fusion constructs,
and the segments from each of FGF19 and FGF21 present in the fusion
peptides. These peptides were analyzed for glucose lowering
activity and statistically significant lipid elevating or
increasing activity (Table 1-9, FIGS. 1 and 9, and the Sequence
Listing).
[0311] Mice (db/db) were injected with viral vector expressing
FGF19, FGF21 or variants, and analyzed after injection.
Glucose-lowering activity of each sequence is represented by a "+"
symbol (a "-" symbol means no glucose lowering activity, a "+/-"
symbol means variants retain minimal glucose-lowering activity);
lipid elevating activity is represented by a "+" symbol (a "-"
symbol means no lipid elevating activity, a "+/-" symbol means
variants retain minimal lipid-elevating activity, FIG. 2).
[0312] Two fusions of FGF21 and FGF19, denoted variant M5 and
variant 45 (M45), exhibited glucose lowering activity and an
absence of statistically significant lipid elevating or increasing
activity. Variants denoted M1, M2 and M69, respectively (FIG. 1),
also exhibited glucose lowering activity (FIGS. 3B and 3C, Table
5). Data comparing M5, M1, M2 and M69 glucose lowering activity and
lipid elevating or increasing activity to FGF19 and FGF21 are
illustrated in FIGS. 3A-3C and 4A-4C.
Example 3
[0313] The following is a description of studies showing that
variants M5, M1, M2 and M69 are not tumorigenic, as determined by
HCC formation, and that variants M5, M2 and M69 also do not reduce
lean muscle and fat mass.
[0314] Animals (db/db) were injected with AAV vectors expressing
FGF19, FGF21, M5, M1, M2, or M69, or injected with saline, and
analyzed 6 months after injection. The data indicate that variants
M5, M1, M2, and M69 did not induce HCC formation significantly
(FIGS. 5A-5C).
[0315] Animals (db/db mice) were also injected with viral vector
expressing FGF19, FGF21, M5, M1, M2 or M69, or injected with
saline, and analyzed 6 months after injection for the effect of on
lean mass and fat mass. The data indicate that M5, M2 and M69
peptides did not cause a statistically significant reduction in
lean mass or fat mass, in contrast to FGF21, and that M1 peptide
reduces lean mass (FIGS. 6A-6C).
Example 4
[0316] The following is a data summary of 25 additional variant
peptides analyzed for lipid elevating activity and tumorigenesis.
The data clearly show a positive correlation between lipid
elevation and tumorigenesis, as determined by HCC formation in
db/db mice.
[0317] Tables 1 to 3 summarize data for 26 different variant
peptides. Such exemplified variant peptides have FGF19 C-terminal
sequence:
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGL
LQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188) at the
C-terminal portion, e.g., following the "TSG" amino acid residues.
Notably, variant peptides (7 total, including M5) that did not
cause a statistically significant elevation of lipids did not
induce HCC formation. In contrast, all variant peptides (17 total)
that caused a statistically significant elevation of lipids also
caused HCC formation in mice. This data indicates that there is a
strong positive correlation between lipid elevating activity and
HCC formation. Accordingly, lipid elevating activity can be used as
an indicator and/or predictor of HCC formation in animals.
TABLE-US-00010 TABLE 1 Elevated Triglyceride and Cholesterol in
db/db Mice Appears to Positively Correlate With HCC Formation (see
SEQ ID NOs: 99, 5 and 74 to 81). ##STR00001## SEQ ID NO. Core SEQ
ID NO. Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI 99
RLRHLYTSG 185 + + (aa 1-20) FGF21 HPIPDSSPLLQ--FGGQV 100 RQRYLYTDD
186 - - (aa 1-16) M5 R-HPIPDSSPLLQ--FGGQV 5 RLRHLYTSG 185 - - (aa
1-17) M74 R------------------DAGPHVHYGWGDPI 74 RLRHLYTSG 185 + +
(aa 1-15) M75 R---------------------------VHYGWGDPI 75 RLRHLYTSG
185 - - (aa 1-10) M76 R------------------------------------GDPI 76
RLRHLYTSG 185 - - (aa 1-5) M77
R--------------------------------------------- 77 RLRHLYTSG 185 - -
(aa 1) M78 R-------------------AGPHVHYGWGDPI 78 RLRHLYTSG 185 + +
(aa 1-14) M79 R---------------------GPHVHYGWGDPI 79 RLRHLYTSG 185 +
+ (aa 1-13) M80 R-----------------------PHVHYGWGDPI 80 RLRHLYTSG
185 - - (aa 1-12) M81 R-------------------------HVHYGWGDPI 81
RLRHLYTSG 185 - - (aa 1-11)
TABLE-US-00011 TABLE 2 Elevated Triglyceride and Cholesterol in
db/db Mice Appears to Positively Correlate with HCC Formation (see
SEQ ID NOs: 99, 100 and 82 to 98). ##STR00002## SEQ ID NO. Core SEQ
ID NO. Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI 99
(aa 1-20) RLRHLYTSG 185 + + FGF21 HPIPDSSPLLQ--FGGQV 100 (aa 1-16)
RQRYLYTDD 186 - - M82 RPLAFSAAGPHVHYGWGDPI 82 (aa 1-20) RLRHLYTSG
185 + + M83 RPLAFSDAAPHVHYGWGDPI 83 (aa 1-20) RLRHLYTSG 185 +/- +/
M84 RPLAFSDAGAHVHYGWGDPI 84 (aa 1-20) RLRHLYTSG 185 +/- +/ M85
RPLAFSDAGPHVHYGAGDPI 85 (aa 1-20) RLRHLYTSG 185 - - M86
RPLAFSDAGPHVHYGWGAPI 86 (aa 1-20) RLRHLYTSG 185 + + M87
RPLAFSDAGPHVHYGWGDAI 87 (aa 1-20) RLRHLYTSG 185 + +
TABLE-US-00012 TABLE 3 Elevated Triglyceride and Cholesterol in
db/db Mice Appears to Positively Correlate with HCC Formation (see
SEQ ID NOs: 99, 100 and 88 to 98) ##STR00003## Core SEQ ID NO Lipid
Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa
1-29) + + FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) - -
H31A/S141A(M88) FGF19 + + H31A/H142A(M89) FGF19 + +
K127A/R129A(M90) FGF19 + + K127A/S141A(M91) FGF19 + +
K127A/H142A(M92) FGF19 + + R129A/S141A(M93) FGF19 + +
S141A/H142A(M94) FGF19 + + K127A/H142A(M95) FGF19 + +
K127A/R129A/S141A(M96) FGF19 + + K127A/R129A/H142A(M97) FGF19 + +
K127A/R129A/S141A/H142A(M98) FGF19 + +
TABLE-US-00013 M88 (H31A/S141A): (SEQ ID NO: 88)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M89
(H31A/H142A): (SEQ ID NO: 89)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M90
(K127A/R129A): (SEQ ID NO: 90)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M91
(K127A/5141A): (SEQ ID NO: 91)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M92
(K127A/H142A): (SEQ ID NO: 92)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M93
(R129A/5141A): (SEQ ID NO: 93)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQAQLYKN
RGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M94
(5141A/H142A): (SEQ ID NO: 94)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M95
(K127A/H142A): (SEQ ID NO: 95)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M96
(K127A/R129A/5141A): (SEQ ID NO: 96)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M97
(K127A/R129A/H142A): (SEQ ID NO: 97)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M98
(K127A/R129A/5141A/H142A): (SEQ ID NO: 98)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK
Example 5
[0318] The following is a data summary of additional FGF19 variant
peptides analyzed for glucose lowering activity and lipid elevating
activity.
[0319] Table 4 illustrates the peptide "core sequences" of 35
additional FGF19 variants, denoted M5 to M40. Such exemplified
variant peptides have FGF19 C-terminal sequence,
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGL
LQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188) at the
C-terminal portion, e.g., following the "TSG" amino acid residues
of the core sequence. The data clearly show that variants M6, M7,
M8, mM38 and M39 have the desired characteristics of glucose
lowering activity and not statistically significant lipid elevating
activity in db/db mice.
TABLE-US-00014 TABLE 4 Additional Variants and Fine Mapping of the
N-terminal Domain (see SEQ ID NOs: 99, 100, and 5 to 40) SEQ ID NO
of N-term- SEQ ID Glucose Lipid N-terminal Domain Domain Core NO.
Lowering Elevation FGF19 RPLAFSDAGPHVHYGWGDPI 99 (aa 1-20)
RLRHLYTSG 185 + + FGF21 -HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD
186 + - M5 RHPIPDSSPLLQ--FGGQV 5 (aa 1-17) RLRHLYTSG 185 + - M6
R----DSSPLLQ--FGGQV 6 (aa 1-18) RLRHLYTSG 185 + - M7
RPLAFSDSSPLLQ--FGGQV 7 (aa 1-18) RLRHLYTSG 185 + - M8
R-HPIPDSSPLLQ--WGDPI 8 (aa 1-17) RLRHLYTSG 185 + - M9
R-HPIPDSSPLLQFGWGDPI 9 (aa 1-19) RLRHLYTSG 185 + + M10
R-HPIPDSSPHVHYGWGDPI 10 (aa 1-19) RLRHLYTSG 185 - + Mll
RPLAFSDAGPLLQ--WGDPI 11 (aa 1-18) RLRHLYTSG 185 N/D N/D M12
RPLAFSDAGPLLQFGWGDPI 12 (aa 1-20) RLRHLYTSG 185 - + M13
RPLAFSDAGPLLQ--FGGQV 13 (aa 1-18) RLRHLYTSG 185 - - M14
R-HPIPDSSPHVHYG--GQV 14 (aa 1-17) RLRHLYTSG 185 - - M15
RPLAFSDAGPHVHYG--GQV 15 (aa 1-18) RLRHLYTSG 185 + + M16
RPLAFSDAGPHVH--WGDPI 16 (aa 1-18) RLRHLYTSG 185 N/D N/D M17
RPLAFSDAGPHV--GWGDPI 17 (aa 1-18) RLRHLYTSG 185 N/D N/D M18
RPLAFSDAGPH--YGWGDPI 18 (aa 1-18) RLRHLYTSG 185 N/D N/D M19
RPLAFSDAGP-V-YGWGDPI 19 (aa 1-18) RLRHLYTSG 185 N/D N/D M20
RPLAFSDAGP-VH-GWGDPI 20 (aa 1-18) RLRHLYTSG 185 N/D N/D M21
RPLAFSDAGP-VHY-WGDPI 21 (aa 1-18) RLRHLYTSG 185 N/D N/D M22
RPLAFSDAGPHVH-GWGDPI 22 (aa 1-18) RLRHLYTSG 185 N/D N/D M23
RPLAFSDAGPH-H-GWGDPI 23 (aa 1-18) RLRHLYTSG 185 N/D N/D M24
RPLAFSDAGPH-HY-WGDPI 24 (aa 1-18) RLRHLYTSG 185 N/D N/D M25
RPLAFSDAGPHV-Y-WGDPI 25 (aa 1-18) RLRHLYTSG 185 N/D N/D M26
RPLAFSDSSPLVH--WGDPI 26 (aa 1-18) RLRHLYTSG 185 N/D N/D M27
RPLAFSDSSPHVH--WGDPI 27 (aa 1-18) RLRHLYTSG 185 N/D N/D M28
RPLAFSDAPHV----WGDPI 28 (aa 1-16) RLRHLYTSG 185 N/D N/D M29
RPLAFSDAGPHVHY-WGDPI 29 (aa 1-19) RLRHLYTSG 185 N/D N/D M30
RPLAFSDAGPHVHYAWGDPI 30 (aa 1-20) RLRHLYTSG 185 N/D N/D M31
R-HPIPDSSPLLQ--FGAQV 31 (aa 1-17) RLRHLYTSG 185 +/- - M32
R-HPIPDSSPLLQ-- 32 (aa 1-18) RLRHLYTSG 185 - - FGIYQV M33
R-HPIPDSSPLLQ--FGGQV 33 (aa 1-17) RLRHLYTSG 185 - - M34
R-HPIPDSSPLLQ--FGGAV 34 (aa 1-17) RLRHLYTSG 185 +/- - M35
R-HPIPDSSPLLQ--FGGEV 35 (aa 1-17) RLRHLYTSG 185 +/- +/ M36
R-HPIPDSSPLLQ--FGGQV 36 (aa 1-17) RLRHLYTSG 185 +/- - M37
R-HPIPDSSPLLQ--FGGUA 37 (aa 1-17) RLRHLYTSG 185 - - M38
R-HPIPDSSPLLQ--FGGQT 38 (aa 1-17) RLRHLYTSG 185 + - M39
R-HPIPDSSPLLQ--FGGQT 39 (aa 1-17) RLRHLYTSG 185 + - M40
R-HPIPDSSPLLQFGWGQP 40 (aa 1-16) RLRHLYTSG 185 - +
TABLE-US-00015 TABLE 4a (see SEQ ID NOs: 99, 100, 5, 9, 8, 12, 10,
13, 15, 14, 43, 6 and 7) ##STR00004## Core SEQ ID NO. Glucose
Lowering Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI
RLRHLYTSG 99 (aa 1-29) + + + FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD 100
(aa 1-25) + - - M5 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + - -
M9 R-HPIPDSSPLLQFGWGDPI RLRHLYTSG 9 (aa 1-28) + + + M8
R-HPIPDSSPLLQ--WGDPI RLRHLYTSG 8 (aa 1-26) + + + M12
RPLAFSDAGPLLQFGWGDPI RLRHLYTSG 12 (aa 1-29) - + + M10
R-HPIPDSSPHVHYGWGDPI RLRHLYTSG 10 (aa 1-28) - + + M13
RPLAFSDAGPLLQ--FGGQV RLRHLYTSG 13 (aa 1-27) - + + M15
RPLAFSDAGPHVHYG--GQV RLRHLYTSG 15 (aa 1-27) - - +/- M14
R-HPIPDSSPHVHYG--GQV RLRHLYTSG 14 (aa 1-26) - - +/- M43
RPLAFSDAGPHVHYG-GD-I RLRHLYTSG 43 (aa 1-27) + - +/- M6
R-----DSSPLLQ--FGGQV RLRHLYTSG 6 (aa 1-22) + - - M7
RPLAFSDSSPLLQ--FGGQV RLRHLYTSG 7 (aa 1-27) - - -
TABLE-US-00016 TABLE 4b (see SEQ ID NOs: 99, 5 and 31 to 40)
##STR00005## Core SEQ ID NO. Glucose Lowering Lipid Elevation HCC
Formation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa 1-29) + + +
FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + - - M5
R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + - - M31
R-HPIPDSSPLLQ--FGAQV RLRHLYTSG 31 (aa 1-26) + - + M32
R-HPIPDSSPLLQ--FGDQV RLRHLYTSG 32 (aa 1-26) + - - M33
R-HPIPDSSPLLQ--FGPQV RLRHLYTSG 33 (aa 1-26) - - + M34
R-HPIPDSSPLLQ--FGGAV RLRHLYTSG 34 (aa 1-26) - - + M35
R-HPIPDSSPLLQ--FGGEV RLRHLYTSG 35 (aa 1-26) - - + M36
R-HPIPDSSPLLQ--FGGNV RLRHLYTSG 36 (aa 1-26) + - +/- M37
R-HPIPDSSPLLQ--FGGQA RLRHLYTSG 37 (aa 1-26) - - + M38
R-HPIPDSSPLLQ--FGGQI RLRHLYTSG 38 (aa 1-26) - - + M39
R-HPIPDSSPLLQ--FGGQT RLRHLYTSG 39 (aa 1-26) - - + M40
R-HPIPDSSPLLQFGWGQPV RLRHLYTSG 40 (aa 1-28) - + +
TABLE-US-00017 TABLE 4c (see SEQ ID NOs: 99, 100, 5, 52, 54, to 68,
4, 69, 70 and 53) ##STR00006## Core SEQ ID NO. Glucose Lowering
Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG
99 (aa 1-29) + + + FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25)
+ - - M5 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + - - M52
R-----DSSPLLQ--WGDPI RLRHLYTSG 52 (aa 1-22) + + - M54
RPLAFSDAGPLLQ--WGDPI RLRHLYTSG 54 (aa 1-27) - + + M55
RPLAFSDAGPH--YGWGDPI RLRHLYTSG 55 (aa 1-27) - + + M56
RPLAFSDAGP-V-YGWGDPI RLRHLYTSG 56 (aa 1-27) - + + M57
RPLAFSDAGP-VT-GWGDPI RLRHLYTSG 57 (aa 1-27) - + + M58
RPLAFSDAGP-VHY-WGDPI RLRHLYTSG 58 (aa 1-27) - + + M59
RPLAFSDAGPH-H-GWGDPI RLRHLYTSG 59 (aa 1-27) - + + M60
RPLAFSDAGPH-HY-WGDPI RLRHLYTSG 60 (aa 1-27) - + + M61
RPLAFSDAGPHV--GWGDPI RLRHLYTSG 61 (aa 1-27) - + + M62
RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG 62 (aa 1-27) - + + M63
RPLAFSDAGPHVH--WGDPI RLRHLYTSG 63 (aa 1-27) + + + M64
RPLAFSDSSPLVH--WGDPI RLRHLYTSG 64 (aa 1-27) + + + M65
RPLAFSDSSPHVH--WGDPI RLRHLYTSG 65 (aa 1-27) - + + M66
RPLAFSDAGPHLQ--WGDPI RLRHLYTSG 66 (aa 1-27) + + + M67
RPLAFSDAGPHV---WGDPI RLRHLYTSG 67 (aa 1-26) - - +/- M68
RPLAFSDAGPHVHY-WGDPI RLRHLYTSG 68 (aa 1-28) - + - M4
RPLAFSDAGPHVHYAWGDPI RLRHLYTSG 4 (aa 1-29) + + + M69
R-----DSSPLVHYGWGDPI RLRHLYTSG 69 (aa 1-24) + + - M70
MR----DSSPLVHYGWGDPI RLRHLYTSG 70 (aa 1-25) + + - M53
M-----DSSPLLQ--WGDPI RLRHLYTSG 192 (aa 1-22) + + -
[0320] Table 5 illustrates the peptide sequences of 3 FGFT9
variants, denoted M1, M2 and M69. The data clearly show that these
three variants have the desired characteristics of glucose lowering
activity in db/db mice. These three variants appear to elevate
lipids in db/db mice.
TABLE-US-00018 TABLE 5 Additional Variants (SEQ ID NOs: 1, 2 and
69) M1: RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 1 or 139)
M2: RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 2 or 140)
M69: RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 69)
Example 6
[0321] The following is a data summary showing that FGF19 reduces
body weight in diet-induced obese mice and in ob/ob mice, and liver
tumor formation activity and body weight in db/db mice.
[0322] Mice were injected with FGF19 or FGF21 in AAV vector. Body
weight was recorded 4 weeks after injection.
TABLE-US-00019 TABLE 6 FGF19 reduces body weight in diet-induced
obese mice and in ob/ob mice (sequences correspond to aa 1-29 of
SEQ ID NO: 99 and aa 1-25 of SEQ ID NO: 100, respectively)
##STR00007## Core Body Weight- Lowering in DIO Body Weight-
Lowering in Ob/ob FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21
HPIPDSSPLLQ--FGGQV RQRYLYTDD + +
TABLE-US-00020 TABLE 7 Correlation of body weight and liver tumor
formation of FGF19, FGF21 and selected variants in db/db mice (see,
e.g., SEQ ID NOs: 99, 100, 5, 6, 32, 52 and 69) ##STR00008## core
SEQ ID NO Liver Tumor Nodule Body Weight FGF19 RPLAFSDAGPHVHYGWGDPI
RLRHLYTSG 99 (aa 1-29) + Increased FGF21 HPIPDSSPLLQ--FGGQV
RQRYLYTDD 100 (aa 1-25) - Decreased M5 R-HPIPDSSPLLQ--FGGQV
RLRHLYTSG 5 (aa 1-26) - Increased M6 R-----DSSPLLQ--FGGQV RLRHLYTSG
6 (aa 1-22) - Decreased M32 R-HPIPDSSPLLQ--FGDQV RLRHLYTSG 32 (aa
1-26) - Decreased M52 R-----DSSPLLQ--WGDPI RLRHLYTSG 52 (aa 1-22) -
Decreased M69 R-----DSSPLVHYGWGDPI RLRHLYTSG 69 (aa 1-24) -
Increased
Example 7
[0323] The following is a study showing that variant M5 and variant
M69 peptides reduce blood glucose.
[0324] Mice (ob/ob) were injected (subcutaneously) with M5 (0.1 and
1 mg/kg, s.c.) or FGF19 (1 mg/kg, s.c.), or variant M69 (0.1 and 1
mg/kg, s.c.) or FGF19 (1 mg/kg, s.c.). Plasma glucose levels were
measured at 2, 4, 7, and 24 hours after injection, and the results
are shown in FIG. 7. M5 (FIG. 7A) and variant M69 (FIG. 7B) showed
similar glucose lowering effects as wild type FGF19.
Example 8
[0325] This example sets forth several variant polypeptides and
particular characteristics thereof, including the variants' effect
on glucose lowering, lipid profile parameters, and HCC
formation.
[0326] In particular, Table 8 compares data generated for variants
M5 (SEQ ID NO:5), M6 (SEQ ID NO:6) and M50 (SEQ ID NO:50) with data
generated for corresponding variant polypeptides (denoted as M144,
M145, and M146, respectively) having N-terminal Arg (R) deletions.
Only certain sequence domains for each variant are listed:
N-terminal domain, Core, and Sheet-8/Loop-8/Sheet-9 region.
TABLE-US-00021 TABLE 8 ##STR00009## Core Sheet-8/Loop8/ Sheet-9
region Glucose Lowering Body Weight Re- duction HDL Elevation Tri-
glyceride Elevation HCC For- mation FGF19 RPLAFSDAGPHVHYGWGDPI
RLRHLYTSG //EEIRPDGYNVY// + - + + + (aa 1-20 of (aa 21-29 of (aa
102-112 of SEQ ID NO: 99) SEQ ID NO: SEQ ID NO: 99) 99) FGF21
HPIPDSSPLLQ--FGGQV RQRYLYTDD //ELLLEDGYNVY// + + - - - (aa 1-20 of
(aa 21-29 of (aa 97-107 of SEQ ID NO: 100) SEQ ID NO: SEQ ID NO:
100) 100) M5 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG //EEIRPDGYNVY// + - - -
- (aa 1-17 of (aa 18-26 of (aa 99-109 of SEQ ID NO: 5) SEQ ID NO:
SEQ ID NO: 5) 5) M6 R-------DSSPLLQ--FGGQV RLRHLYTSG
//EEIRPDGYNVY// + - - - - (aa 1-14 of (aa 15-23 of (aa 95-105 of
SEQ ID NO: 6) SEQ ID NO: SEQ ID NO: 6) 6) M50 R-HPIPDSSPLLQ--FGDQV
RLRHLYTSG //EEIRPDGYNVY// + + - - - (aa 1-17 of (aa 18-26 of (aa
99-109 of SEQ ID NO: 50) SEQ ID NO: SEQ ID NO: 50) 50) M144
--HPIPDSSPLLQ--FGGQV RLRHLYTSG //EEIRPDGYNVY// + - - - - (aa 2-17
of (aa 18-26 of (aa 99-109 of SEQ ID NO: 5) SEQ ID NO: SEQ ID NO:
5) 5) M145 ------DSSPLLQ-FGGQV RLRHLYTSG //EEIRPDGYNVY// + - - - -
(aa 2-14 of (aa 15-23 of (aa 95-105 of SEQ ID NO: 6) SEQ ID NO: SEQ
ID NO: 6) 6) M146 --HPIPDSSPLLQ--FGDQV RLRHLYTSG //EEIRPDGYNVY// +
+ - - - (aa 2-17 of (aa 18-26 of (aa 99-109 of SEQ ID NO: 50) SEQ
ID NO: SEQ ID NO: 50) 50)
[0327] As the data in Table 8 indicate, the deletion of the
N-terminal Arg (R) did not significantly impact glucose lowering,
body weight reduction, HDL and triglyceride elevation, and HCC
formation.
Example 9
[0328] This example sets forth several variant peptides having
amino acid substitutions in the Loop 8 region of FGF19, along with
the variants' effect on body weight, certain metabolic parameters,
and HCC formation.
[0329] The data in Table 9 are associated with variant polypeptides
denoted as M3, M139, M140, M141 and M160. The amino acid sequence
for M3 is set forth elsewhere herein, and the amino acid sequences
for M139, M140, M141 and M160 are as follows:
TABLE-US-00022 (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139); (SEQ ID NO:
194) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140); (SEQ ID NO:
195) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141); and (SEQ ID
NO: 196) RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160).
[0330] Only the following sequence domains for each of the
aforementioned variants are listed in Table 9: N-terminal domain,
Core, and Sheet-8/Loop-8/Sheet-9 region. While the particular amino
acid residues making up the Loop 8 region are not universally
accepted in the literature, FGF19 residues 127-129 are defined
herein as constituting the Loop-8 region.
TABLE-US-00023 TABLE 9 ##STR00010## Core Glucose Lowering Body
Weight Reduction HDL Elevation Tri- glyceride Elevation HCC For-
mation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEIRPDGYNVY// + - + +
+ (aa 1-20 of (aa 21-29 of (aa 102-112 of SEQ ID NO: 99) SEQ ID NO:
99) SEQ ID NO: 99) FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD
//ELLLEDGYNVY// + + - - - (aa 1-20 of (aa 21-29 of (aa 97-107 of
SEQ ID NO: 100) SEQ ID SEQ ID NO: 100) NO: 100) M3
RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + + +/- (aa 1-20
of (aa 21-29 of (aa 102-112 of SEQ ID NO: 3) SEQ ID NO: 3) SEQ ID
NO: 3) M139 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILPDGYNVY// + - + +
+ (aa 1-20 of (aa 21-29 of (aa 102-112 of SEQ ID N0193) SEQ ID SEQ
ID NO: 193) NO: 193) M140 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG
//EEIREDGYNVY// + + + + +/- (aa 1-20 of (aa 21-29 of (aa 102-112 of
SEQ ID NO: 194) SEQ ID SEQ ID NO: 194) NO: 194) M141
RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILCDGYNVY// + - + + + (aa 1-20
of (aa 21-29 of (aa 102-112 of SEQ ID NO: 195) SEQ ID SEQ ID NO:
195) NO: 195) M160 RPLAFSDAGPHVHYGWGDPI RQRHLYTSG //EEILEDGYNVY// +
+ + + - (aa 1-20 of (aa 21-29 of (aa 102-112 of SEQ ID NO: 196) SEQ
ID SEQ ID NO: 196) NO: 196)
[0331] Referring to Table 9, the P128E substitution appears
necessary to significantly prevent HCC formation, but is
insufficient by itself to prevent HCC formation. In particular, an
improvement in preventing HCC formation is observed with the P128E
substitution in M140. Conversely, by itself the R127L substitution
does not prevent HCC formation (see M139). As indicated in
comparison to M3, a combination of the R127L and P128E
substitutions decreases HCC formation but does not eliminate HCC
formation. Surprisingly, however, a combination of the R127L and
P128E substitutions along with a substitution of Gln (Q) for Leu
(L) in the FGF19 core region does significantly prevent HCC
formation (see M160).
[0332] These data indicate that the FGF19 Loop 8 region plays a
role in HCC formation. Amino acid residues outside of the Loop 8
region (e.g., substitutions in the core region) may enhance the
prevention of HCC formation.
TABLE-US-00024 M1 (SEQ ID NO: 1)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M2 (SEQ ID NO: 2)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK M3 (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M5 (SEQ ID NO: 5)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M5-R (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
M48 (SEQ ID NO: 48)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M49 (SEQ ID NO: 49)
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL
RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA
KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR
SPSFEK M50 (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M51 (SEQ ID NO: 51)
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M52 (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M53 (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVA
IKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M69 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
M70 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M71 (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M72 (SEQ
ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M73 (SEQ
ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPE
NCKTLLTDIDRTHTEKPVWDGITGE M75 (SEQ ID NO: 75)
RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKG
VHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLY
KNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M76
(SEQ ID NO: 76)
RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVR
YLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFL
PLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK FGF19 (SEQ
ID NO: 99)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK
Example 10
[0333] This example shows activation of mouse FGFR4-.beta.-klotho
signaling by FGF19, M3, and M70 in a rat myoblast cell line.
[0334] Methods:
[0335] An ELK luciferase assay was performed in L6 cells
transiently transfected with mouse FGFR4, .beta.-klotho, and
reporter constructs containing 5.times.UAS 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 were incubated
with ligands for 6 hours before lysed for luciferase activity
measurements.
[0336] A cell-based receptor activation assay was 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, was
transfected with DNAs encoding FGFR4 and .beta.-klotho from mouse,
as well as plasmids containing an Elk1-dependent chimeric
transcription factor-based reporter system.
[0337] Following transfection, concentration response of
ligand-dependent luciferase expression was analyzed in whole-cell
lysates in the presence of luciferin substrate.
[0338] Results:
[0339] Co-expression of FGFR4 and .beta.-klotho in L6 cells was
found to potentiate activation of intracellular signaling pathways
by both M3, M70 and FGF19 (EC.sub.50=20, 38 and 53 pM, respectively
(see Table 10 and FIG. 8).
TABLE-US-00025 TABLE 10 Co-expression of Mouse FGFR4/.beta.-klotho
complex in L6 Cells Potentiates Activation of Intracellular
Signaling Pathways by FGF19, M3 and M70. FGFR4/.beta.klotho Ligand
EC.sub.50 (pM) E.sub.max (fold potentiation) FGF19 52.5 .+-. 0.01
1.82 .+-. 0.09 M3 19.8 + 0.04 1.68 + 0.04 M70 38.3 .+-. 0.12 1.85
.+-. 0.14 EC.sub.50 = half-maximal effective concentration;
E.sub.max = maximum efficacy. Data are expressed as mean .+-.
SD
[0340] These data 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.
Example 11
[0341] This example sets forth several variant peptides having
amino acid substitutions in the Loop 8 region of FGF19, along with
the variants' effect on body weight, certain metabolic parameters,
and HCC formation.
[0342] The data in Table 11 are associated with variant
polypeptides denoted as M3, M200, M201, M202, M203, M204, M205, and
M206. The amino acid sequence for each of M3, M200, M201, M202,
M203, M204, M205, and M206 (FIG. 9) are as follows:
TABLE-US-00026 M3 (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M200 (SEQ ID NO: 197)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
M201 (SEQ ID NO: 198)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK M202 (SEQ ID NO: 199)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M203 (SEQ ID NO: 200)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M204 (SEQ ID NO: 201)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M205 (SEQ ID NO: 202)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M206 (SEQ ID NO: 203)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK
[0343] Table 11 shows a summary of fine mapping of amino acid
residues involved in the regulation of glucose, lipid levels and
HCC formation in db/db mice. Only the following sequence domains
for each of the aforementioned variants are listed in Table 11:
N-terminal domain, Core, and Sheet-8/Loop-8/Sheet-9 region. While
the particular amino acid residues making up the Loop 8 region are
not universally accepted in the literature, FGF19 residues 127-129
are defined herein as constituting the Loop-8 region.
TABLE-US-00027 TABLE 11 ##STR00011## Core Glucose Lowering Body
Weight Reduction HDL Elevation Tri- glyceride Elevation HCC For-
mation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEIRPDGYNVY// + - + +
+ (aa 1-20 of SEQ ID (SEQ ID (SEQ ID NO: NO: 99) NO: 185) 190)
FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD //ELLLEDGYNVY// + + - - - (aa
1-20 of SEQ ID (SEQ ID (SEQ ID NO: NO: 100) NO: 186) 191) M3
RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + + +/- (aa 1-20
of SEQ ID (SEQ ID (SEQ ID NO: NO: 3) NO: 185) 205) M200
R-----DSSPLVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + - - (aa 1-15
of SEQ ID (SEQ ID (SEQ ID NO: NO: 69) NO: 185) 205) M201
RPLAFSDSSPLVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + - - (aa 1-20
of SEQ ID (SEQ ID (SEQ ID NO: NO: 2) NO: 185) 205) M202
RPLAFSDASPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + - - (aa 1-20
of SEQ ID (SEQ ID (SEQ ID NO: NO: 1) NO: 185) 205) M203
R-----DSSPLLQ--WGDPI RLRHLYTSG //EEILEDGYNVY// + + + + - (aa 1-13
of SEQ ID (SEQ ID (SEQ ID NO: NO: 52) NO: 185) 205) M204
R-HPIPDSSPLLQ--FGDQV RLRHLYTSG //EEILEDGYNVY// + + - - - (aa 1-17
of SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO :185) 205) M205
R-----DSSPLLQ--FGGQV RLRHLYTSG //EEILEDGYNVY// + + - - - (aa 1-13
of SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 185) 205) M206
R-HPIPDSSPLLQ--FGGQV RLRHLYTSG //EEILEDGYNVY// + - - - - (aa 1-17
of SEQ ID (SEQ ID (SEQ ID NO: NO: 5) NO: 185) 205)
[0344] As shown in Table 11, a combination of the R127L and P128E
substitutions decreases HCC formation, and these data indicate that
the FGF19 Loop 8 region plays a role in HCC formation.
Example 12
[0345] The ability of M200 to activate its receptors in a
cell-based assay using 293T cells transfected with an
FGF-responsive GAL-Elk1 luciferase reporter was evaluated.
[0346] Methods:
[0347] 293T cells (ATCC) were cultured in DMEM supplemented with
10% FBS at 37.degree. C. under 5% CO2. Cells were transiently
transfected with expression vectors encoding FGFR1c, FGFR4, KLB,
GAL4-Elk-1 transcriptional activator (pFA2-Elk1, Stratagene), and
firefly luciferase reporter driven GAL4 binding sites (pFR-luc,
Stratagene) using FuGENE.RTM. 6 transfection reagent (Roche Applied
Science). Luciferase activity was determined 12 hours after ligand
addition.
[0348] Results:
[0349] In this assay, effective binding of a ligand to FGFR and KLB
co-receptor results in the activation of the endogenous ERK kinase
pathway, leading to subsequent activation of a chimeric
transcriptional activator comprising of an Elk-1 activation domain
and a GAL4 DNA-binding domain. As shown in FIG. 10, the variant
denoted M200 activated intracellular signaling pathways in 293T
cells expressing FGFR4/KLB receptor complexes (FIGS. 10B-10C) or
FGFR1c/KLB receptor complexes (FIG. 10A) as effectively as
FGF19.
[0350] In addition, the activation of endogenous receptors was
evaluated in H4IIE cells, a rat hepatomas cell line that expresses
KLB and, among the FGFR isoforms, predominantly FGFR4. As shown in
FIG. 10D, recombinant V130 protein induced phosphorylation and
activation of ERK with a similar potency and efficacy as wild type
FGF19.
Example 13
[0351] The following is a description of studies showing the
glucose lowering activity of variant peptides having amino acid
substitutions in the Loop 8 region of FGF19.
[0352] Mice (db/db) were injected with viral vector expressing
FGF19, FGF21 or variants, and analyzed after injection. Variants
denoted M200 and M202 (FIG. 11A), M201 (FIG. 11B) and M203, M204,
M205 and M206 (FIG. 11C) exhibited glucose lowering activity.
[0353] Data comparing M200, M201, M202, M203, M204, M205 and M205
glucose lowering activity and lipid elevating or increasing
activity to FGF19 and FGF21 are illustrated in FIGS. 11 and 12.
Example 14
[0354] The following is a description of studies showing that
variants M200-M206 are not tumorigenic, as determined by HCC
formation, and the effect on variants M200, M201, M202, M203, M204
M205 and M206 on the reduction of lean muscle and fat mass.
[0355] Animals (db/db) were injected with AAV vectors expressing
FGF19, FGF21, M200, M201, M202, M203, M204, M205 or M206, or
injected with saline, and analyzed 6 months after injection. The
data indicate that variants M200, M201, M202, M203, M204 M205 and
M206 did not induce HCC formation significantly (FIGS.
13A-13C).
[0356] Animals (db/db mice) were also injected with viral vector
expressing FGF19, FGF21, variants M200, M201, M202, M203, M204 M205
or M206, or injected with saline, and analyzed 6 months after
injection for the effect of on lean mass and fat mass. Data for
variants M200, M201, M202, M203, M204 M205 and M206 are illustrated
in FIGS. 14A-14C.
SEQUENCE LISTING
[0357] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing. The CRF entitled
13370-107-999_Sequence_Listing.txt, which was created on Jun. 7,
2019, and is 255,674 bytes in size, is identical to the paper copy
of the Sequence Listing and is incorporated herein by reference in
its entirety.
Sequence CWU 1
1
2051194PRTHomo 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
10
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