U.S. patent application number 16/917284 was filed with the patent office on 2020-12-17 for methods for treatment of bile acid-related disorders.
The applicant listed for this patent is NGM Biopharmaceuticals, Inc.. Invention is credited to Lei Ling, Jian Luo, Hui Tian.
Application Number | 20200390859 16/917284 |
Document ID | / |
Family ID | 1000005059422 |
Filed Date | 2020-12-17 |
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United States Patent
Application |
20200390859 |
Kind Code |
A1 |
Ling; Lei ; et al. |
December 17, 2020 |
METHODS FOR TREATMENT OF BILE ACID-RELATED DISORDERS
Abstract
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). In some embodiments, these variants and
fusions modulate bile acid homeostasis, and are useful in treatment
of bile acid related and associated disorders. In some embodiments,
these variants and fusions have glucose lowering activity, and are
useful in treatment of hyperglycemia and other disorders.
Inventors: |
Ling; Lei; (Foster City,
CA) ; Luo; Jian; (Albany, CA) ; Tian; Hui;
(Foster City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NGM Biopharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005059422 |
Appl. No.: |
16/917284 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15773120 |
May 2, 2018 |
10744185 |
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PCT/US2016/060944 |
Nov 8, 2016 |
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16917284 |
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62252939 |
Nov 9, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/575 20130101;
A61P 1/16 20180101; C07K 14/50 20130101; A61K 38/1825 20130101;
A61K 9/0019 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/50 20060101 C07K014/50; A61P 1/16 20060101
A61P001/16 |
Claims
1.-76. (canceled)
77. A method of preventing the induction or exacerbation of
pruritus associated with non-alcoholic steatohepatitis (NASH) in a
subject being treated for NASH, comprising administering to the
subject an effective amount of a peptide, wherein the peptide
comprises: TABLE-US-00066 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70).
78. The method of claim 77, wherein the peptide consists of SEQ ID
NO:70.
79. The method of claim 77, wherein the effective amount of the
peptide is 0.3 mg.
80. The method of claim 77, wherein the effective amount of the
peptide is 1 mg.
81. The method of claim 77, wherein the effective amount of the
peptide is 2 mg.
82. The method of claim 77, wherein the effective amount of the
peptide is 3 mg.
83. The method of claim 77, wherein the effective amount of the
peptide is 5 mg.
84. The method of claim 77, wherein the effective amount of the
peptide is 10 mg.
85. The method of claim 77, wherein the peptide is for
administration once a day.
86. The method of claim 77, wherein the peptide is for
administration twice a day.
87. The method of claim 77, wherein the peptide is for subcutaneous
administration.
88. The method of claim 77, wherein the peptide is for
administration for 7 days or longer.
89. The method of claim 77, wherein the peptide is for
administration for 14 days or longer.
90. The method of claim 77, wherein the peptide is for
administration for 21 days or longer.
91. The method of claim 77, wherein the peptide is for
administration for 28 days or longer.
92. The method of claim 77, wherein the peptide is for
administration for 1 to 12 months.
93. The method of claim 77, wherein the peptide is for
administration for 12 months.
94. The method of claim 77, wherein the peptide is for
administration for more than 12 months.
95. The method of claim 77, wherein the peptide is administered in
combination with ursodeoxycholic acid (UDCA).
96. The method of claim 77, wherein the subject is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
15/773,120, filed May 2, 2018, which is a 371 national stage
application of international application Serial No.
PCT/US2016/060944, filed Nov. 8, 2016, which claims the benefit of
priority to U.S. Ser. No. 62/252,939 filed Nov. 9, 2015, the
contents of each of which are incorporated herein by reference in
entirety.
1. 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). In some embodiments, these variants and
fusions modulate bile acid homeostasis, and are useful in treatment
of bile acid related and associated disorders. In some embodiments,
these variants and fusions have glucose lowering activity, and are
useful in treatment of hyperglycemia and other disorders.
2. SUMMARY
[0003] 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. In one embodiment, the
activity is glucose lowering activity. In another embodiment, the
activity is bile acid homeostasis modulating 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.
[0004] In one embodiment, provide herein is a method of modulating
bile acid homeostasis, comprising administering a chimeric peptide
sequence provided herein. Also provided herein is a method of
treating a bile acid-related disorder, comprising administering a
chimeric peptide sequence provided herein. In another embodiment,
provided herein is a method of treating a bile acid-associated
disorder comprising administering a chimeric peptide sequence
provided herein. In specific embodiments, an effective amount of
the chimeric peptide sequence is administered.
[0005] 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.
[0006] In another embodiment, the treatment peptide, comprises: 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 (i) a first
C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids
16 to 29 of SEQ ID NO:99 [FGF19]), wherein the W residue
corresponds to the first amino acid position of the C-terminal
region; and (ii) a second C-terminal region sequence comprising
TABLE-US-00001 PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYL
CMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG LVTGLEAVRSPSFEK
(amino acid residues 30 to 194 of SEQ ID NO: 99 [FGF19]).
In certain embodiments, the peptide (i) binds to fibroblast growth
factor receptor 4 (FGFR4) with an affinity equal to or greater than
FGF19 binding affinity for FGFR4; (ii) activates FGFR4 to an extent
or amount equal to or greater than FGF19 activates FGFR4; (iii) has
at least one of reduced hepatocellular carcinoma (HCC) formation;
greater glucose lowering activity, less lipid increasing activity,
less triglyceride activity, less cholesterol activity, less non-HDL
activity or less HDL increasing activity, as compared to FGF19, or
as compared to 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 (SEQ ID NO:99); and/or (iv) has less lean mass
reducing activity as compared to FGF21.
[0007] In some embodiments, the second C-terminal region sequence
of the treatment peptide comprises from 1 to 5 amino acid
substitutions, deletions or insertions. In some embodiments, the
treatment peptide is less than about 250 amino acids in length.
[0008] In one embodiment, the treatment peptide has an amino acid
sequence comprising or consisting of
TABLE-US-00002 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.
In certain embodiments, the treatment peptide has an amino acid
sequence comprising SEQ ID NO:70. In other embodiments, the
treatment peptide has an amino acid sequence consisting of SEQ ID
NO:70. In some embodiments, the treatment peptide is fused with an
immunoglobulin Fc region.
[0009] In another embodiment, the treatment peptide has an amino
acid sequence comprising or consisting of
TABLE-US-00003 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.
In certain embodiments, the treatment peptide has an amino acid
sequence comprising SEQ ID NO:69. In other embodiments, the
treatment peptide has an amino acid sequence consisting of SEQ ID
NO:69. In some embodiments, the treatment peptide is fused with an
immunoglobulin Fc region.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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 residues GQV.
[0014] In some embodiments, the peptide comprises i) a FGF19
sequence variant having one or more amino acid substitutions,
insertions or deletions compared to a reference or wild type FGF19;
ii) a FGF21 sequence variant having one or more amino acid
substitutions, insertions or deletions compared to a reference or
wild type FGF21; iii) a portion of a FGF19 sequence fused to a
portion of a FGF21 sequence; or iv) a portion of a FGF19 sequence
fused to a portion of a 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.
[0015] 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).
[0016] 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.
[0017] 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 1-11, or
a peptide sequence that comprises or consists of any sequence set
forth in the Sequence Listing herein.
[0018] 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.
[0019] 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).
[0020] Methods and uses provided herein can be practiced using a
peptide or chimeric sequence of any suitable length. In particular
embodiments, the N-terminal or C-terminal region of the peptide or
chimeric sequence is from about 20 to about 200 amino acid residues
in length. In other particular aspects, a peptide or chimeric
sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more amino acid deletions from the amino
terminus, the carboxy-terminus or internally. In further particular
embodiments, a peptide or chimeric sequence has an N-terminal
region, or a C-terminal region that 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.
[0021] In yet additional embodiments, a peptide sequence or a
chimeric peptide sequence has a WGDPI (SEQ ID NO:170) sequence
motif corresponding to the WGDPI sequence of amino acids 16-20 of
SEQ ID NO:99 (FGF19); has a substituted, mutated or absent WGDPI
(SEQ ID NO:170) sequence motif corresponding to FGF19 WGDPI (SEQ ID
NO:170) sequence of amino acids 16-20 of FGF19; has a WGDPI (SEQ ID
NO:170) sequence with one or more amino acids substituted, mutated
or absent. In various other further aspects, the peptide sequence
is distinct from an FGF19 variant sequence having any of GQV, GDI,
WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173),
GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID
NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ
ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ
ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184)
substituted for the FGF19 WGDPI (SEQ ID NO:170) sequence at amino
acids 16-20.
[0022] In yet further embodiments, a peptide sequence or a chimeric
peptide sequence has N-terminal region comprises amino acid
residues VHYG (SEQ ID NO:101), wherein the N-terminal region
comprises amino acid residues DASPHVHYG (SEQ ID NO:102), or the
N-terminal region comprises amino acid residues DSSPLVHYG (SEQ ID
NO:103). More particularly, in one aspect the G corresponds to the
last position of the N-terminal region.
[0023] In various additional aspects, the N-terminal region
comprises amino acid residues DSSPLLQ (SEQ ID NO:104), where the Q
residue is the last amino acid position of the N-terminal region,
or comprises amino acid residues DSSPLLQFGGQV (SEQ ID NO:105),
where the V residue corresponds to the last position of the
N-terminal region.
[0024] In certain embodiments, an N-terminal region comprises or
consists of (or further comprises or consists of): RHPIP (SEQ ID
NO:106), where R is the first amino acid position of the N-terminal
region; or HPIP (SEQ ID NO:107), where H is the first amino acid
position of the N-terminal region; or RPLAF (SEQ ID NO:108), where
R is the first amino acid position of the N-terminal region; or
PLAF (SEQ ID NO:109), where P is the first amino acid position of
the N-terminal region; or R, where R is the first amino acid
position of the N-terminal region.
[0025] In various other aspects, a peptide or chimeric sequence
has: amino acid residues HPIP (SEQ ID NO:107), which are the first
4 amino acid residues of the N-terminal region. In various still
further aspects, a peptide or chimeric sequence has: an R residue
at the first position of the N-terminal region, or the first
position of the N-terminal region is an M residue, or the first and
second positions of the N-terminal region is an MR sequence, or the
first and second positions of the N-terminal region is an RM
sequence, or the first and second positions of the N-terminal
region is an RD sequence, or the first and second positions of the
N-terminal region is an DS sequence, or the first and second
positions of the N-terminal region is an MD sequence, or the first
and second positions of the N-terminal region is an MS sequence, or
the first through third positions of the N-terminal region is an
MDS sequence, or the first through third positions of the
N-terminal region is an RDS sequence, or the first through third
positions of the N-terminal region is an MSD sequence, or the first
through third positions of the N-terminal region is an MSS
sequence, or the first through third positions of the N-terminal
region is an DSS sequence, or the first through fourth positions of
the N-terminal region is an RDSS (SEQ ID NO:115), sequence, or the
first through fourth positions of the N-terminal region is an MDSS
(SEQ ID NO:116), sequence, or the first through fifth positions of
the N-terminal region is an MRDSS (SEQ ID NO:117), sequence, or the
first through fifth positions of the N-terminal region is an MSSPL
(SEQ ID NO:113) sequence, or the first through sixth positions of
the N-terminal region is an MDSSPL (SEQ ID NO:110) sequence, or the
first through seventh positions of the N-terminal region is an
MSDSSPL (SEQ ID NO:111) sequence.
[0026] In various other particular aspects, a peptide or chimeric
sequence has at the N-terminal region first amino acid position an
"M" residue, an "R" residue, a "S" residue, a "H" residue, a "P"
residue, a "L" residue or an "D" residue. In various alternative
particular aspects, a peptide or chimeric sequence peptide sequence
does not have a "M" residue or an "R" residue at the first amino
acid position of the N-terminal region.
[0027] In further various other aspects, a peptide or chimeric
sequence has an N-terminal region with any one of the following
sequences: MDSSPL (SEQ ID NO:110), MSDSSPL (SEQ ID NO:111), SDSSPL
(SEQ ID NO:112), MSSPL (SEQ ID NO:113) or SSPL (SEQ ID NO:114).
[0028] In 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 a residue at the last position of the C-terminal
region that corresponds to about residue 194 of SEQ ID NO:99
(FGF19). In further other embodiments, a chimeric peptide sequence
or peptide sequence comprises all or a portion of an FGF19 sequence
(e.g., SEQ ID NO:99), positioned at the C-terminus of the peptide,
or where the amino terminal "R" residue is deleted from the
peptide.
[0029] In more particular embodiments, a chimeric peptide sequence
or peptide sequence comprises or consists of any of M1 to M98, M101
to M160, or M200 to M207 variant peptide sequences, or a
subsequence or fragment of any of the M1 to M98, M101 to M160, or
M200 to M207 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 1-11 or a peptide sequence that
comprises or consists of any sequence set forth in the Sequence
Listing herein.
[0030] In various more particular aspects, a peptide sequence
comprises or consists of any one of the following sequences:
TABLE-US-00004 (M3) (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M140) (SEQ ID NO:
194) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M160) (SEQ ID NO:
196) RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M69) (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M52) (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M5) (SEQ ID NO: 5)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M5-R) (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M71) (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYAS; (M72) (SEQ ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYAS; (M73) (SEQ ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTH TEKPVWDGITGE;
(M1) (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M2) (SEQ ID NO: 2 or
140) RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M48) (SEQ ID NO: 48
or 6 or 148) RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M49) (SEQ ID NO: 49 or 7 or
149) RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M50) (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M51) (SEQ ID NO: 51 or
36 or 155) RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M53) (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M70) (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M139) (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or (M141) (SEQ ID NO:
195) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment of any of the foregoing peptide
sequences. In certain embodiments of any of the foregoing peptide
sequences, the R terminal residue (R residue at the N-terminus) is
deleted.
[0031] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00005 (M200) (SEQ ID NO: 197)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIL
EDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0032] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00006 (M201) (SEQ ID NO: 198)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0033] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00007 (M202) (SEQ ID NO: 199)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0034] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00008 (M203) (SEQ ID NO: 200)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0035] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00009 (M204) (SEQ ID NO: 201)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0036] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00010 (M205) (SEQ ID NO: 202)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0037] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00011 (M206) (SEQ ID NO: 203)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0038] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00012 (M207) (SEQ ID NO:204)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
LEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof.
[0039] In some embodiments, the peptide is a variant peptide
designated M139. In some embodiments, the peptide comprises an
amino acid sequence set forth in SEQ ID NO:193. In other
embodiments, the peptide consists of an amino acid sequence set
forth in SEQ ID NO:193. In some embodiments, the peptide is a
variant peptide designated M140. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:194. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:194. In some embodiments, the peptide is a
variant peptide designated M141. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:195. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:195. In some embodiments, the peptide is a
variant peptide designated M160. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:196. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:196. In some embodiments, the peptide is a
variant peptide designated M200. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:197. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:197. In some embodiments, the peptide is a
variant peptide designated M201. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:198. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:198. In other embodiments, the peptide is a
variant peptide designated M202. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:199. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:199. In certain embodiments, the peptide is
a variant peptide designated M203. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:200. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:200. In some embodiments, the peptide is a
variant peptide designated M204. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:201. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:201. In another embodiment, the peptide is a
variant peptide designated M205. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:202. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:202. In other embodiments, the peptide is a
variant peptide designated M206. In some embodiments, the peptide
comprises an amino acid sequence set forth in SEQ ID NO:203. In
other embodiments, the peptide consists of an amino acid sequence
set forth in SEQ ID NO:203. In yet other embodiments, the peptide
is a variant peptide designated M207. In some embodiments, the
peptide comprises an amino acid sequence set forth in SEQ ID
NO:204. In other embodiments, the peptide consists of an amino acid
sequence set forth in SEQ ID NO:204.
[0040] In various additional particular aspects, the N-terminus of
the peptide sequence includes or consists of any of:
TABLE-US-00013 (M5-R) (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG; (M6-R) (amino acids 2-22 of SEQ ID NO:
6) DSSPLLQFGGQVRLRHLYTSG; (M7) (amino acids 1-27 of SEQ ID NO: 7)
RPLAFSDSSPLLQFGGQVRLRHLYTSG; (M8-R) (amino acids 2-26 of SEQ ID NO:
8) HPIPDSSPLLQWGDPIRLRHLYTSG; (M9-R) (amino acids 2-28 of SEQ ID
NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG; (M10-R) (amino acids 2-28 of
SEQ ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG; (M11) (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG; (M12) (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M13) (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14-R)
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG;
(M15) (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG; (M16) (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG; (M17) (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG; (M18) (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG; (M19) (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG; (M20) (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG; (M21) (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG; (M22) (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG; (M23)
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG;
(M24) (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHHYWGDPIRLRHLYTSG; (M25) (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG; (M26) (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG; (M27) (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG; (M28) (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG; (M29) (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG; (M30) (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG; (M31) (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG; (M32)
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG;
(M33) (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG; (M34) (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG; (M35) (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG; (M36) (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG; (M37) (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG; (M38) (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG; (M39) (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG; (M40) (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG; (M74-R)
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG;
(M75-R) (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG;
(M77-R) (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG; (M9) (amino
acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG; (M8)
(amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG;
(M12) (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M10) (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG; (M13) (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14) (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG; (M43) amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG; or (M6) (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG.
In certain embodiments, the peptide comprises or consists of any
of:
TABLE-US-00014 (M5-R) (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG; (M6-R) (amino acids 2-22 of SEQ ID NO:
6) DSSPLLQFGGQVRLRHLYTSG; (M7) (amino acids 1-27 of SEQ ID NO: 7)
RPLAFSDSSPLLQFGGQVRLRHLYTSG; (M8-R) (amino acids 2-26 of SEQ ID NO:
8) HPIPDSSPLLQWGDPIRLRHLYTSG; (M9-R) (amino acids 2-28 of SEQ ID
NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG; (M10-R) (amino acids 2-28 of
SEQ ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG; (M11) (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG; (M12) (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M13) (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14-R)
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG;
(M15) (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG; (M16) (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG; (M17) (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG; (M18) (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG; (M19) (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG; (M20) (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG; (M21) (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG; (M22) (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG; (M23)
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG;
(M24) (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHHYWGDPIRLRHLYTSG; (M25) (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG; (M26) (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG; (M27) (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG; (M28) (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG; (M29) (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG; (M30) (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG; (M31) (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG; (M32)
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG;
(M33) (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG; (M34) (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG; (M35) (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG; (M36) (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG; (M37) (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG; (M38) (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG; (M39) (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG; (M40) (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG; (M74-R)
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG;
(M75-R) (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG;
(M77-R) (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG; (M9) (amino
acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG; (M8)
(amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG;
(M12) (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M10) (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG; (M13) (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14) (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG; (M43) amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG; or (M6) (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG.
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.
[0041] In various further particular aspects, a peptide sequence
includes or consists of:
TABLE-US-00015 (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 138 or 161)
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 2 or 140)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPM
VPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or (SEQ ID NO: 141)
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPED
LRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof of any of the foregoing
peptide sequences. In certain embodiments of any of the foregoing
peptide sequences, the R terminal residue is deleted.
[0042] 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.
[0043] 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.
[0044] 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).
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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. In one embodiment, a
composition, such as a pharmaceutical composition includes chimeric
peptide sequence or peptide sequence and an agent that improves
bile acid homeostasis.
[0053] 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.
[0054] 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.
[0055] In one embodiment, provided herein is a method of preventing
a disease or disorder in a subject having, or at risk of having, a
disease or disorder preventable by a peptide sequence provided
herein, comprising administering a pharmaceutical composition
comprising a peptide provided herein to a subject in an amount
effective for preventing the disease or disorder. In another
embodiment, provided herein is a method of treating a disease or
disorder in a subject having, or at risk of having, a disease or
disorder treatable by a peptide sequence provided herein,
comprising administering a pharmaceutical composition comprising a
peptide provided herein to a subject in an amount effective for
treating the disease or disorder. In yet another embodiment,
provided herein is a method of managing a disease or disorder in a
subject having, or at risk of having, a disease or disorder
manageable by a peptide sequence provided herein, comprising
administering a pharmaceutical composition comprising a peptide
provided herein to a subject in an amount effective for managing
the disease or disorder. In one embodiment, the disease or disorder
is a bile acid-related disease or associated disorder.
[0056] Non-limiting exemplary bile acid-related or associated
disorders preventable, treatable or manageable according to the
methods and uses provided herein include: cholestasis, including,
for example diseases of intrahepatic cholestasis (e.g., primary
biliary cirrhosis (PBC), primary familial intrahepatic cholestasis
(PFIC) (e.g., progressive PFIC), primary sclerosing choangitis
(PSC), pregnancy intrahepatic cholestasis (PIC), neonatal
cholestasis, and drug-induced cholestasis (e.g., estrogen)), and
diseases of extrahepatic cholestasis (e.g., bile cut compression
from tumor, bile duct blockade by gall stones); bile acid
malabsorption and other disorders involving the distal small
intestine, including ileal resection, inflammatory bowel diseases
(e.g., Crohn's disease and ulcerative colitis), short bowel
syndrome, disorders impairing absorption of bile acids not
otherwise characterized (idiopathic)) leading to diarrhea (e.g.,
bile acid diarrhea (BAD)) and GI symptoms, and GI, liver, and/or
biliary cancers (e.g., colon cancer and hepatocellular cancer);
and/or bile acid synthesis abnormalities, such as those
contributing to non-alcoholic steatohepatitis (NASH), cirrhosis and
portal hypertension; e.g., in mammals, such as humans. Additional
bile acid-related or associated disorders include metabolic
syndrome; a lipid or glucose disorder; cholesterol or triglyceride
metabolism; type 2 diabetes.
[0057] In one particular embodiment, the bile acid-related or
associated disorder is bile acid malabsorption. In another
particular embodiment, the bile acid-related or associated disorder
is diarrhea. In another particular embodiment, the bile
acid-related or associated disorder is bile acid diarrhea. In a
still further particular embodiment, the bile acid-related or
associated disorder is cholestasis. In one embodiment, the
cholestasis is intrahepatic cholestasis. In another embodiment, the
cholestasis is extrahepatic cholestasis. In another, further
particular embodiment, the bile acid-related or associated disorder
is an error in bile acid synthesis. In another further particular
embodiment, the bile acid-related or associated disorder is primary
biliary cirrhosis (PBC). In other particular embodiments, the bile
acid-related or associated disorder is primary sclerosing
cholangitis (PSC). In another embodiment, the bile acid-related or
associated disorder is PFIC (e.g., progressive PFIC). In another
embodiment, the bile acid-related or associated disorder is NASH.
In another embodiment, the bile acid-related or associated disorder
is a hyperglycemic condition. In a specific embodiment, the bile
acid-related or associated disorder is type 2 diabetes.
[0058] In some embodiments, the pharmaceutical composition further
comprises at least one additional agent effective in modulating
bile acid homeostasis or treating a bile acid-related or associated
disorder, wherein the additional agent is: a glucocorticoid; CDCA;
UDCA; insulin, an insulin secretagogues, an insulin mimetic, a
sulfonylurea and a meglitinide; a biguanide; an alpha-glucosidase
inhibitors; a DPP-IV inhibitor, GLP-1, a GLP-1 agonists and a GLP-1
analog; a DPP-IV-resistant analogue; a PPAR gamma agonist, a
dual-acting PPAR agonist, a pan-acting PPAR agonist; a PTP1B
inhibitor; an SGLT inhibitor; an RXR agonist; a glycogen synthase
kinase-3 inhibitor; an immune modulator; a beta-3 adrenergic
receptor agonist; an 11beta-HSD1 inhibitor; amylin and an amylin
analogue; a bile acid sequestrant; or an SGLT-2 inhibitor. In
certain embodiments, the at least one additional agent effective in
modulating PBC is UDCA, an FXR agonist, OCA, an ASBT inhibitor, an
autoimmune agent, an anti-IL-12 agent, an anti-CD80 agent, an
anti-CD20 agent, a CXCL10 neutralizing antibody, a ligand for
CXCR3, a fibrate, fish oil, colchicine, methotrexate, azathioprine,
cyclosporine, or an anti-retroviral therapy. In particular
embodiments, the at least one additional agent effective in
modulating PBC is UDCA, OCA, an ASBT inhibitor, an anti-IL-12
agent, an anti-CD20 agent, or a fibrate.
[0059] Non-limiting exemplary disorders or conditions preventable,
treatable or manageable with the peptide formulations, methods and
uses thereof provided herein, include metabolic diseases and
disorders. Non-limiting examples of diseases and disorders include:
metabolic syndrome; a lipid- or glucose-related disorder;
cholesterol or triglyceride metabolism; type 2 diabetes;
cholestasis, including, for example diseases of intrahepatic
cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and
drug induced cholestasis (e.g., estrogen)), and diseases of
extrahepatic cholestasis (e.g., bile cut compression from tumor,
bile duct blockade by gall stones); bile acid malabsorption and
other disorders involving the distal small intestine, including
ileal resection, inflammatory bowel diseases (e.g., Crohn's disease
and ulcerative colitis), disorders impairing absorption of bile
acids not otherwise characterized (idiopathic)) leading to diarrhea
(e.g., BAD) and GI symptoms, and GI, liver, and/or biliary cancers
(e.g., colon cancer and hepatocellular cancer); and/or bile acid
synthesis abnormalities, such as those contributing to NASH,
cirrhosis and portal hypertension. For treatment, peptide provided
herein can be administered to subjects in need of modulation of
bile acid homeostasis or having a bile-acid related or associated
disorder. Peptides 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; dyslipidemias and their sequelae such as, for example,
atherosclerosis, coronary artery disease, cerebrovascular disorders
and the like.
[0060] Other conditions which may be associated with metabolic
syndrome, such as obesity and elevated body mass (including the
co-morbid conditions thereof such as, but not limited to,
nonalcoholic fatty liver disease (NAFLD), nonalcoholic
steatohepatitis (NASH), and polycystic ovarian syndrome (PCOS)),
and also include thromboses, hypercoagulable and prothrombotic
states (arterial and venous), hypertension (including portal
hypertension (defined as a hepatic venous pressure gradient (HVPG)
greater than 5 mm Hg), cardiovascular disease, stroke and heart
failure; disorders or conditions in which inflammatory reactions
are involved, including atherosclerosis, chronic inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), asthma,
lupus erythematosus, arthritis, or other inflammatory rheumatic
disorders; Disorders of cell cycle or cell differentiation
processes such as adipose cell tumors, lipomatous carcinomas
including, for example, liposarcomas, solid tumors, and neoplasms;
Neurodegenerative diseases and/or demyelinating disorders of the
central and peripheral nervous systems and/or neurological diseases
involving neuroinflammatory processes and/or other peripheral
neuropathies, including Alzheimer's disease, multiple sclerosis,
Parkinson's disease, progressive multifocal leukoencephalopathy and
Guillian-Barre syndrome; Skin and dermatological disorders and/or
disorders of wound healing processes, including erythemato-squamous
dermatoses; and other disorders such as syndrome X, osteoarthritis,
and acute respiratory distress syndrome.
[0061] In one embodiment, of the various methods provided herein,
the subject is a human. In certain embodiments, the subject is a
subject in need thereof.
[0062] In some embodiments, the chimeric peptide sequence or a
peptide sequence described herein, either alone or in combination
with at least one additional therapeutic agent or treatment
modality, is assessed to ensure that it does not cause untoward
adverse effects in the subject. In a particular aspect, the
combination of a chimeric peptide sequence or a peptide sequence
described herein and at least one additional therapeutic agent or
treatment modality is assessed to ensure that it does not induce
HCC in the subject. Such assessments may be performed before
initiation of therapy (e.g., in a dose escalation study), during
therapy, (e.g., by evaluating a marker correlating with HCC
activity), or subsequent to termination of therapy (e.g., by
performing a liver biopsy). In some aspects, the assessment is
performed in a suitable test environment (e.g., a validated animal
model). One of ordinary skill in the art is familiar with
additional means for ensuring that the combination therapy
described herein is suitable for the particular subject, or a
subject population representative of the particular subject, taking
into consideration all relevant factors including, for example, the
severity of the subject's bile acid-related or associated disorder
(e.g., PBC) and the other medications be taken by the subject.
[0063] In one 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. 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. 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.
[0064] In particular aspects of the invention methods and uses, a
chimeric peptide sequence or peptide sequence is administered to a
subject in an amount effective to improve or provide bile acid
homeostasis. Non-limiting exemplary bile acid related or associated
disorders treatable according to the invention methods and uses
include: metabolic syndrome; a lipid- or glucose-related disorder;
cholesterol or triglyceride metabolism; type 2 diabetes;
cholestasis, including, for example diseases of intrahepatic
cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and
drug induced cholestasis (e.g., estrogen)), and diseases of
extrahepatic cholestasis (e.g., bile cut compression from tumor,
bile duct blockade by gall stones); bile acid malabsorption and
other disorders involving the distal small intestine, including
ileal resection, inflammatory bowel diseases (e.g., Crohn's disease
and ulcerative colitis), disorders impairing absorption of bile
acids not otherwise characterized (idiopathic)) leading to diarrhea
(e.g., BAD) and GI symptoms, and GI, liver, and/or biliary cancers
(e.g., colon cancer and hepatocellular cancer); and/or bile acid
synthesis abnormalities, such as those contributing to NASH,
cirrhosis and portal hypertension. In one embodiment, the bile acid
related or associated disorder is bile acid malabsorption. In
another embodiment, the bile acid related or associated disorder is
diarrhea. In another embodiment, the bile acid related or
associated disorder is cholestasis (e.g., intrahepatic or
extrahepatic cholestasis). In another embodiment, the bile acid
related or associated disorder is primary billiary cirrhosis. In
another embodiment, the bile acid related or associated disorder is
primary sclerosing cholangitis. In another embodiment, the bile
acid related or associated disorder is PFIC (e.g., progressive
PFIC).
3. DESCRIPTION OF DRAWINGS
[0065] FIG. 1 shows cyp7a1 expression in db/db mice dosed
intraperitoneally with the indicated concentrations of FGF19 and
FGF21 (SEQ ID NOs:99 and 100).
[0066] FIG. 2A-2D show cyp7a1 expression in human primary
hepatocytes following dosing of A) variant M1 (SEQ ID NO:1); B)
variant M2 (SEQ ID NO:2); C) variant M5 (SEQ ID NO:5); and D)
variant M32 (SEQ ID NO:32).
[0067] FIG. 3A-3D show cyp7a1 expression in human primary
hepatocytes following dosing of A) variant M69 (SEQ ID NO:69); B)
variant M75 (SEQ ID NO:75); C) variant M70 (SEQ ID NO:70); and D)
variant M76 (SEQ ID NO:76).
[0068] FIG. 4A-4D show cyp7a1 expression in human primary
hepatocytes following dosing of A) variant M85 (SEQ ID NO:85); B)
variant M96 (SEQ ID NO:96); C) variant M90 (SEQ ID NO:90); and D)
variant M98 (SEQ ID NO:98).
[0069] FIG. 5 is a table showing the cyp7a1 IC.sub.50 (pM),
relative cyp7a1 expression and HCC core of the indicated variants:
M1, M2, M5, M32, M69, M70, M75, M76, M85, M90, M96 and M98.
[0070] FIG. 6 depicts the results of a human clinical trial,
showing administration of M70 is able to suppress
7a-hydroxy-4-cholsten-3-one (C4), a marker of bile acid synthesis,
as compared to a placebo.
[0071] FIG. 7 depicts that the expression of FGFR4/.beta.-klotho
complex in L6 cells potentiates activation of intracellular
signaling pathways by FGF19, M3 and M70.
[0072] FIG. 8 depicts that administration of M70 is able to
suppress C4 as compared to a placebo.
[0073] FIG. 9 depicts that mice treated with M70 showed a
statistically significant improvement in biochemical markers of
liver damage, such as alkaline phosphatase (ALP), alkaline
aminotransferase (ALT), aspartate aminotransfease (AST) and
gamma-glutamyltransferase (GGT), following bile duct ligation (BDL)
surgery.
[0074] FIG. 10 depicts that continuous expression of M70 in Mdr2
knockout mouse normalized liver enzymes such as ALP, ALT, and
AST.
[0075] FIG. 11 depicts the results of a human clinical trial,
showing administration of M70 was able to promote body weight loss
and to reduce serum triglycerides in type 2 diabetes patients.
4. DETAILED DESCRIPTION
[0076] Before the present disclosure is further described, it is to
be understood that the disclosure is not limited to the particular
embodiments set forth herein, and it is also to be understood that
the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to be
limiting.
4.1 Definitions
[0077] The terms "patient" or "subject" are used interchangeably to
refer to a human or a non-human animal (e.g., a mammal).
[0078] The terms "treat", "treating", treatment" and the like refer
to a course of action (such as administering a polypeptide or a
pharmaceutical composition comprising a polypeptide) initiated
after a disease, disorder or condition, or a symptom thereof, has
been diagnosed, observed, and the like so as to eliminate, reduce,
suppress, mitigate, or ameliorate, either temporarily or
permanently, at least one of the underlying causes of a disease,
disorder, or condition afflicting a subject, or at least one of the
symptoms associated with a disease, disorder, condition afflicting
a subject. Thus, treatment includes inhibiting (i.e., arresting the
development or further development of the disease, disorder or
condition or clinical symptoms association therewith) an active
disease.
[0079] The term "in need of treatment" as used herein refers to a
judgment made by a physician or other medical professional that a
subject requires or will benefit from treatment.
[0080] The terms "prevent", "preventing", "prevention" and the like
refer to a course of action (such as administering a polypeptide or
a pharmaceutical composition comprising a polypeptide) initiated in
a manner (e.g., prior to the onset of a disease, disorder,
condition or symptom thereof) so as to prevent, suppress, inhibit
or reduce, either temporarily or permanently, a subject's risk of
developing a disease, disorder, condition or the like (as
determined by, for example, the absence of clinical symptoms) or
delaying the onset thereof, generally in the context of a subject
predisposed to having a particular disease, disorder or condition.
In certain instances, the terms also refer to slowing the
progression of the disease, disorder or condition or inhibiting
progression thereof to a harmful or otherwise undesired state.
[0081] The term "in need of prevention" as used herein refers to a
judgment made by a physician or other medical professional that a
subject requires or will benefit from preventative care.
[0082] The phrase "therapeutically effective amount" refers to the
administration of an agent to a subject, either alone or as a part
of a pharmaceutical composition and either in a single dose or as
part of a series of doses, in an amount that is capable of having
any detectable, positive effect on any symptom, aspect, or
characteristics of a disease, disorder or condition when
administered to a patient. The therapeutically effective amount can
be ascertained by measuring relevant physiological effects. For
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 an agent is
effective to treat the hyperglycemic condition. For example, a
therapeutically effective amount is an amount sufficient to reduce
or decrease any level (e.g., a baseline level) of fasting plasma
glucose (FPG), wherein, for example, the amount is sufficient to
reduce a FPG level greater than 200 mg/dl to less than 200 mg/dl,
wherein the amount is sufficient to reduce a FPG level between 175
mg/dl and 200 mg/dl to less than the starting level, wherein the
amount is sufficient to reduce a FPG level between 150 mg/dl and
175 mg/dl to less than the starting level, wherein the amount is
sufficient to reduce a FPG level between 125 mg/dl and 150 mg/dl to
less than the starting 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.). Moreover, in the case of
HbAIc levels, the effective amount is 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
contemplated by the present disclosure. The therapeutically
effective amount can be adjusted in connection with the dosing
regimen and diagnostic analysis of the subject's condition and the
like.
[0083] The phrase "in a sufficient amount to effect a change" means
that there is a detectable difference between a level of an
indicator measured before (e.g., a baseline level) and after
administration of a particular therapy. Indicators include any
objective parameter (e.g., level of glucose or insulin) or
subjective parameter (e.g., a subject's feeling of well-being).
[0084] The phrase "glucose tolerance", as used herein, refers to
the ability of a subject to control the level of plasma glucose
and/or plasma insulin when glucose intake fluctuates. For example,
glucose tolerance encompasses the subject's ability to reduce,
within about 120 minutes, the level of plasma glucose back to a
level determined before the intake of glucose.
[0085] Broadly speaking, the terms "diabetes" and "diabetic" refer
to a progressive disease of carbohydrate metabolism involving
inadequate production or utilization of insulin, frequently
characterized by hyperglycemia and glycosuria. The terms
"pre-diabetes" and "pre-diabetic" refer to a state wherein a
subject does not have the characteristics, symptoms and the like
typically observed in diabetes, but does have characteristics,
symptoms and the like that, if left untreated, can progress to
diabetes. The presence of these conditions can be determined using,
for example, either the fasting plasma glucose (FPG) test or the
oral glucose tolerance test (OGTT). Both usually require a subject
to fast for at least 8 hours prior to initiating the test. In the
FPG test, a subject's blood glucose is measured after the
conclusion of the fasting; generally, the subject fasts overnight
and the blood glucose is measured in the morning before the subject
eats. A healthy subject would generally have a FPG concentration
between about 90 and about 100 mg/dl, a subject with "pre-diabetes"
would generally have a FPG concentration between about 100 and
about 125 mg/dl, and a subject with "diabetes" would generally have
a FPG level above about 126 mg/dl. In the OGTT, a subject's blood
glucose is measured after fasting and again two hours after
drinking a glucose-rich beverage. Two hours after consumption of
the glucose-rich beverage, a healthy subject generally has a blood
glucose concentration below about 140 mg/dl, a pre-diabetic subject
generally has a blood glucose concentration about 140 to about 199
mg/dl, and a diabetic subject generally has a blood glucose
concentration about 200 mg/dl or above. While the aforementioned
glycemic values pertain to human subjects, normoglycemia, moderate
hyperglycemia and overt hyperglycemia are scaled differently in
murine subjects. A healthy murine subject after a four-hour fast
would generally have a FPG concentration between about 100 and
about 150 mg/dl, a murine subject with "pre-diabetes" would
generally have a FPG concentration between about 175 and about 250
mg/dl and a murine subject with "diabetes" would generally have a
FPG concentration above about 250 mg/dl.
[0086] The term "insulin resistance" as used herein refers to a
condition where a normal amount of insulin is unable to produce a
normal physiological or molecular response. In some cases, a
hyper-physiological amount of insulin, either endogenously produced
or exogenously administered, is able to overcome the insulin
resistance, in whole or in part, and produce a biologic
response.
[0087] The term "metabolic syndrome" refers to an associated
cluster of traits that includes, but is not limited to,
hyperinsulinemia, abnormal glucose tolerance, obesity,
redistribution of fat to the abdominal or upper body compartment,
hypertension, dysfibrinolysis, and dyslipidemia characterized by
high triglycerides, low high density lipoprotein (HDL)-cholesterol,
and high small dense low density lipoprotein (LDL) particles.
Subjects having metabolic syndrome are at risk for development of
type 2 diabetes and/or other disorders (e.g., atherosclerosis).
[0088] The phrase "glucose metabolism disorder" encompasses any
disorder characterized by a clinical symptom or a combination of
clinical symptoms that is associated with an elevated level of
glucose and/or an elevated level of insulin in a subject relative
to a healthy individual. Elevated levels of glucose and/or insulin
can be manifested in the following diseases, disorders and
conditions: hyperglycemia, type II diabetes, gestational diabetes,
type I diabetes, insulin resistance, impaired glucose tolerance,
hyperinsulinemia, impaired glucose metabolism, pre-diabetes, other
metabolic disorders (such as metabolic syndrome, which is also
referred to as syndrome X), and obesity, among others. The
polypeptides of the present disclosure, and compositions thereof,
can be used, for example, to achieve and/or maintain glucose
homeostasis, e.g., to reduce glucose level in the bloodstream
and/or to reduce insulin level to a range found in a healthy
subject.
[0089] The term "hyperglycemia", as used herein, refers to a
condition in which an elevated amount of glucose circulates in the
blood plasma of a subject relative to a healthy individual.
Hyperglycemia can be diagnosed using methods known in the art,
including measurement of fasting blood glucose levels as described
herein.
[0090] The term "hyperinsulinemia", as used herein, refers to a
condition in which there are elevated levels of circulating insulin
when, concomitantly, blood glucose levels are either elevated or
normal. Hyperinsulinemia can be caused by insulin resistance which
is associated with dyslipidemia, such as high triglycerides, high
cholesterol, high low-density lipoprotein (LDL) and low
high-density lipoprotein (HDL); high uric acids levels; polycystic
ovary syndrome; type II diabetes and obesity. Hyperinsulinemia can
be diagnosed as having a plasma insulin level higher than about 2
.mu.U/mL.
[0091] As used herein, the phrase "body weight disorder" and
similar terms refer to conditions associated with excessive body
weight and/or enhanced appetite. Various parameters are used to
determine whether a subject is overweight compared to a reference
healthy individual, including the subject's age, height, sex and
health status. For example, a subject can be considered overweight
or obese by assessment of the subject's Body Mass Index (BMI),
which is calculated by dividing a subject's weight in kilograms by
the subject's height in meters squared. An adult having a BMI in
the range of -18.5 to -24.9 kg/m' is considered to have a normal
weight; an adult having a BMI between .about.25 and -29.9 kg/m' can
be considered overweight (pre-obese); and an adult having a BMI of
-30 kg/m' or higher can be considered obese. Enhanced appetite
frequently contributes to excessive body weight. There are several
conditions associated with enhanced appetite, including, for
example, night eating syndrome, which is characterized by morning
anorexia and evening polyphagia often associated with insomnia, but
which can be related to injury to the hypothalamus.
[0092] The terms "polypeptide," "peptide," and "protein", used
interchangeably herein, refer to a polymeric form of amino acids of
any length, which can include genetically coded and non-genetically
coded amino acids, chemically or biochemically modified or
derivatized amino acids, and polypeptides having modified
polypeptide backbones. The terms include fusion proteins,
including, but not limited to, fusion proteins with a heterologous
amino acid sequence, fusion proteins with heterologous and
homologous leader sequences, with or without N-terminus methionine
residues; immunologically tagged proteins; and the like. It will be
appreciated that throughout this disclosure reference is made to
amino acids according to the single letter or three letter
codes.
[0093] As used herein, the term "variant" encompasses
naturally-occurring variants (e.g., homologs and allelic variants)
and non-naturally-occurring variants (e.g., muteins).
Naturally-occurring variants include homologs, i.e., nucleic acids
and polypeptides that differ in nucleotide or amino acid sequence,
respectively, from one species to another. Naturally-occurring
variants include allelic variants, i.e., nucleic acids and
polypeptides that differ in nucleotide or amino acid sequence,
respectively, from one individual to another within a species.
Non-naturally-occurring variants include nucleic acids and
polypeptides that comprise a change in nucleotide or amino acid
sequence, respectively, where the change in sequence is
artificially introduced, e.g., the change is generated in the
laboratory or other facility by human intervention ("hand of
man").
[0094] The term "native", in reference to FGF19, refers to
biologically active, naturally-occurring FGF19, including
biologically active, naturally-occurring FGF19 variants. The term
includes the 194 amino acid human FGF19 mature sequence.
[0095] The terms "label", "labeling" and the like, when use in the
context of a polypeptide or nucleic acid (or antibody, as
appropriate) of the present disclosure are meant to refer broadly
to any means useful in, for example, polypeptide purification,
identification, isolation and synthesis. Labels are generally
covalently bound to the polypeptide of interest and can be
introduced in any manner known in the art, including attachment to
a mature polypeptide (generally at the N- or C-terminus),
incorporation during solid-phase peptide synthesis, or through
recombinant means. Examples include, but are not limited to,
fluorescence, biotinylation, and radioactive isotopes. Polypeptide
and nucleic acid molecules can be labeled by both in vitro and in
vivo methods. Labeling reagents and kits can be obtained from a
number of commercial sources (e.g., Thermo Fischer Scientific,
Rockford, Ill.; and Molecular Probes/Life Technologies; Grand
Island, N.Y.).
[0096] The term "muteins" as used herein refers broadly to mutated
recombinant proteins, i.e., a polypeptide comprising an
artificially introduced change in amino acid sequence, e.g., a
change in amino acid sequence generated in the laboratory or other
facility by human intervention ("hand of man"). These proteins
usually carry single or multiple amino acid substitutions and are
frequently derived from cloned genes that have been subjected to
site-directed or random mutagenesis, or from completely synthetic
genes.
[0097] As used herein in reference to native human FGF19 or a FGF19
mutein, the terms "modified", "modification" and the like refer to
one or more changes that enhance a desired property of human FGF19,
a naturally-occurring FGF19 variant, or a FGF19 mutein, wherein the
change(s) does not alter the primary amino acid sequence of the
FGF19. Such desired properties include, for example, enhancing
solubility, prolonging the circulation half-life, increasing the
stability, reducing the clearance, altering the immunogenicity or
allergenicity, improving aspects of manufacturability (e.g., cost
and efficiency), and enabling the raising of particular antibodies
(e.g., by introduction of unique epitopes) for use in detection
assays. Changes to human FGF19, a naturally-occurring FGF19
variant, or a FGF19 mutein that can be carried out include, but are
not limited to, pegylation (covalent attachment of one or more
molecules of polyethylene glycol (PEG), or derivatives thereof);
glycosylation (e.g., N-glycosylation), polysialylation and
hesylation; albumin fusion; albumin binding through, for example, a
conjugated fatty acid chain (acylation); Fc-fusion; and fusion with
a PEG mimetic. Some particular embodiments entail modifications
involving polyethylene glycol, other particular embodiments entail
modifications involving albumin, and still other particular
modifications entail modifications involving glycosylation.
[0098] The terms "DNA", "nucleic acid", "nucleic acid molecule",
"polynucleotide" and the like are used interchangeably herein to
refer to a polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof.
Non-limiting examples of polynucleotides include linear and
circular nucleic acids, messenger RNA (mRNA), complementary DNA
(cDNA), recombinant polynucleotides, vectors, probes, primers and
the like.
[0099] The term "probe" refers to a fragment of DNA or RNA
corresponding to a gene or sequence of interest, wherein the
fragment has been labeled radioactively (e.g., by incorporating
.sup.32P or .sup.35S) or with some other detectable molecule, such
as biotin, digoxygen or fluorescein. As stretches of DNA or RNA
with complementary sequences will hybridize, a probe can be used,
for example, to label viral plaques, bacterial colonies or bands on
a gel that contain the gene of interest. A probe can be cloned DNA
or it can be a synthetic DNA strand; the latter can be used to
obtain a cDNA or genomic clone from an isolated protein by, for
example, microsequencing a portion of the protein, deducing the
nucleic acid sequence encoding the protein, synthesizing an
oligonucleotide carrying that sequence, radiolabeling the sequence
and using it as a probe to screen a cDNA library or a genomic
library.
[0100] The term "heterologous" refers to two components that are
defined by structures derived from different sources. For example,
in the context of a polypeptide, a "heterologous" polypeptide can
include operably linked amino acid sequences that are derived from
different polypeptides. Similarly, in the context of a
polynucleotide encoding a chimeric polypeptide, a "heterologous"
polynucleotide can include operably linked nucleic acid sequences
that can be derived from different genes. Exemplary "heterologous"
nucleic acids include expression constructs in which a nucleic acid
comprising a coding sequence is operably linked to a regulatory
element (e.g., a promoter) that is from a genetic origin different
from that of the coding sequence (e.g., to provide for expression
in a host cell of interest, which can be of different genetic
origin than the promoter, the coding sequence or both). In the
context of recombinant cells, "heterologous" can refer to the
presence of a nucleic acid (or gene product, such as a polypeptide)
that is of a different genetic origin than the host cell in which
it is present.
[0101] The term "operably linked" refers to linkage between
molecules to provide a desired function. For example, "operably
linked" in the context of nucleic acids refers to a functional
linkage between nucleic acid sequences. By way of example, a
nucleic acid expression control sequence (such as a promoter,
signal sequence, or array of transcription factor binding sites)
can be operably linked to a second polynucleotide, wherein the
expression control sequence affects transcription and/or
translation of the second polynucleotide. In the context of a
polypeptide, "operably linked" refers to a functional linkage
between amino acid sequences (e.g., different domains) to provide
for a described activity of the polypeptide.
[0102] As used herein in the context of the structure of a
polypeptide, "N-terminus" (or "amino terminus") and "C-terminus"
(or "carboxyl terminus") refer to the extreme amino and carboxyl
ends of the polypeptide, respectively, while the terms "N-terminal"
and "C-terminal" refer to relative positions in the amino acid
sequence of the polypeptide toward the N-terminus and the
C-terminus, respectively, and can include the residues at the
N-terminus and C-terminus, respectively. "Immediately N-terminal"
or "immediately C-terminal" refers to a position of a first amino
acid residue relative to a second amino acid residue where the
first and second amino acid residues are covalently bound to
provide a contiguous amino acid sequence.
[0103] "Derived from", in the context of an amino acid sequence or
polynucleotide sequence (e.g., an amino acid sequence "derived
from" a FGF19 polypeptide), is meant to indicate that the
polypeptide or nucleic acid has a sequence that is based on that of
a reference polypeptide or nucleic acid (e.g., a naturally
occurring FGF19 polypeptide or a FGF19-encoding nucleic acid), and
is not meant to be limiting as to the source or method in which the
protein or nucleic acid is made. By way of example, the term
"derived from" includes homologues or variants of reference amino
acid or DNA sequences.
[0104] In the context of a polypeptide, the term "isolated" refers
to a polypeptide of interest that, if naturally occurring, is in an
environment different from that in which it can naturally occur.
"Isolated" is meant to include polypeptides that are within samples
that are substantially enriched for the polypeptide of interest
and/or in which the polypeptide of interest is partially or
substantially purified. Where the polypeptide is not naturally
occurring, "isolated" indicates the polypeptide has been separated
from an environment in which it was made by either synthetic or
recombinant means.
[0105] "Enriched" means that a sample is non-naturally manipulated
(e.g., by a scientist or a clinician) so that a polypeptide of
interest is present in a) a greater concentration (e.g., at least
3-fold greater, at least 4-fold greater, at least 8-fold greater,
at least 64-fold greater, or more) than the concentration of the
polypeptide in the starting sample, such as a biological sample
(e.g., a sample in which the polypeptide naturally occurs or in
which it is present after administration), or b) a concentration
greater than the environment in which the polypeptide was made
(e.g., as in a bacterial cell).
[0106] "Substantially pure" indicates that a component (e.g., a
polypeptide) makes up greater than about 50% of the total content
of the composition, and typically greater than about 60% of the
total polypeptide content. More typically, "substantially pure"
refers to compositions in which at least 75%, at least 85%, at
least 90% or more of the total composition is the component of
interest. In some cases, the polypeptide will make up greater than
about 90%, or greater than about 95% of the total content of the
composition.
[0107] The terms "measuring" or "assaying" 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.
[0108] The terms "antibodies" (Abs) and "immunoglobulins" (Igs)
refer to glycoproteins having the same structural characteristics.
While antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which lack antigen specificity.
[0109] 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
can be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site. In
contrast to polyclonal antibody preparations, which can include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen.
[0110] In the context of an antibody, the term "isolated" refers to
an antibody that has been separated and/or recovered from
contaminant components of its natural environment; such contaminant
components include materials which might interfere with diagnostic
or therapeutic uses for the antibody, and can include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes.
[0111] As used herein, the term "FGF19-dependent" and similar
terms, as used in the context of a disease, disorder or condition,
refers to a disease, disorder or other condition that is caused
all, or in part, by the expression of FGF19. In certain
embodiments, the expression of FGF19 is amplified as compared to a
control. In some embodiments, the expression of FGF19 is amplified
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or more, or any numerical range
thereof. In some embodiments, the amplified expression of FGF19
directly results in the disease, disorder or condition, or a
symptom thereof. In other embodiments, the amplified expression of
FGF19 indirectly results in the disease disorder or condition, or a
symptom thereof.
4.2 Peptides
[0112] In certain embodiments, the pharmaceutical compositions,
formulations and dosage forms provided herein comprise one or more
peptides or peptide sequences provided herein. In certain
embodiments, the pharmaceutical compositions, formulations and
dosage forms provided herein comprise one or more variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of a bile acid-related or associated
disorder (e.g., PBC), a metabolic disorder or a cancer or tumor. In
certain embodiments, the activity is a glucose lowering activity.
Such variants and fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences include sequences that do not substantially increase or
induce HCC formation or HCC tumorigenesis and/or do not induce a
substantial elevation or increase in lipid profile.
[0113] In one embodiment, a chimeric peptide sequence includes or
consists of an N-terminal region having at least seven amino acid
residues and the N-terminal region having a first amino acid
position and a last amino acid position, where the N-terminal
region has a DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122)
sequence; and a C-terminal region having a portion of FGF19 and the
C-terminal region having a first amino acid position and a last
amino acid position, where the C-terminal region includes amino
acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG; SEQ ID NO:169) and
the W residue corresponds to the first amino acid position of the
C-terminal region. In particular embodiments, the variant is
M70:
TABLE-US-00016 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK.
In other particular embodiments, the variant is M69:
TABLE-US-00017 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK.
[0114] In another embodiment, the treatment peptide, comprises: 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; 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 (i) a first C-terminal region
sequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID
NO:99 [FGF19]), wherein the W residue corresponds to the first
amino acid position of the C-terminal region; and (ii) a second
C-terminal region sequence comprising
TABLE-US-00018 PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRY
LCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD
PFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ ID NO: 99
[FGF19]).
[0115] In another embodiment, the treatment peptide, comprises: 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 (i) a first
C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids
16 to 29 of SEQ ID NO:99 [FGF19]), wherein the W residue
corresponds to the first amino acid position of the C-terminal
region; and (ii) a second C-terminal region sequence comprising
TABLE-US-00019 PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRY
LCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD
PFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ ID NO: 99
[FGF19]).
[0116] In certain embodiments, the peptide (i) binds to FGFR4 with
an affinity equal to or greater than FGF19 binding affinity for
FGFR4; (ii) activates FGFR4 to an extent or amount equal to or
greater than FGF19 activates FGFR4; (iii) has at least one of
reduced HCC formation; greater glucose lowering activity, less
lipid increasing activity, less triglyceride activity, less
cholesterol activity, less non-HDL activity or less HDL increasing
activity, as compared to FGF19, or as compared to 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 (SEQ ID NO:99);
and/or (iv) has less lean mass reducing activity as compared to
FGF21.
[0117] In certain embodiments, the second C-terminal region
sequence comprises at least one amino acid substitution to the
EIRPD (amino acids 2-6 of SEQ ID NO:190) sequence. In some
embodiments, the at least one amino acid substitution is to the IRP
sequence of the EIRPD (amino acids 2-6 of SEQ ID NO:190) sequence.
In some embodiments, the at least one amino acid substitution is to
the RP sequence of the EIRPD sequence (amino acids 2-6 of SEQ ID
NO:190). In some embodiments, the at least one amino acid
substitution is R to L substitution. In other embodiments, the at
least one amino acid substitution is P to E substitution. In yet
other embodiments, the at least one amino acid substitution is RP
to LE substitution.
[0118] In some embodiments, the second C-terminal region sequence
comprises from 2 to 5 amino acid substitutions, deletions or
insertions. In other embodiments, the peptide is less than about
250 amino acids in length.
[0119] In one embodiment, the treatment peptide has an amino acid
sequence comprising or consisting of
TABLE-US-00020 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK.
In certain embodiments, the treatment peptide has an amino acid
sequence comprising SEQ ID NO:70. In other embodiments, the
treatment peptide has an amino acid sequence consisting of SEQ ID
NO:70. In some embodiments, the treatment peptide is fused with an
immunoglobulin Fc region.
[0120] In another embodiment, the treatment peptide has an amino
acid sequence comprising or consisting of
TABLE-US-00021 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK.
In certain embodiments, the treatment peptide has an amino acid
sequence comprising SEQ ID NO:69. In other embodiments, the
treatment peptide has an amino acid sequence consisting of SEQ ID
NO:69. In some embodiments, the treatment peptide is fused with an
immunoglobulin Fc region.
[0121] In another embodiment, the treatment peptide, comprises: 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; and b) a C-terminal region comprising a first
amino acid position and a last amino acid position, wherein the
C-terminal region comprises (i) a first C-terminal region sequence
comprising WGDPIRQRHLYTSG (SEQ ID NO:169 with a L7Q substitution),
wherein the W residue corresponds to the first amino acid position
of the C-terminal region; and (ii) a second C-terminal region
sequence comprising
TABLE-US-00022 (SEQ ID NO: 188)
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYL
CMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG
LVTGLEAVRSPSFEK.
[0122] In another embodiment, the treatment peptide, comprises: 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), DASPH (SEQ ID NO:122), or DAGPH (amino acids 7 to
11 of SEQ ID NO:99 [FGF19]); and b) a C-terminal region comprising
a first amino acid position and a last amino acid position, wherein
the C-terminal region comprises (i) a first C-terminal region
sequence comprising WGDPIRQRHLYTSG (SEQ ID NO:169 with a L7Q
substitution), wherein the W residue corresponds to the first amino
acid position of the C-terminal region; and (ii) a second
C-terminal region sequence comprising
TABLE-US-00023 (SEQ ID NO: 188)
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYL
CMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQR
QLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG
LVTGLEAVRSPSFEK.
In some embodiments, the peptide (i) binds to FGFR4 with an
affinity equal to or greater than FGF19 binding affinity for FGFR4;
(ii) activates FGFR4 to an extent or amount equal to or greater
than FGF19 activates FGFR4; (iii) has at least one of reduced
hepatocellular carcinoma (HCC) formation; greater glucose lowering
activity, less lipid increasing activity, less triglyceride
activity, less cholesterol activity, less non-HDL activity or less
HDL increasing activity, as compared to FGF19, or as compared to 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; and/or
(iv) has less lean mass reducing activity as compared to FGF21.
[0123] In certain embodiments, the second C-terminal region
sequence comprises at least one amino acid substitution to the
EIRPD (amino acids 2-6 of SEQ ID NO:190) sequence. In some
embodiments, the at least one amino acid substitution is to the IRP
sequence of the EIRPD (amino acids 2-6 of SEQ ID NO:190) sequence.
In some embodiments, the at least one amino acid substitution is to
the RP sequence of the EIRPD sequence (amino acids 2-6 of SEQ ID
NO:190). In some embodiments, the at least one amino acid
substitution is R to L substitution. In other embodiments, the at
least one amino acid substitution is P to E substitution. In yet
other embodiments, the at least one amino acid substitution is RP
to LE substitution.
[0124] In some embodiments, the second C-terminal region sequence
comprises from 2 to 5 amino acid substitutions, deletions or
insertions. In other embodiments, the peptide is less than about
250 amino acids in length.
[0125] In another embodiment, a chimeric peptide sequence includes
or consists of an N-terminal region having a portion of FGF21 and
the N-terminal region having a first amino acid position and a last
amino acid position, where the N-terminal region has a GQV sequence
and the V residue corresponds to the last amino acid position of
the N-terminal region; and a C-terminal region having a portion of
FGF19 and the C-terminal region having a first amino acid position
and a last amino acid position where the C-terminal region includes
amino acid residues 21-29 of FGF19 (RLRHLYTSG; SEQ ID NO: 185) and
the R residue corresponds to the first position of the C-terminal
region.
[0126] In particular aspects, modifications to the Loop-8 region of
FGF19 are disclosed herein that possess favorable metabolic
parameters without exhibiting substantial tumorigenicity. Herein,
FGF19 residues 127-129 are defined as constituting the Loop-8
region, although in the literature the Loop-8 region is sometimes
defined as including or consisting of other residues (e.g.,
residues 125-129). Certain combinations of R127L and P128E
substitutions to the FGF19 framework had an unexpectedly positive
effect on HCC formation. Even more surprisingly, a combination of
R127L and P128E substitutions and a substitution of Gln (Q) for Leu
(L) in the FGF19 core region had an even more significant effect on
preventing HCC formation.
[0127] 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, such as the region corresponding to
amino acids 21-29 of SEQ ID NO:99). In some embodiments, the Loop-8
modified variant comprises a substitution in the FGF19 Loop-8
region corresponding to amino acids 127-129 of SEQ ID NO:99. In
certain embodiments, the Loop-8 modified variant comprises a
substitution in the FGF19 Loop-8 region corresponding to (i) a
R127L substitution, (ii) a P128E substitution, or (iii) a R127L
substitution and a P128E substitution.
[0128] 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.
[0129] In some embodiments, the FGF19 variant comprises or further
comprises a substitution in the core region corresponding to amino
acids 21-29 of SEQ ID NO:99. In certain embodiments, the FGF19
variant comprises or further comprises a substitution in the core
region corresponding to a L22Q substitution.
[0130] In some embodiments, the Loop-8 modified variant is M70:
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV
SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDNIFSSPLETDS16MDPFG
LVTGLEAVRSPSFEK (SEQ ID NO:70), comprising a substitution in the
FGF19 Loop-8 region (underlined). In certain embodiments, the
Loop-8 modified M70 variant comprises a substitution in the FGF19
Loop-8 region (RPD; underlined) corresponding to (i) an R to L
substitution, (ii) a P to E substitution, or (iii) an R to L
substitution and a P to E substitution (SEQ. ID NO:204). In certain
embodiments, the Loop-8 modified M70 variant further comprises or
further comprises a substitution in the FGF19 core region. In some
embodiments, the Loop-8 modified M70 variant comprises a L18Q
substitution (i.e., SEQ ID NO:70 with an L18Q substitution).
[0131] In some embodiments, the Loop-8 modified variant is M69:
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA
LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVS
LSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDNIFSSPLETDS16MDPFGL
VTGLEAVRSPSFEK (SEQ ID NO:69), comprising a substitution in the
FGF19 Loop-8 region (underlined). In certain embodiments, the
Loop-8 modified M69 variant comprises a substitution in the FGF19
Loop-8 region (RPD; underlined) corresponding to (i) an R to L
substitution, (ii) a P to E substitution, or (iii) an R to L
substitution and a P to E substitution. In certain embodiments, the
Loop-8 modified M69 variant further comprises or further comprises
a substitution in the FGF19 core region. In some embodiments, the
Loop-8 modified M69 variant comprises a L17Q substitution (i.e.,
SEQ ID NO:69 with an L17Q substitution).
[0132] Other counterpart modifications in other variants provided
herein are also contemplated. 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.
[0133] In further embodiments, a peptide sequence includes or
consists of a FGF19 variant having one or more amino acid
substitutions, insertions or deletions compared to a reference or
wild type FGF19. In additional embodiments, a peptide sequence
includes or consists of a FGF21 sequence variant having one or more
amino acid substitutions, insertions or deletions compared to a
reference or wild type FGF21. In yet additional embodiments, a
peptide sequence includes or consists of a portion of a FGF19
sequence fused to a portion of a FGF21 sequence. In still
additional embodiments, a peptide sequence includes or consists of
a portion of a FGF19 sequence fused to a portion of a FGF21
sequence, where the FGF19 and/or FGF21 sequence portion(s) have one
or more amino acid substitutions, insertions or deletions compared
to a reference or wild type FGF19 and/or FGF21. Examples of such
sequences are disclosed in PCT Pub. No. WO 2013/006486 and US Pub.
No. 2013/0023474, as well as PCT Publ. No. WO 2014/085365,
published Jun. 5, 2014. Tables 1-11 and the Sequence Listing also
sets forth representative sequences that may be used in the methods
provided herein.
[0134] In some embodiments, the treatment peptides provided herein
include variants and fusions of FGF19 and/or FGF21 peptide
sequences. In one embodiment, the treatment peptides include one or
more variant or fusion FGF19 and/or FGF21 peptide. In other
embodiments, the methods provided herein include 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 a bile
acid-related or associated disorder.
[0135] A representative reference or wild type FGF19 sequence is
set forth as:
TABLE-US-00024 (SEQ ID NO: 99)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCA
RGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDC
AFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPML
PMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.
[0136] A representative reference or wild type FGF21 sequence is
set forth as:
TABLE-US-00025 (SEQ ID NO: 100)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSP
ESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREL
LLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALP
EPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS. FGF21 allelic variants include,
e.g., M70, M71 and M72.
[0137] 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.
[0138] In various particular aspects, a peptide or chimeric
sequence provided herein has at the N-terminal region first amino
acid position an "M" residue, an "R" residue, a "S" residue, a "H"
residue, a "P" residue, a "L" residue or an "D" residue. In various
alternative particular aspects, a peptide or chimeric sequence
peptide sequence does not have a "M" residue or an "R" residue at
the first amino acid position of the N-terminal region.
[0139] Also provided herein are subsequences, variants and modified
forms of the exemplified peptide sequences (including the FGF19 and
FGF21 variants and subsequences listed in the Sequence Listing, or
Tables 1-11), so long as the foregoing retains at least a
detectable or measurable activity or function. Also, certain
exemplified variant peptides, for example, those having all or a
portion of FGF21 sequence at the amino-terminus, have an "R"
residue positioned at the N-terminus, which can be omitted.
Similarly, certain exemplified variant peptides, include an "M"
residue positioned at the N-terminus, which can be appended to or
further substituted for an omitted residue, such as an "R" residue.
More particularly, in various embodiments peptide sequences at the
N-terminus include any of: RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID
NO:116) or MRDSS (SEQ ID NO:117). Furthermore, when a "M" residue
is adjacent to a "S" residue, the "M" residue may be cleaved such
that the "M" residue is deleted from the peptide sequence, whereas
when the "M" residue is adjacent to a "D" residue, the "M" residue
may not be cleaved. Thus, by way of example, in various embodiments
peptide sequences include those with the following residues at the
N-terminus: MDSSPL (SEQ ID NO:119), MSDSSPL (SEQ ID NO:120)
(cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL (SEQ ID NO:113)
(cleaved to SSPL (SEQ ID NO:114)).
[0140] Exemplified herein are peptide sequences, distinct from
reference FGF19 and FGF21 polypeptides set forth herein, that
modulate bile acid homeostasis, hyperglycemic conditions, insulin
resistance, hyperinsulinemia, glucose intolerance, metabolic
syndrome, or related disorders, in vivo (e.g., Tables 1-11 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.
[0141] 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
FGF19 WGDPI sequence of amino acids 16-20 of FGF19 (SEQ ID
NO:99).
[0142] Particular peptide sequences provided herein also include
sequences distinct from FGF19 and FGF21 (e.g., as set forth
herein), and FGF 19 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).
[0143] 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),
M101 to M160, or M200 to M207. More particular non-limiting
examples of peptide sequences include or consist of all or a part
of a sequence set forth as:
TABLE-US-00026 (M5-R) (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK (FGF21 sequences can also
include an residue at the amino terminus); (SEQ ID NO: 138 and 161)
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLR
GHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M1) (SEQ ID NO: 1 or 139)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M2) (SEQ ID NO: 2
or 140) RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 141)
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPED
LRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M69) (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M52) (SEQ ID NO: 52)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M5-R) (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M71) (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYAS; (M72) (SEQ ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYAS; (M73) (SEQ ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTH TEKPVWDGITGE;
(M3) (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M48) (SEQ ID NO:
48, 6 or 148) RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M49) (SEQ ID NO: 49, 7 or
149) RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M50) (SEQ ID NO: 50)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M51) (SEQ ID NO: 51,
36 or 155) RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M53) (SEQ ID NO: 192)
MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M70) (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M139) (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M140) (SEQ ID NO:
194) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M141) (SEQ ID NO:
195) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or (M160) (SEQ ID
NO: 196) RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof any of the foregoing peptide
sequences. In certain embodiments of any of the foregoing peptide
sequences, the R terminal residue is deleted.
[0144] 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:
TABLE-US-00027 (M5-R) (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG; (M6) (M6-R) (amino acids 2-22 of SEQ ID
NO: 6) DSSPLLQFGGQVRLRHLYTSG; (M7) (amino acids 1-27 of SEQ ID NO:
7) RPLAFSDSSPLLQFGGQVRLRHLYTSG; (M8-R) (amino acids 2-26 of SEQ ID
NO: 8) HPIPDSSPLLQWGDPIRLRHLYTSG; (M9-R) (amino acids 2-28 of SEQ
ID NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG; (M10-R) (amino acids 2-28 of
SEQ ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG; (M11) (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG; (M12) (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M13) (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14-R)
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG;
(M15) (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG; (M16) (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG; (M17) (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG; (M18) (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG; (M19) (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG; (M20) (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG; (M21) (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG; (M22) (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG; (M23)
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG;
(M24) (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHEYWGDPIRLRHLYTSG; (M25) (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG; (M26) (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG; (M27) (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG; (M28) (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG; (M29) (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG; (M30) (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG; (M31) (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG; (M32)
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG;
(M33) (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG; (M34) (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG; (M35) (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG; (M36) (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG; (M37) (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG; (M38) (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG; (M39) (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG; (M40) (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG; (M74-R)
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG;
(M75-R) (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG;
(M77-R) (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG; (M9) (amino
acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG; (M8)
(amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG;
(M12) (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M10) (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG; (M13) (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14) (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG; (M43) amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG; or (M6) (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG;
and for any of the foregoing peptide sequences the amino terminal R
residue may be deleted.
[0145] In certain embodiments, the peptide comprises or consists of
any of:
TABLE-US-00028 (M5-R) (amino acids 1-25 of SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLRHLYTSG; (M6) (M6-R) (amino acids 2-22 of SEQ ID
NO: 6) DSSPLLQFGGQVRLRHLYTSG; (M7) (amino acids 1-27 of SEQ ID NO:
7) RPLAFSDSSPLLQFGGQVRLRHLYTSG; (M8-R) (amino acids 2-26 of SEQ ID
NO: 8) HPIPDSSPLLQWGDPIRLRHLYTSG; (M9-R) (amino acids 2-28 of SEQ
ID NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG; (M10-R) (amino acids 2-28 of
SEQ ID NO: 10) HPIPDSSPHVHYGWGDPIRLRHLYTSG; (M11) (amino acids 1-27
of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG; (M12) (amino acids
1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M13) (amino
acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14-R)
(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG;
(M15) (amino acids 1-27 of SEQ ID NO: 15)
RPLAFSDAGPHVHYGGQVRLRHLYTSG; (M16) (amino acids 1-27 of SEQ ID NO:
16) RPLAFSDAGPHVHWGDPIRLRHLYTSG; (M17) (amino acids 1-27 of SEQ ID
NO: 17) RPLAFSDAGPHVGWGDPIRLRHLYTSG; (M18) (amino acids 1-27 of SEQ
ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG; (M19) (amino acids 1-27 of
SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG; (M20) (amino acids 1-27
of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPIRLRHLYTSG; (M21) (amino acids
1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG; (M22) (amino
acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG; (M23)
(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG;
(M24) (amino acids 1-27 of SEQ ID NO: 24)
RPLAFSDAGPHEYWGDPIRLRHLYTSG; (M25) (amino acids 1-27 of SEQ ID NO:
25) RPLAFSDAGPHVYWGDPIRLRHLYTSG; (M26) (amino acids 1-27 of SEQ ID
NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG; (M27) (amino acids 1-27 of SEQ
ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG; (M28) (amino acids 1-26 of
SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG; (M29) (amino acids 1-28
of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPIRLRHLYTSG; (M30) (amino acids
1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG; (M31) (amino
acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG; (M32)
(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG;
(M33) (amino acids 1-26 of SEQ ID NO: 33)
RHPIPDSSPLLQFGPQVRLRHLYTSG; (M34) (amino acids 1-26 of SEQ ID NO:
34) RHPIPDSSPLLQFGGAVRLRHLYTSG; (M35) (amino acids 1-26 of SEQ ID
NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG; (M36) (amino acids 1-26 of SEQ
ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG; (M37) (amino acids 1-26 of
SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG; (M38) (amino acids 1-26
of SEQ ID NO: 38) RHPIPDSSPLLQFGGQIRLRHLYTSG; (M39) (amino acids
1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG; (M40) (amino
acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG; (M74-R)
(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG;
(M75-R) (amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG;
(M77-R) (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG; (M9) (amino
acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG; (M8)
(amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG;
(M12) (amino acids 1-29 of SEQ ID NO: 12)
RPLAFSDAGPLLQFGWGDPIRLRHLYTSG; (M10) (amino acids 1-28 of SEQ ID
NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG; (M13) (amino acids 1-27 of
SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG; (M14) (amino acids 1-26
of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG; (M43) amino acids
1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG; or (M6) (amino
acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG.
In some embodiments, the peptide comprise one of the foregoing
sequences. In another embodiment, the peptide consists of one of
the foregoing sequences. In some embodiments, the peptide comprises
a C-terminal region comprising a portion of SEQ ID NO:99 (FGF19),
the C-terminal region having a first amino acid position and a last
amino acid position, 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.
[0146] In a specific embodiment, a peptide sequence comprises or
consists of:
TABLE-US-00029 (M70) (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK,
or a subsequence or fragment thereof.
[0147] In another embodiment, a peptide sequence comprises or
consists of:
TABLE-US-00030 (M69) (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK,
or a subsequence or fragment thereof.
[0148] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00031 (M200) (SEQ ID NO: 197)
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIL
EDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0149] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00032 (M201) (SEQ ID NO: 198)
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0150] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00033 (M202) (SEQ ID NO: 199)
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV
PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0151] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00034 (M203) (SEQ ID NO: 200)
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0152] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00035 (M204) (SEQ ID NO: 201)
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0153] In certain embodiments, the peptide comprises or consists
of:
TABLE-US-00036 (M205) (SEQ ID NO: 202)
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILED
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0154] In some embodiments, the peptide comprises or consists
of:
TABLE-US-00037 (M206) (SEQ ID NO: 203)
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
ILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof. In one embodiment, the
N-terminal R residue is deleted.
[0155] In other embodiments, the peptide comprises or consists
of:
TABLE-US-00038 (M207) (SEQ ID NO: 204)
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
LEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;
or a subsequence or fragment thereof.
[0156] 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.
[0157] Peptide sequences provided herein additionally include those
with reduced or absent induction or formation of HCC compared to
FGF19, or a FGF 19 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 a FGF 19
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 a FGF 19 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.
[0158] 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.
[0159] 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 a FGF19 sequence and a portion of a
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.
[0160] In some embodiments, the treatment peptides provided herein
also include subsequences, variants and modified forms of the
exemplified peptide sequences (including the FGF19 and FGF21
variants and subsequences listed in Tables 1-11 and Sequence
Listing), so long as the foregoing retains at least a detectable or
measurable activity or function. For example, certain exemplified
variant peptides have FGF19 C-terminal sequence,
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKM
QGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPL SHFLPML
PMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at
the C-terminal portion, e.g., following the "TSG" amino acid
residues of the variant.
[0161] 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 "M" residue may not be
cleaved. Thus, by way of example, in various embodiments peptide
sequences include those with the following residues at the
N-terminus: MDSSPL (SEQ ID NO:119), MSDSSPL (SEQ ID NO:120)
(cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL (SEQ ID NO:113)
(cleaved to SSPL (SEQ ID NO:114)).
[0162] Accordingly, in some embodiments, 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 1-11 and Sequence
Listing, and the FGF19/FGF21 fusions and chimeras listed in Tables
1-11 and Sequence Listing, so long as the subsequence, variant or
modified form (e.g., fusion or chimera) retains at least a
detectable activity or function, e.g., glucose lowering activity
and/or modulation of bile acid homeostasis.
[0163] 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.
[0164] Particular examples of such subsequences, variants and
modified forms of the peptide sequences exemplified herein (e.g., a
peptide sequence listed in the Sequence Listing or Tables 1-11)
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.
[0165] 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, a 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, a 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 a
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.
[0166] 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.
[0167] Additional specific examples are non-conservative and
conservative substitutions. A "conservative substitution" is a
replacement of one amino acid by a biologically, chemically or
structurally similar residue. Biologically similar means that the
substitution is compatible with a biological activity, e.g.,
activity that improves PBC and/or the manifestations thereof.
Structurally similar means that the amino acids have side chains
with similar length, such as alanine, glycine and serine, or having
similar size, or the structure of a first, second or additional
peptide sequence is maintained. Chemical similarity means that the
residues have the same charge or are both hydrophilic and
hydrophobic. Particular examples include the substitution of one
hydrophobic residue, such as isoleucine, valine, leucine or
methionine, for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acids, or glutamine for asparagine, serine
for threonine, etc. Routine assays can be used to determine whether
a subsequence, variant or modified form has activity, e.g.,
activity that improves PBC and/or the manifestations thereof.
[0168] Particular examples of subsequences, variants and modified
forms of the peptide sequences exemplified herein have 50%-60%,
60%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 96%, 97%,
98%, or 99% identity to a reference peptide sequence. The term
"identity" and "homology" and grammatical variations thereof mean
that two or more referenced entities are the same. Thus, where two
amino acid sequences are identical, they have the identical amino
acid sequence. "Areas, regions or domains of identity" mean that a
portion of two or more referenced entities are the same. Thus,
where two amino acid sequences are identical or homologous over one
or more sequence regions, they share identity in those regions.
[0169] 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)).
[0170] In the peptide sequences, including subsequences, variants
and modified forms of the peptide sequences exemplified herein, an
"amino acid" or "residue" includes conventional alpha-amino acids
as well as beta-amino acids; alpha, alpha disubstituted amino
acids; and N-substituted amino acids, wherein at least one side
chain is an amino acid side chain moiety as defined herein. An
"amino acid" further includes N-alkyl alpha-amino acids, wherein
the N-terminus amino group has a C.sub.1 to C.sub.6 linear or
branched alkyl substituent. The term "amino acid" therefore
includes stereoisomers and modifications of naturally occurring
protein amino acids, non-protein amino acids, post-translationally
modified amino acids (e.g., by glycosylation, phosphorylation,
ester or amide cleavage, etc.), enzymatically modified or
synthesized amino acids, derivatized amino acids, constructs or
structures designed to mimic amino acids, amino acids with a side
chain moiety modified, derivatized from naturally occurring
moieties, or synthetic, or not naturally occurring, etc. Modified
and unusual amino acids are included in the peptide sequences
provided herein (see, for example, in Synthetic Peptides: A User's
Guide; Hruby et al., Biochem. J. 268:249 (1990); and Toniolo C.,
Int. J. Peptide Protein Res. 35:287 (1990)).
[0171] 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.
[0172] A "derivative of an amino acid side chain moiety" is
included within the definition of an amino acid side chain moiety.
Non-limiting examples of derivatized amino acid side chain moieties
include, for example: (a) adding one or more saturated or
unsaturated carbon atoms to an existing alkyl, aryl, or aralkyl
chain; (b) substituting a carbon in the side chain with another
atom, such as oxygen or nitrogen; (c) adding a terminal group to a
carbon atom of the side chain, including methyl (--CH.sub.3),
methoxy (--OCH.sub.3), nitro (--NO.sub.2), hydroxyl (--OH), or
cyano (--C.dbd.N); (d) for side chain moieties including a hydroxy,
thiol or amino groups, adding a suitable hydroxy, thiol or amino
protecting group; or (e) for side chain moieties including a ring
structure, adding one or more ring substituents, including
hydroxyl, halogen, alkyl, or aryl groups attached directly or
through, e.g., an ether linkage. For amino groups, suitable
protecting groups are known to the skilled artisan. Provided such
derivatization provides a desired activity in the final peptide
sequence (e.g., activity that improves PBC and/or the
manifestations thereof).
[0173] 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.
[0174] A single amino acid, including stereoisomers and
modifications of naturally occurring protein amino acids,
non-protein amino acids, post-translationally modified amino acids,
enzymatically-synthesized amino acids, non-naturally occurring
amino acids including derivatized amino acids, an alpha, alpha
disubstituted amino acid derived from any of the foregoing (i.e.,
an alpha, alpha disubstituted amino acid, wherein at least one side
chain is the same as that of the residue from which it is derived),
a beta-amino acid derived from any of the foregoing (i.e., a
beta-amino acid which, other than for the presence of a
beta-carbon, is the same as the residue from which it is derived)
etc., including all of the foregoing can be referred to herein as a
"residue." Suitable substituents, in addition to the side chain
moiety of the alpha-amino acid, include C.sub.1 to C.sub.6 linear
or branched alkyl. Aib is an example of an alpha, alpha
disubstituted amino acid. While alpha, alpha disubstituted amino
acids can be referred to using conventional L- and D-isomeric
references, it is to be understood that such references are for
convenience, and that where the substituents at the alpha-position
are different, such amino acid can interchangeably be referred to
as an alpha, alpha disubstituted amino acid derived from the L- or
D-isomer, as appropriate, of a residue with the designated amino
acid side chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can
be referred to as either an alpha, alpha disubstituted amino acid
derived from L-Nle (norleucine) or as an alpha, alpha disubstituted
amino acid derived from D-Ala. Similarly, Aib can be referred to as
an alpha, alpha disubstituted amino acid derived from Ala. Whenever
an alpha, alpha disubstituted amino acid is provided, it is to be
understood as including all (R) and (S) configurations thereof.
[0175] 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.
[0176] In certain embodiments, covalent modifications of the
peptide sequences, including subsequences, variants and modified
forms of the peptide sequences exemplified herein are provided. An
exemplary type of covalent modification includes reacting targeted
amino acid residues with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues of the peptide. Derivatization with
bifunctional agents is useful, for instance, for cross-linking
peptide to a water-insoluble support matrix or surface for use in
the method for purifying anti-peptide antibodies, and vice-versa.
Commonly used cross linking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio] propioimidate.
[0177] 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.
[0178] Exemplified peptide sequences, and subsequences, variants
and modified forms of the peptide sequences exemplified herein can
also include alterations of the backbone for stability,
derivatives, and peptidomimetics. The term "peptidomimetic"
includes a molecule that is a mimic of a residue (referred to as a
"mimetic"), including but not limited to piperazine core molecules,
keto-piperazine core molecules and diazepine core molecules. Unless
otherwise specified, an amino acid mimetic of a peptide sequence
provided herein includes both a carboxyl group and amino group, and
a group corresponding to an amino acid side chain, or in the case
of a mimetic of Glycine, no side chain other than hydrogen.
[0179] 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.
[0180] The term "bind," or "binding," when used in reference to a
peptide sequence, means that the peptide sequence interacts at the
molecular level. Specific and selective binding can be
distinguished from non-specific binding using assays known in the
art (e.g., competition binding, immunoprecipitation, ELISA, flow
cytometry, Western blotting).
[0181] 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.
[0182] 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.
[0183] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Tables
1-11), can also be modified to form a chimeric molecule. In certain
embodiments, provided herein are peptide sequences that include a
heterologous domain. Such domains can be added to the
amino-terminus or at the carboxyl-terminus of the peptide sequence.
Heterologous domains can also be positioned within the peptide
sequence, and/or alternatively flanked by FGF19 and/or FGF21
derived amino acid sequences.
[0184] The term "peptide" also includes dimers or multimers
(oligomers) of peptides. In certain embodiments, dimers or
multimers (oligomers) of the exemplified peptide sequences are
provided herein, as well as subsequences, variants and modified
forms of the exemplified peptide sequences, including sequences
listed in the Sequence Listing or Tables 1-11.
[0185] In certain embodiments, a peptide sequence provided herein
comprises an amino acid sequence set forth in Table 1. In other
embodiments, a peptide sequence provided herein consists of an
amino acid sequence set forth in Table 1.
TABLE-US-00039 TABLE 1 SEQ ID NO. Amino Acid Sequence 1.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 2.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 3.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 4.
RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 5.
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 6.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 7.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 8.
RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 9.
RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 10.
RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 11.
RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 12.
RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 13.
RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 14.
RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 15.
RPLAFSDAGPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 16.
RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 17.
RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 18.
RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 19.
RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 20.
RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 21.
RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 22.
RPLAFSDAGPHVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 23.
RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 24.
RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 25.
RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 26.
RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 27.
RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 28.
RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 29.
RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 30.
RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 31.
RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 32.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 33.
RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 34.
RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 35.
RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 36.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 37.
RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 38.
RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 39.
RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 40.
RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 41.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGI
LAPQPPDVGSSDPLSMVGPSQGRSPSYAS 42.
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQP
PDVGSSDPLSMVGPSQGRSPSYAS 43.
RPLAFSDAGPHVHYGGDIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 44.
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAAD
QSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREL
LLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 45.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDG
YNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPMVPEEPE
DLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 46.
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAAD
QSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREL
LLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPP
GILAPQPPDVGSSDPLSMVGPSQGRSPSYASPMVPEEPEDLRGHLESDMFSS
PLETDSMDPFGLVTGLEAVRSPSFEK 47.
HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 48.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 49.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 50.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILE
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 51.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 52.
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 53.
MDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 54.
RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 55.
RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 56.
RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 57.
RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 58.
RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 59.
RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 60.
RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 61.
RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 62.
RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 63.
RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 64.
RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 65.
RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 66.
RPLAFSDAGPHLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 67.
RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 68.
RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 69.
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 70.
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 71.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDG
YNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAP
QPPDVGSSDPLSMVGPSQGRSPSYAS 72.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDG
YNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAP
QPPDVGSSDPLSMVGPSQGRSPSYAS 73.
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES
LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDG
YNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAP
QPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPV WDGITGE 74.
RDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 75.
RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK
AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN
VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHL
ESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 76.
RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKH
RLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSP
LETDSMDPFGLVTGLEAVRSPSFEK 77.
RRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL
PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLE
TDSMDPFGLVTGLEAVRSPSFEK 78.
RAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 79.
RGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 80.
RPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLE
IKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGY
NVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGH
LESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 81.
RHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGY
NVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGH
LESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 82.
RPLAFSAAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 83.
RPLAFSDAAPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 84.
RPLAFSDAGAHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVP
EEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 85.
RPLAFSDAGPHVHYGAGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 86.
RPLAFSDAGPHVHYGWGAPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 87.
RPLAFSDAGPHVHYGWGDAICARGQSAHSLLEIKAVALRTVAIKGVHSVR
YLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF
GLVTGLEAVRSPSFEK 88.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 89.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 90.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 91.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLAHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 92.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 93.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQAQLYKNRGFLPLAHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 94.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 95.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 96.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 97.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 98.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAAFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 138.
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGY
NVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGH
LESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 139.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 140.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 141.
DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL
LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPD
GYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLR
GHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 142.
RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 143.
RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 144.
RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 145.
RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 146.
RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 147.
RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 148.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 149.
RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 150.
RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 151.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 152.
RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 153.
RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 154.
RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 155.
RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 156.
RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 157.
RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 158.
RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 159.
RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQ
SAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 160.
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 161.
DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI
KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGY
NVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGH
LESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 162.
HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 163.
HPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPE
DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 164.
HPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQS
AHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEE
IRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 165.
HPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 166.
DAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRP
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 167.
VHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNV
YRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLE
SDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 168.
RLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIK
GVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLP
VSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLET
DSMDPFGLVTGLEAVRSPSFEK 188.
PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLC
MGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV TGLEAVRSPSFEK
192. MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 193.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 194.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 195.
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 196.
RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVP
EEPEDLRGHLESDNIFSSPLETDSMDPFGLVTGLEAVRSPSFEK 197.
RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS
LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILE
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 198.
RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 199.
RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFE
EEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPE
EPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 200.
RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 201.
RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILE
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 202.
RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL
EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDG
YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRG
HLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 203.
RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILE
DGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDL
RGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 204.
MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIL
EDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPED
LRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK
[0186] In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:1. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:2. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:3. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:4. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:5. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:6. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:7. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:8. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:9. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:10. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:11. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:12. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:13. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:14. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:15. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:16. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:17. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:18. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:19. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:20. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:21. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:22. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:23. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:24. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:25. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:26. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:27. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:28. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:29. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:30. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:31. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:32. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:33. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:34. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:35. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:36. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:37. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:38. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:39. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:40. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:41. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:42. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:43. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:44. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:45. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:46. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:47. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:48. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:49. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:50. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:51. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:52. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:53. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:54. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:55. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:56. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:57. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:58. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:59. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:60. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:61. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:62. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:63. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:64. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:65. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:66. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:67. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:68. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:69. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:70. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:71. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:72. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:73. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:74. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:75. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:76. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:77. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:78. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:79. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:80. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:81. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:82. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:83. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:84. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:85. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:86. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:87. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:88. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:89. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:90. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:91. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:92. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:93. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:94. In another embodiment, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:95. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:96. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:97. In another embodiment, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:98. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:138. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:139. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:140. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:141. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:142. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:143. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:144. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:145. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:146. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:147. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:148. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:149. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:150. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:151. In another embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:152. In
other embodiments, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:153. In one embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:154. In another embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:155. In other
embodiments, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:156. In one embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:157. In
another embodiment, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:158. In other embodiments, the
peptide sequence comprises an amino acid sequence set forth in SEQ
ID NO:159. In one embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:160. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:161. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:162. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:163. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:164. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:165. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:166. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:167. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:168. In another
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:192. In other embodiments, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:193. In one embodiment, the peptide sequence comprises an amino
acid sequence set forth in SEQ ID NO:194. In another embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:195. In other embodiments, the peptide sequence comprises
an amino acid sequence set forth in SEQ ID NO:196. In one
embodiment, the peptide sequence comprises an amino acid sequence
set forth in SEQ ID NO:197. In another embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:198. In other embodiments, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:199. In one embodiment,
the peptide sequence comprises an amino acid sequence set forth in
SEQ ID NO:200. In another embodiment, the peptide sequence
comprises an amino acid sequence set forth in SEQ ID NO:201. In
other embodiments, the peptide sequence comprises an amino acid
sequence set forth in SEQ ID NO:202. In one embodiment, the peptide
sequence comprises an amino acid sequence set forth in SEQ ID
NO:203. In another embodiment, the peptide sequence comprises an
amino acid sequence set forth in SEQ ID NO:204. In certain
embodiments of the various peptide sequences provided herein, the R
residue at the N-terminus is deleted.
[0187] In yet other embodiments, the peptide sequence consists of
an amino acid sequence set forth in SEQ ID NO:1. In another
embodiment, the peptide sequence consists of an amino acid sequence
set forth in SEQ ID NO:2. In other embodiments, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:3. In one embodiment, the peptide sequence consists of an amino
acid sequence set forth in SEQ ID NO:4. In another embodiment, the
peptide sequence consists of an amino acid sequence set forth in
SEQ ID NO:5. In other embodiments, the peptide sequence consists of
an amino acid sequence set forth in SEQ ID NO:6. In one embodiment,
the peptide sequence consists of an amino acid sequence set forth
in SEQ ID NO:7. In another embodiment, the peptide sequence
consists of an amino acid sequence set forth in SEQ ID NO:8. In
other embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:9. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:10. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:11. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:12. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:13. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:14. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:15. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:16. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:17. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:18. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:19. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:20. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:21. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:22. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:23. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:24. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:25. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:26. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:27. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:28. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:29. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:30. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:31. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:32. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:33. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:34. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:35. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:36. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:37. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:38. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:39. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:40. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:41. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:42. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:43. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:44. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:45. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:46. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:47. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:48. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:49. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:50. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:51. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:52. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:53. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:54. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:55. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:56. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:57. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:58. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:59. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:60. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:61. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:62. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:63. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:64. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:65. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:66. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:67. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:68. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:69. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:70. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:71. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:72. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:73. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:74. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:75. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:76. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:77. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:78. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:79. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:80. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:81. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:82. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:83. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:84. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:85. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:86. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:87. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:88. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:89. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:90. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:91. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:92. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:93. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:94. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:95. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:96. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:97. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:98. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:138. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:139. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:140. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:141. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:142. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:143. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:144. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:145. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:146. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:147. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:148. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:149. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:150. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:151. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:152. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:153. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:154. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:155. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:156. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:157. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:158. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:159. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:160. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:161. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:162. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:163. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:164. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:165. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:166. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:167. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:168. In another embodiment, the
peptide sequence consists of an amino acid sequence set forth in
SEQ ID NO:192. In other embodiments, the peptide sequence consists
of an amino acid sequence set forth in SEQ ID NO:193. In one
embodiment, the peptide sequence consists of an amino acid sequence
set forth in SEQ ID NO:194. In another embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:195. In other embodiments, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:196. In one embodiment,
the peptide sequence consists of an amino acid sequence set forth
in SEQ ID NO:197. In another embodiment, the peptide sequence
consists of an amino acid sequence set forth in SEQ ID NO:198. In
other embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:199. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:200. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:201. In other
embodiments, the peptide sequence consists of an amino acid
sequence set forth in SEQ ID NO:202. In one embodiment, the peptide
sequence consists of an amino acid sequence set forth in SEQ ID
NO:203. In another embodiment, the peptide sequence consists of an
amino acid sequence set forth in SEQ ID NO:204. In certain
embodiments of the various peptide sequences provided herein, the R
residue at the N-terminus is deleted.
4.3 Particular Modifications to Enhance Peptide Function
[0188] It is frequently beneficial, and sometimes imperative, to
improve one of more physical properties of the treatment modalities
disclosed herein and/or the manner in which they are administered.
Improvements of physical properties include, for example,
modulating immunogenicity; methods of increasing solubility,
bioavailability, serum half-life, and/or therapeutic half-life;
and/or modulating biological activity. Certain modifications may
also be useful to, for example, raise of antibodies for use in
detection assays (e.g., epitope tags) and to provide for ease of
protein purification. Such improvements must generally be imparted
without adversely impacting the bioactivity of the treatment
modality and/or increasing its immunogenicity.
[0189] Pegylation of is one particular modification contemplated
herein, while other modifications include, but are not limited to,
glycosylation (N- and O-linked); polysialylation; albumin fusion
molecules comprising serum albumin (e.g., human serum albumin
(HSA), cyano serum albumin, or bovine serum albumin (BSA)); albumin
binding through, for example a conjugated fatty acid chain
(acylation); and Fc-fusion proteins.
[0190] 4.3.1 Pegylation
[0191] The clinical effectiveness of protein therapeutics is often
limited by short plasma half-life and susceptibility to protease
degradation. Studies of various therapeutic proteins (e.g.,
filgrastim) have shown that such difficulties may be overcome by,
for example, conjugating or linking the protein to any of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes. This is frequently
effected by a linking moiety covalently bound to both the protein
and the nonproteinaceous polymer, e.g., a PEG. Such PEG-conjugated
biomolecules have been shown to possess clinically useful
properties, including better physical and thermal stability,
protection against susceptibility to enzymatic degradation,
increased solubility, longer in vivo circulating half-life and
decreased clearance, reduced immunogenicity and antigenicity, and
reduced toxicity. In addition to the beneficial effects of
pegylation on pharmacokinetic parameters, pegylation itself may
enhance activity.
[0192] PEGs suitable for conjugation to a polypeptide sequence are
generally soluble in water at room temperature, and have the
general formula R(O--CH.sub.2--CH.sub.2).sub.nO--R, where R is
hydrogen or a protective group such as an alkyl or an alkanol
group, and where n is an integer from 1 to 1000. When R is a
protective group, it generally has from 1 to 8 carbons. The PEG
conjugated to the polypeptide sequence can be linear or branched.
Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are
contemplated by the present disclosure. A molecular weight of the
PEG used in embodiments provided herein is not restricted to any
particular range, and examples are set forth elsewhere herein; by
way of example, certain embodiments have molecular weights between
500 Da and 20 kDa, while other embodiments have molecular weights
between 4 kDa and 10 kDa.
[0193] In other embodiments, provided herein are compositions of
conjugates wherein the PEGs have different n values, and thus the
various different PEGs are present in specific ratios. For example,
some compositions comprise a mixture of conjugates where n=1, 2, 3
and 4. In some compositions, the percentage of conjugates where n=1
is 18-25%, the percentage of conjugates where n=2 is 50-66%, the
percentage of conjugates where n=3 is 12-16%, and the percentage of
conjugates where n=4 is up to 5%. Such compositions can be produced
by reaction conditions and purification methods know in the art.
Cation exchange chromatography may be used to separate conjugates,
and a fraction is then identified which contains the conjugate
having, for example, the desired number of PEGs attached, purified
free from unmodified protein sequences and from conjugates having
other numbers of PEGs attached.
[0194] Pegylation most frequently occurs at the alpha amino group
at the N-terminus of the polypeptide, the epsilon amino group on
the side chain of lysine residues, and the imidazole group on the
side chain of histidine residues. Since most recombinant
polypeptides possess a single alpha and a number of epsilon amino
and imidazole groups, numerous positional isomers can be generated
depending on the linker chemistry.
[0195] General pegylation strategies known in the art can be
applied herein. PEG may be bound to a polypeptide provided herein
via a terminal reactive group (a "spacer" or "linker") which
mediates a bond between the free amino or carboxyl groups of one or
more of the polypeptide sequences and polyethylene glycol. The PEG
having the spacer which may be bound to the free amino group
includes N-hydroxysuccinylimide polyethylene glycol which may be
prepared by activating succinic acid ester of polyethylene glycol
with N-hydroxysuccinylimide. Another activated polyethylene glycol
which may be bound to a free amino group is
2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which may
be prepared by reacting polyethylene glycol monomethyl ether with
cyanuric chloride. The activated polyethylene glycol which is bound
to the free carboxyl group includes polyoxyethylenediamine.
[0196] Conjugation of one or more of the polypeptide sequences
provided herein to PEG having a spacer may be carried out by
various conventional methods. For example, the conjugation reaction
can be carried out in solution at a pH of from 5 to 10, at
temperature from 4.degree. C. to room temperature, for 30 minutes
to 20 hours, utilizing a molar ratio of reagent to protein of from
4:1 to 30:1. Reaction conditions may be selected to direct the
reaction towards producing predominantly a desired degree of
substitution. In general, low temperature, low pH (e.g., pH=5), and
short reaction time tend to decrease the number of PEGs attached,
whereas high temperature, neutral to high pH (e.g., pH.gtoreq.7),
and longer reaction time tend to increase the number of PEGs
attached. Various means known in the art may be used to terminate
the reaction. In some embodiments, the reaction is terminated by
acidifying the reaction mixture and freezing at, e.g., -20.degree.
C. Pegylation of various molecules is discussed in, for example,
U.S. Pat. Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and
5,985,263.
[0197] In some embodiments, also provided herein are uses of PEG
mimetics. Recombinant PEG mimetics have been developed that retain
the attributes of PEG (e.g., enhanced serum half-life) while
conferring several additional advantageous properties. By way of
example, simple polypeptide chains (comprising, for example, Ala,
Glu, Gly, Pro, Ser and Thr) capable of forming an extended
conformation similar to PEG can be produced recombinantly already
fused to the peptide or protein drug of interest (e.g., XTEN
technology; Amunix; Mountain View, Calif.). This obviates the need
for an additional conjugation step during the manufacturing
process. Moreover, established molecular biology techniques enable
control of the side chain composition of the polypeptide chains,
allowing optimization of immunogenicity and manufacturing
properties.
[0198] 4.3.2 Glycosylation
[0199] As used herein, "glycosylation" is meant to broadly refer to
the enzymatic process by which glycans are attached to proteins,
lipids or other organic molecules. The use of the term
"glycosylation" herein is generally intended to mean adding or
deleting one or more carbohydrate moieties (either by removing the
underlying glycosylation site or by deleting the glycosylation by
chemical and/or enzymatic means), and/or adding one or more
glycosylation sites that may or may not be present in the native
sequence. In addition, the phrase includes qualitative changes in
the glycosylation of the native proteins involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0200] Glycosylation can dramatically affect the physical
properties (e.g., solubility) of polypeptides and can also be
important in protein stability, secretion, and subcellular
localization. Glycosylated polypeptides may also exhibit enhanced
stability or may improve one or more pharmacokinetic properties,
such as half-life. In addition, solubility improvements can, for
example, enable the generation of formulations more suitable for
pharmaceutical administration than formulations comprising the
non-glycosylated polypeptide.
[0201] Addition of glycosylation sites can be accomplished by
altering the amino acid sequence. The alteration to the polypeptide
may be made, for example, by the addition of, or substitution by,
one or more serine or threonine residues (for O-linked
glycosylation sites) or asparagine residues (for N-linked
glycosylation sites). The structures of N-linked and O-linked
oligosaccharides and the sugar residues found in each type may be
different. One type of sugar that is commonly found on both is
N-acetylneuraminic acid (hereafter referred to as sialic acid).
Sialic acid is usually the terminal residue of both N-linked and
O-linked oligosaccharides and, by virtue of its negative charge,
may confer acidic properties to the glycoprotein. A particular
embodiment comprises the generation and use of N-glycosylation
variants.
[0202] The polypeptide sequences provided herein may optionally be
altered through changes at the nucleic acid level, particularly by
mutating the nucleic acid encoding the polypeptide at preselected
bases such that codons are generated that will translate into the
desired amino acids.
[0203] 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.
[0204] 4.3.3 Polysialylation
[0205] In certain embodiments, also provided herein is the use of
polysialylation, the conjugation of polypeptides to the naturally
occurring, biodegradable .alpha.-(2.fwdarw.8) linked polysialic
acid ("PSA") in order to improve the polypeptides' stability and in
vivo pharmacokinetics.
[0206] Albumin Fusion:
[0207] Additional suitable components and molecules for conjugation
include albumins such as human serum albumin (HSA), cyno serum
albumin, and bovine serum albumin (BSA).
[0208] In some embodiments, albumin is conjugated to a drug
molecule (e.g., a polypeptide described herein) at the carboxyl
terminus, the amino terminus, both the carboxyl and amino termini,
and internally (see, e.g., U.S. Pat. Nos. 5,876,969 and
7,056,701).
[0209] In the HSA-drug molecule conjugates embodiments provided
herein, various forms of albumin may be used, such as albumin
secretion pre-sequences and variants thereof, fragments and
variants thereof, and HSA variants. Such forms generally possess
one or more desired albumin activities. In additional embodiments,
fusion proteins are provided herein comprising a polypeptide drug
molecule fused directly or indirectly to albumin, an albumin
fragment, an albumin variant, etc., wherein the fusion protein has
a higher plasma stability than the unfused drug molecule and/or the
fusion protein retains the therapeutic activity of the unfused drug
molecule. In some embodiments, the indirect fusion is effected by a
linker, such as a peptide linker or modified version thereof.
[0210] As alluded to above, fusion of albumin to one or more
polypeptides provided herein can, for example, be achieved by
genetic manipulation, such that the nucleic acid coding for HSA, or
a fragment thereof, is joined to the nucleic acid coding for the
one or more polypeptide sequences.
[0211] 4.3.4 Alternative Albumin Binding Strategies
[0212] Several albumin--binding strategies have been developed as
alternatives to direct fusion and may be used with the agents
described herein. By way of example, in certain embodiments,
provided herein is albumin binding through a conjugated fatty acid
chain (acylation) and fusion proteins which comprise an albumin
binding domain (ABD) polypeptide sequence and the sequence of one
or more of the polypeptides described herein.
[0213] Fusion of albumin to a peptide sequence 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.
[0214] 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.
[0215] 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.
[0216] 4.3.5 Conjugation with Other Molecules
[0217] Additional suitable components and molecules for conjugation
include, for example, thyroglobulin; tetanus toxoid; Diphtheria
toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6
polypeptides of rotaviruses; influenza virus hemagglutinin,
influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and
hepatitis B virus core protein and surface antigen; or any
combination of the foregoing.
[0218] Thus, in certain embodiments, conjugation of one or more
additional components or molecules at the N- and/or C-terminus of a
polypeptide sequence, such as another polypeptide (e.g., a
polypeptide having an amino acid sequence heterologous to the
subject polypeptide), or a carrier molecule is also contemplated.
Thus, an exemplary polypeptide sequence can be provided as a
conjugate with another component or molecule.
[0219] A polypeptide may also be conjugated to large, slowly
metabolized macromolecules such as proteins; polysaccharides, such
as sepharose, agarose, cellulose, or cellulose beads; polymeric
amino acids such as polyglutamic acid, or polylysine; amino acid
copolymers; inactivated virus particles; inactivated bacterial
toxins such as toxoid from diphtheria, tetanus, cholera, or
leukotoxin molecules; inactivated bacteria; and dendritic cells.
Such conjugated forms, if desired, can be used to produce
antibodies against a polypeptide provided herein.
[0220] 4.3.6 Fc-fusion Molecules
[0221] In certain embodiments, the amino- or carboxyl-terminus of a
polypeptide sequence provided herein is fused with an
immunoglobulin Fc region to form a fusion conjugate (or fusion
molecule). In a specific embodiment, the immunoglobuling Fc region
is a human Fc region. Fusion conjugates have been shown to increase
the systemic half-life of biopharmaceuticals, and thus the
biopharmaceutical product may require less frequent administration.
In certain embodiments, the half-life is increased as compared to
the same polypeptide that is not fused to an immunoglobuling Fc
region.
[0222] Fc binds to the neonatal Fc receptor (FcRn) in endothelial
cells that line the blood vessels, and, upon binding, the Fc fusion
molecule is protected from degradation and re-released into the
circulation, keeping the molecule in circulation longer. This Fc
binding is believed to be the mechanism by which endogenous IgG
retains its long plasma half-life. More recent Fc-fusion technology
links a single copy of a biopharmaceutical to the Fc region of an
antibody to optimize the pharmacokinetic and pharmacodynamic
properties of the biopharmaceutical as compared to traditional
Fc-fusion conjugates.
[0223] 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 the Fc region of an antibody to optimize the
pharmacokinetic and pharmacodynamic properties of the
biopharmaceutical as compared to traditional Fc-fusion
conjugates.
[0224] In some embodiments, provided herein is a fusion of M70 to a
human antibody Fc fragment. In some embodiments, provided herein is
a fusion of M69 to a human antibody Fc fragment. Such fusions can
be useful in the treatment of bile acid related disorders and other
metabolic disorders provided herein. In some embodiments, the
Fc-fusion of M70 has a longer half-life. In specific embodiments,
the longer half-life of the Fc-fusion of M70 is as compared to M70
that is not an Fc-fusion. In some embodiments, the Fc-fusion of M69
has a longer half-life. In specific embodiments, the longer half
life of the Fc-fusion of M69 is as compared to M69 that is not an
Fc-fusion. Such a long half-life makes these fusions suitable for
once weekly, or less frequent dosing.
[0225] In some embodiments, the Fc-fusion comprises a linker.
Exemplary flexible linkers include glycine polymers (G)n,
glycine-serine polymers, glycine-alanine polymers, alanine-serine
polymers, and other flexible linkers. In certain embodiments, the
linker is (G).sub.4S. In some embodiments, the linker is
((G).sub.4S).sub.n, where n is an integer of at least one. In some
embodiments, the linker is ((G).sub.4S).sub.2. Glycine and
glycine-serine polymers are relatively unstructured, and therefore
may serve as a neutral tether between components. In some
embodiments, the glycine-serine polymer is (GS).sub.n, where n is
an integer of at least one. In some embodiments, the glycine-serine
polymer is GSGGS.sub.n (SEQ ID NO:129), where n is an integer of at
least one. In some embodiments, the glycine-serine polymer is
GGGS.sub.n (SEQ ID NO:130), where n is an integer of at least one.
In certain embodiments, the linker comprises an additional G
residue at the N' terminus of SEQ ID NO:130. In one embodiment, the
linker is GGSG (SEQ ID NO:131). In one embodiment, the linker is
GGSGG (SEQ ID NO:132). In one embodiment, the linker is GSGSG (SEQ
ID NO:133). In one embodiment, the linker is GSGGG (SEQ ID NO:134).
In one embodiment, the linker is GGGSG (SEQ ID NO:189). In one
embodiment, the linker is GSSSG (SEQ ID NO:135).
[0226] 4.3.7 Purification
[0227] 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.
[0228] Purification methods such as cation exchange chromatography
may be used to separate conjugates by charge difference, which
effectively separates conjugates into their various molecular
weights. For example, the cation exchange column can be loaded and
then washed with .about.20 mM sodium acetate, pH.about.4, and then
eluted with a linear (0 M to 0.5 M) NaCl gradient buffered at a pH
from 3 to 5.5, such as at pH.about.4.5. The content of the
fractions obtained by cation exchange chromatography may be
identified by molecular weight using conventional methods, for
example, mass spectroscopy, SDS-PAGE, or other known methods for
separating molecular entities by molecular weight. A fraction is
then identified which contains the conjugate having the desired
number of PEGs attached, purified free from unmodified protein
sequences and from conjugates having other numbers of PEGs
attached.
[0229] 4.3.8 Other Modifications
[0230] In certain embodiments, also provided herein is the use of
other modifications, currently known or developed in the future, to
improve one or more properties. Examples include hesylation,
various aspects of which are described in, for example, U.S. Patent
Appln. Nos. 2007/0134197 and 2006/0258607, and fusion molecules
comprising SUMO as a fusion tag (LifeSensors, Inc.; Malvern,
Pa.).
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 4.3.9 Linkers
[0235] Linkers and their use have been described above. Any of the
foregoing components and molecules used to modify the polypeptide
sequences provided herein may optionally be conjugated via a
linker. Suitable linkers include "flexible linkers" which are
generally of sufficient length to permit some movement between the
modified polypeptide sequences and the linked components and
molecules. The linker molecules are generally about 6-50 atoms
long. The linker molecules may also be, for example, aryl
acetylene, ethylene glycol oligomers containing 2-10 monomer units,
diamines, diacids, amino acids, or combinations thereof. Suitable
linkers can be readily selected and can be of any suitable length,
such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,
10-20, 20-30, 30-50 or more than 50 amino acids.
[0236] Exemplary flexible linkers include glycine polymers (Con,
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). In certain embodiments, the
linker is (G).sub.4S. In some embodiments, the linker is
((G).sub.4S).sub.n), where n is an integer of at least one. In some
embodiments, the linker is ((G).sub.4S).sub.2). In some
embodiments, the glycine-serine polymer is (GS).sub.n, where n is
an integer of at least one. In some embodiments, the glycine-serine
polymer is GSGGS.sub.n (SEQ ID NO:129), where n is an integer of at
least one. In some embodiments, the glycine-serine polymer is
GGGS.sub.n (SEQ ID NO:130), where n is an integer of at least one.
In certain embodiments, the linker comprises an additional G
residue at the N' terminus of SEQ ID NO:130. In one embodiment, the
linker is GGSG (SEQ ID NO:131). In one embodiment, the linker is
GGSGG (SEQ ID NO:132). In one embodiment, the linker is GSGSG (SEQ
ID NO:133). In one embodiment, the linker is GSGGG (SEQ ID NO:134).
In one embodiment, the linker is GGGSG (SEQ ID NO:189). In one
embodiment, the linker is GSSSG (SEQ ID NO:135).
[0237] Peptide sequences provided herein, including the FGF19 and
FGF21 variants and subsequences and the FGF19/FGF21 fusions and
chimeras listed in Tables 1-11 and Sequence Listing, as well as
subsequences, sequence variants and modified forms of the sequences
listed in Tables 1-11 and Sequence Listing have one or more
activities as set forth herein. One example of an activity is
modulating bile acid homeostasis. 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 treating a bile acid-related or associated
disorder. Activities such as, for example, modulation of bile acid
homeostasis, glucose lowering activity, analysis of a bile
acid-related or associated disorder, HCC formation or
tumorigenesis, 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 or known to the skilled
artisan.
[0238] Various methodologies can be used in the screening and
diagnosis of HCC and are well known to the skilled artisan.
Indicators for HCC include detection of a tumor maker such as
elevated alpha-fetoprotein (AFP) or des-gamma carboxyprothrombin
(DCP) levels. A number of different scanning and imaging techniques
are also helpful, including ultrasound, CT scans and Mill. In
certain embodiments, evaluation of whether a peptide (e.g., a
candidate peptide) exhibits evidence of inducing HCC may be
determined in vivo by, for example, quantifying HCC nodule
formation in an animal model, such as db/db mice, administered a
peptide, compared to HCC nodule formation by wild type FGF19.
Macroscopically, liver cancer may be nodular, where the tumor
nodules (which are round-to-oval, grey or green, well circumscribed
but not encapsulated) appear as either one large mass or multiple
smaller masses. Alternatively, HCC may be present as an
infiltrative tumor which is diffuse and poorly circumscribed and
frequently infiltrates the portal veins. 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).
[0239] More particularly, peptide sequences provided herein,
including the FGF19 and FGF21 variants and subsequences and the
FGF19/FGF21 fusions and chimeras listed in Tables 1-11 and Sequence
Listing, as well as subsequences, variants and modified forms of
the sequences listed in Tables 1-11 and Sequence Listing include
those with the following activities: peptide sequences modulating
bile acid homeostasis or treating a bile acid-related or associated
disorder while having reduced HCC formation compared to FGF19, or a
FGF 19 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 bile acid modulating activity
compared to FGF19, or FGF 19 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 a FGF 19 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.
[0240] More particularly, peptide sequences provided herein,
including the FGF19 and FGF21 variants and subsequences and the
FGF19/FGF21 fusions and chimeras listed in Tables 1-11 and Sequence
Listing, as well as subsequences, variants and modified forms of
the sequences listed in Tables 1-11 and the Sequence Listing
include those with the following activities: peptide sequences that
modulate bile acid homeostasis; peptide sequences that treat a bile
acid-related or associated disorder, 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.
[0241] 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.
[0242] 4.4 Dosing and Administration
[0243] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Tables
1-11), may be formulated in a unit dose or unit dosage form. In a
particular embodiment, a peptide sequence is in an amount effective
to treat a subject in need of treatment, e.g., due to abnormal or
aberrant bile acid homeostasis, such as metabolic syndrome; a
lipid- or glucose-related disorder; cholesterol or triglyceride
metabolism; type 2 diabetes; cholestasis, including, for example
diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,
neonatal cholestasis, and drug induced cholestasis (e.g.,
estrogen)), and diseases of extrahepatic cholestasis (e.g., bile
cut compression from tumor, bile duct blockade by gall stones);
bile acid malabsorption and other disorders involving the distal
small intestine, including ileal resection, inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), disorders
impairing absorption of bile acids not otherwise characterized
(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and
GI, liver, and/or biliary cancers (e.g., colon cancer and
hepatocellular cancer); and/or bile acid synthesis abnormalities,
such as those contributing to NASH, cirrhosis and portal
hypertension. Exemplary unit doses range from about 25-250,
250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,
25,000-50,000 ng; from about 25-250, 250-500, 500-1000, 1000-2500
or 2500-5000, 5000-25,000, 25,000-50,000 .mu.g; and from about
25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,
25,000-50,000 mg.
[0244] Peptide sequences provided herein including subsequences,
sequence variants and modified forms of the exemplified peptide
sequences (e.g., sequences listed in the Sequence Listing or Tables
1-11) 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).
[0245] Peptide sequences provided herein including subsequences,
variants and modified forms of the exemplified peptide sequences
(e.g., sequences listed in the Sequence Listing or Tables 1-11) can
be administered and methods may be practiced via systemic, regional
or local administration, by any route. For example, a peptide
sequence can be administered parenterally (e.g., subcutaneously,
intravenously, intramuscularly, or intraperitoneally), orally
(e.g., ingestion, buccal, or sublingual), inhalation,
intradermally, intracavity, intracranially, transdermally
(topical), transmucosally or rectally. Peptide sequences provided
herein including subsequences, variants and modified forms of the
exemplified peptide sequences (e.g., sequences listed in the
Sequence Listing or Tables 1-11) and methods provided herein
including pharmaceutical compositions can be administered via a
(micro)encapsulated delivery system or packaged into an implant for
administration.
[0246] 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.
[0247] Another non-limiting example of parenteral administration
entails the use of DUROS.RTM.-type implantable osmotic pumps (from,
e.g., DURECT Corp.). The DUROS.RTM. system can be used for
therapies requiring systemic or site-specific administration of a
drug. To deliver drugs systemically, the DUROS.RTM. system is
placed just under the skin, for example in the upper arm, in an
outpatient procedure that is completed in just a few minutes using
local anesthetic. To deliver a drug to a specific site,
miniaturized catheter technology can be used. The catheter can be
attached to the DUROS.RTM. system to direct the flow of a drug to
the target organ, tissue or synthetic medical structure, such as a
graft. Site-specific delivery enables a therapeutic concentration
of a drug to be administered to the desired target without exposing
the entire body to a similar concentration. The precision, size and
performance of the DUROS.RTM. system will allow for continuous
site-specific delivery to a variety of precise locations within the
body.
[0248] Yet another non-limiting example of parenteral
administration entails the use of an on-body delivery system (e.g.,
the Neulasta.RTM. Delivery Kit by Amgen). This on-body delivery
system includes an on-body injector, which is a small, lightweight,
injection system applied on the same day as a doctor visit (such as
the day of chemotherapy). It is designed to deliver a dose of the
therapeutic agent the next day, or in the near future of the doctor
visit, so that the patient does not need to return to the doctor's
office to receive the injection.
[0249] Various methods of controlled release is also contemplated
herein. Encapsulation of therapeutic molecules within polymer
particles is a well-established method for achieving controlled
release and can be used in methods provided herein. Also, by taking
advantage of the adsorption of protein therapeutics to
poly(lactic-co-glycolic acid) (PLGA) nanoparticles, controlled
release can also be achieved without encapsulation. In particular,
extended-release for protein therapeutics cam be applied with and
without encapsulation in PLGA nanoparticles embedded within a
hydrogel. The release profile tunable by modifying nanoparticle
concentration, nanoparticle size, or environmental pH. Pakulska et
al., Science Advances 2(5): e1600519 (2016)
4.5 Methods of Preventing, Treating and Managing Diseases and
Disorders
[0250] In one embodiment, provided herein is a method of preventing
a disease or disorder in a subject having, or at risk of having, a
disease or disorder preventable by a peptide sequence provided
herein, comprising administering a pharmaceutical composition
comprising a peptide provided herein to a subject in an amount
effective for preventing the disease or disorder. In another
embodiment, provided herein is a method of treating a disease or
disorder in a subject having, or at risk of having, a disease or
disorder treatable by a peptide sequence provided herein,
comprising administering a pharmaceutical composition comprising a
peptide provided herein to a subject in an amount effective for
treating the disease or disorder. In yet another embodiment,
provided herein is a method of managing a disease or disorder in a
subject having, or at risk of having, a disease or disorder
manageable by a peptide sequence provided herein, comprising
administering a pharmaceutical composition comprising a peptide
provided herein to a subject in an amount effective for managing
the disease or disorder. In one embodiment, the disease or disorder
is a bile acid-related disease or associated disorder. In another
embodiment, the disease or disorder is a metabolic disease or
disorder. In other embodiments, the disease or disorder is a cancer
or tumor.
[0251] Administration of various FGF19 and/FGF21 variants and
fusion peptide sequences to mice successfully modulated bile acid
homeostasis and hyperglycemia (data not shown). Furthermore, in
contrast to FGF19, certain peptide sequences did not stimulate or
induce HCC formation or tumorigenesis in mice (data not shown).
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 Tables 1-11 and the Sequence Listing, and
the FGF19/FGF21 fusions and chimeras listed in Tables 1-11 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, such as bile-acid related or associated disorders, and
metabolic disorders, such as disorders related to high sugar
levels, hyperglycemic conditions, insulin resistance,
hyperinsulinemia, glucose intolerance, metabolic syndrome, or
related disorders, as set forth herein,
[0252] 4.5.1 Methods of Preventing, Treating and Managing Bile
Acid-Related or Associated Disorders
[0253] As used herein, the phrases "bile acid-related disorder,"
"bile acid-related or associated disorder," and the like, when used
in reference to a condition of a subject, means a disruption of
bile acid homeostasis, which may manifest itself as, for example,
an acute, transient or chronic abnormal level of a bile acid or one
or more bile acids. The condition can be caused by inhibition,
reduction or a delay in bile acid synthesis, metabolism or
absorption such that the subject exhibits a bile acid level not
typically found in normal subjects.
[0254] Also provided herein are in vitro, ex vivo and in vivo
(e.g., on or in a subject) methods and uses. Such methods and uses
can be practiced with any of the peptide sequences set forth
herein. In various embodiments, the methods include administering a
peptide sequence, such as a FGF19 or FGF21 variant, fusion or
chimera disclosed herein (e.g., in the Sequence Listing or Tables
1-11), or a subsequence, a variant or modified form of a FGF19 or
FGF21 variant, fusion or chimera disclosed herein (e.g., the
Sequence Listing or Tables 1-11), to a subject in an amount
effective for treating a bile acid-related or associated
disorder.
[0255] In certain embodiments, the peptide is administered in
combination with an additional therapeutic agent(s) and/or
treatment modalities (e.g., an agent useful in the treatment and/or
prevention of PBC). The additional therapeutic agent(s) can be
administered before, with, or following administration of the
peptides described herein.
[0256] Also provided herein are methods of preventing (e.g., in
subjects predisposed to having a particular disorder(s)), delaying,
slowing or inhibiting progression of, the onset of, or treating
(e.g., ameliorating) a bile acid-related or associated disorder
relative to an appropriate matched subject of comparable age,
gender, race, etc.). Thus, in various embodiments, a method
provided herein for, for example, modulating bile acid homeostasis
or treating a bile acid-related or associated disorder includes
contacting or administering one or more peptides provided herein
(e.g., a variant or fusion of FGF19 and/or FGF21 as set forth in
the Sequence Listing or Tables 1-11) in an amount effective to
modulate bile acid homeostasis or treat a bile acid-related or
associated disorder. In certain embodiments the method further
comprises contacting or administering at least one additional
therapeutic agent or treatment modality that is useful in the
treatment or prevention of a bile acid-related or associated
disorder (e.g., PBC).
[0257] 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.
[0258] Subjects include those having a disorder, e.g., a bile
acid-related or associated disorder, such as cholestasis,
including, for example diseases of intrahepatic cholestasis (e.g.,
PBC, PFIC, PSC, PIC, neonatal cholestasis, and drug induced
cholestasis (e.g., estrogen)), and diseases of extrahepatic
cholestasis (e.g., bile cut compression from tumor, bile duct
blockade by gall stones); bile acid malabsorption and other
disorders involving the distal small intestine, including ileal
resection, inflammatory bowel diseases (e.g., Crohn's disease and
ulcerative colitis), short bowel syndrome, disorders impairing
absorption of bile acids not otherwise characterized (idiopathic))
leading to diarrhea (e.g., BAD) and GI symptoms, and GI, liver,
and/or biliary cancers (e.g., colon cancer and hepatocellular
cancer); and/or bile acid synthesis abnormalities, such as those
contributing to NASH, cirrhosis and portal hypertension; or
subjects that do not have a disorder but may be at risk of
developing the disorder.
[0259] Non-limiting exemplary bile acid-related or associated
disorders preventable, treatable or manageable according to the
methods and uses provided herein include: cholestasis, including,
for example diseases of intrahepatic cholestasis (e.g., primary
biliary cirrhosis (PBC), primary familial intrahepatic cholestasis
(PFIC) (e.g., progressive PFIC), primary sclerosing choangitis
(PSC), pregnancy intrahepatic cholestasis (PIC), neonatal
cholestasis, and drug-induced cholestasis (e.g., estrogen)), and
diseases of extrahepatic cholestasis (e.g., bile cut compression
from tumor, bile duct blockade by gall stones); bile acid
malabsorption and other disorders involving the distal small
intestine, including ileal resection, inflammatory bowel diseases
(e.g., Crohn's disease and ulcerative colitis), short bowel
syndrome, disorders impairing absorption of bile acids not
otherwise characterized (idiopathic)) leading to diarrhea (e.g.,
bile acid diarrhea (BAD)) and GI symptoms, and GI, liver, and/or
biliary cancers (e.g., colon cancer and hepatocellular cancer);
and/or bile acid synthesis abnormalities, such as those
contributing to non-alcoholic steatohepatitis (NASH), cirrhosis and
portal hypertension; e.g., in mammals, such as humans. Additional
bile acid-related or associated disorders include metabolic
syndrome; a lipid or glucose disorder; cholesterol or triglyceride
metabolism; type 2 diabetes. In one particular embodiment, the bile
acid-related or associated disorder is bile acid malabsorption. In
another particular embodiment, the bile acid-related or associated
disorder is diarrhea. In a still further particular embodiment, the
bile acid-related or associated disorder is cholestasis (e.g.,
intrahepatic or extrahepatic cholestasis). In another further
particular embodiment, the bile acid-related or associated disorder
is primary biliary cirrhosis (PBC). In other particular
embodiments, the bile acid-related or associated disorder is
primary sclerosing cholangitis. In another embodiment, the bile
acid-related or associated disorder is PFIC (e.g., progressive
PFIC). In another embodiment, the bile acid-related or associated
disorder is NASH. In another embodiment, the bile acid-related or
associated disorder is a hyperglycemic condition. In a specific
embodiment, the bile acid-related or associated disorder is type 2
diabetes.
[0260] In some embodiments, the methods provided herein comprises
administration of at least one additional agent effective in
modulating bile acid homeostasis or treating a bile acid-related or
associated disorder, wherein the additional agent is: a
glucocorticoid; CDCA; UDCA; insulin, an insulin secretagogues, an
insulin mimetic, a sulfonylurea and a meglitinide; a biguanide; an
alpha-glucosidase inhibitors; a DPP-IV inhibitor, GLP-1, a GLP-1
agonists and a GLP-1 analog; a DPP-IV-resistant analogue; a PPAR
gamma agonist, a dual-acting PPAR agonist, a pan-acting PPAR
agonist; a PTP1B inhibitor; an SGLT inhibitor; an RXR agonist; a
glycogen synthase kinase-3 inhibitor; an immune modulator; a beta-3
adrenergic receptor agonist; an 11beta-HSD1 inhibitor; amylin and
an amylin analogue; a bile acid sequestrant; or an SGLT-2
inhibitor. In certain embodiments, the at least one additional
agent effective in modulating PBC is UDCA, an FXR agonist, OCA, an
ASBT inhibitor, an autoimmune agent, an anti-IL-12 agent, an
anti-CD80 agent, an anti-CD20 agent, a CXCL10 neutralizing
antibody, a ligand for CXCR3, a fibrate, fish oil, colchicine,
methotrexate, azathioprine, cyclosporine, or an anti-retroviral
therapy. In particular embodiments, the at least one additional
agent effective in modulating PBC is UDCA, OCA, an ASBT inhibitor,
an anti-IL-12 agent, an anti-CD20 agent, or a fibrate.
[0261] Additional bile acid-related or associated disorders that
may be treated or prevented with the peptide sequences provided
herein include metabolic syndrome, a lipid or glucose disorder,
cholesterol or triglyceride metabolism, diabetes (e.g., type 2
diabetes), other hyperglycemic-related disorders, including kidney
damage (e.g., tubule damage or nephropathy), liver degeneration,
eye damage (e.g., diabetic retinopathy or cataracts), and diabetic
foot disorders, and dyslipidemias and their sequelae such as, for
example, atherosclerosis, coronary artery disease, cerebrovascular
disorders and the like.
[0262] Other conditions which may be associated with metabolic
syndrome, such as obesity and elevated body mass (including the
co-morbid conditions thereof such as, but not limited to,
nonalcoholic fatty liver disease (NAFLD), nonalcoholic
steatohepatitis (NASH), and polycystic ovarian syndrome (PCOS)),
and also include thromboses, hypercoagulable and prothrombotic
states (arterial and venous), hypertension (including portal
hypertension (defined as a hepatic venous pressure gradient (HVPG)
greater than 5 mm Hg), cardiovascular disease, stroke and heart
failure; Disorders or conditions in which inflammatory reactions
are involved, including atherosclerosis, chronic inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), asthma,
lupus erythematosus, arthritis, or other inflammatory rheumatic
disorders; Disorders of cell cycle or cell differentiation
processes such as adipose cell tumors, lipomatous carcinomas
including, for example, liposarcomas, solid tumors, and neoplasms;
Neurodegenerative diseases and/or demyelinating disorders of the
central and peripheral nervous systems and/or neurological diseases
involving neuroinflammatory processes and/or other peripheral
neuropathies, including Alzheimer's disease, multiple sclerosis,
Parkinson's disease, progressive multifocal leukoencephalopathy and
Guillian-Barre syndrome; Skin and dermatological disorders and/or
disorders of wound healing processes, including erythemato-squamous
dermatoses; and Other Disorders such as syndrome X, osteoarthritis,
and acute respiratory distress syndrome.
[0263] Treatment of a bile acid-related or associated disorder
(e.g., NASH) may have the benefit of alleviating or abolishing a
disorder secondary thereto. By way of example, a subject suffering
from NASH may also have depression or anxiety due to NASH; thus,
treating the subject's NASH may also indirectly treat the
depression or anxiety. The use of the therapies disclosed herein to
target such secondary disorders is also contemplated in certain
embodiments.
[0264] In particular embodiments, the subject has or is at risk of
having PBC. In other particular embodiments, the subject has or is
at risk of having NASH.
[0265] Subjects at risk of developing a bile acid-related or
associated disorder (such as the disorders described above)
include, for example, those who may have a family history or
genetic predisposition toward such disorder, as well those whose
diet may contribute to development of such disorders.
[0266] 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 provided herein, for example, as set
forth in the Sequence Listing or Tables 1-11) in an amount
effective to achieve a desired outcome or result in a subject. A
treatment that results in a desired outcome or result includes
decreasing, reducing or preventing the severity or frequency of one
or more symptoms of the condition in the subject, e.g., an
improvement in the subject's condition or a "beneficial effect" or
"therapeutic effect." Therefore, treatment can decrease or reduce
or prevent the severity or frequency of one or more symptoms of the
disorder, stabilize or inhibit progression or worsening of the
disorder, and in some instances, reverse the disorder, transiently
(e.g., for 1-6, 6-12, or 12-24 hours), for medium term (e.g., 1-6,
6-12, 12-24 or 24-48 days) or long term (e.g., for 1-6, 6-12,
12-24, 24-48 weeks, or greater than 24-48 weeks). Thus, in the case
of a bile acid-related or associated disorder, treatment can lower
or reduce one or more symptoms or effects of the bile acid-related
or associated disorders described above.
[0267] In certain embodiments, the various methods provided herein
further include contacting or administering one or more additional
agents or therapeutic modalities useful in the treatment or
prevention of a bile acid-related or associated disorder, such as
those agents or therapeutic modalities described herein, in an
amount effective to achieve a desired outcome or result in a
subject.
[0268] An "effective amount" or a "sufficient amount" for use
and/or for treating a subject refers to an amount that provides, in
single or multiple doses, alone, or in combination with one or more
other agents, treatments, protocols, or therapeutic regimens, a
detectable response of any duration of time (transient, medium or
long term), a desired outcome in or an objective or subjective
benefit to a subject of any measurable or detectable degree or for
any duration of time (e.g., for hours, days, months, years, in
remission or cured). Such amounts typically are effective to
ameliorate a disorder, or one, multiple or all adverse symptoms,
consequences or complications of the disorder, to a measurable
extent, although reducing or inhibiting a progression or worsening
of the disorder, is considered a satisfactory outcome.
[0269] As used herein, the term "ameliorate" means an improvement
in the subject's disorder, a reduction in the severity of the
disorder, or an inhibition of progression or worsening of the
disorder (e.g., stabilizing the disorder). In the case of a bile
acid-related or associated disorder such as those described above,
including cholestasis (e.g., PBC), disorders impairing absorption
of bile acids leading to diarrhea (e.g., BAD) and bile acid
synthesis abnormalities (e.g., NASH), an improvement can be a
lowering or a reduction in one or more symptoms or effects of the
disorder.
[0270] 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.).
[0271] Thus, in the case of a disorder treatable by a peptide
sequence provided herein, either alone or in combination with an
additional agent, the amount of the peptide (and optionally the
additional agent)sufficient to ameliorate a disorder will depend on
the type, severity and extent, or duration of the disorder, the
therapeutic effect or outcome desired, and can be readily
ascertained by the skilled artisan. Appropriate amounts will also
depend upon the individual subject (e.g., the bioavailability
within the subject, gender, age, etc.). For example, a transient,
or partial, restoration of normal bile acid homeostasis in a
subject can reduce the dosage amount or frequency of the peptides
and agents described herein in order to treat the bile acid-related
or associated disorders described previously even though complete
freedom from treatment has not resulted. An effective amount can be
ascertained, for example, by measuring one or more relevant
physiological effects.
[0272] Methods and uses provided herein for treating a subject are
applicable for prophylaxis to prevent or reduce the likelihood of a
disorder in a subject, such as a bile acid-related or associated
disorder. Accordingly, methods and uses provided herein for
treating a subject having, or at risk of developing, a bile
acid-related or associated disorder can be practiced prior to,
substantially contemporaneously with, or following administration
or application of another agent useful for the treatment or
prevention of a bile acid-related or associated disorder, and/or
can be supplemented with other forms of therapy. Supplementary
therapies include other glucose lowering treatments, such as
insulin, an insulin sensitivity enhancer and other drug treatments,
a change in diet (low sugar, fats, etc.), weight loss
surgery--(reducing stomach volume by gastric bypass, gastrectomy),
gastric banding, gastric balloon, gastric sleeve, etc. For example,
a method or use provided herein for treating a hyperglycemic or
insulin resistance disorder can be used in combination with drugs
or other pharmaceutical compositions that lower glucose or increase
insulin sensitivity in a subject.
[0273] In one embodiment, a method or use includes contacting or
administering to a subject one or more variant or fusion FGF19
and/or FGF21 peptide sequences in an amount effective for
preventing a bile-acid related or associated disorder. In one
embodiment, a method or use 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 a bile-acid related
or associated disorder. In one embodiment, a method or use includes
contacting or administering to a subject one or more variant or
fusion FGF19 and/or FGF21 peptide sequences in an amount effective
for managing a bile-acid related or associated disorder.
[0274] 4.5.1.1 PBC and Therapy with Agents Effective in the
Treatment or Prevention Thereof
[0275] Primary biliary cirrhosis (PBC), the most common cholestatic
liver disease, is a progressive hepatic disease that primarily
results from autoimmune destruction of the bile ducts that
transport bile acids out of the liver. As the disease progresses,
persistent toxic build-up of bile acids causes progressive liver
damage marked by chronic inflammation and fibrosis. Because
patients with PBC have an increased risk of HCC, therapy with the
variants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21
peptide sequences and variants of fusions (chimeras) of FGF19
and/or FGF21 peptide sequences described herein is of particular
import, as such sequences do not induce, or do not substantially
increase, HCC formation or HCC tumorigenesis.
[0276] Although patients with PBC are often asymptomatic at the
time of initial diagnosis, most present, or subsequently develop,
one or more of the following: pruritus; fatigue; jaundice;
xanthoma; disorders associated with an extrahepatic autoimmune
disorder (e.g., Sjogren's Syndrome and rheumatoid arthritis); and
complications that result from cirrhosis or portal hypertension
(e.g., ascites, esophageal varices and hepatic encephalopathy).
[0277] While a definitive cause of PBC has not been identified,
most research suggests that it is an autoimmune disorder. There
appears to be a genetic predisposition, and genetic studies have
indicated that part of the IL-12 signaling cascade, including
IL-12A and I-12RB2 polymorphisms, is important in the etiology of
the disease.
[0278] There is no definitive means of diagnosing PBC; rather,
assessment of a number of factors is generally required. Moreover,
diagnosis of PBC requires that other conditions with similar
symptoms (e.g., autoimmune hepatitis and primary sclerosing
cholangitis) be ruled out; by way of example, abdominal ultrasound
or CT scan is usually performed to rule out blockage of the bile
ducts.
[0279] Diagnostic blood tests include deranged liver function tests
(gamma-glutamyl transferase and alkaline phosphatase) and the
presence of particular antibodies (antimitochondrial antibody (AMA)
an antinuclear antibody (ANA)). Antinuclear antibodies are believed
to be prognostic indicators of PBC. When other tests and procedures
are indicative of PBC, a liver biopsy is frequently performed to
confirm disease. Endoscopic retrograde cholangiopancreatography
(ERCP), an endoscopic evaluation of the bile duct, may also be
employed to confirm disease.
[0280] PBC is classified into four stages marking the progression
of disease. Stage 1 (Portal Stage) is characterized by portal
inflammation and mild bile duct damage; Stage 2 (Periportal Stage)
is characterized by enlarged triads, periportal fibrosis or
inflammation; Stage 3 (Septal Stage) is characterized by active
and/or passive fibrous septa; and Stage 4 (Biliary Cirrhosis) is
characterized by the presence of hepatic nodules. Liver biopsy is
required to determine the stage of disease.
[0281] Serum bilirubin is an indicator of PBC progression and
prognosis. Patients with a serum bilirubin level of 2-6 mg/dL have
a mean survival time of 4.1 years, patients with a serum bilirubin
level of 6-10 mg/dL have a mean survival time of 2.1 years, and
patients with a serum bilirubin level above 10 mg/dL have a mean
survival time of 1.4 years. Liver transplantation is an option in
advanced cases of PBC, although the recurrence rate may be as high
as 18% at 5 years, and up to 30% at 10 years.
[0282] Although disease progression may be slowed, pharmaceutical
intervention with currently used therapies is neither curative nor
effective in all patient populations. In order to improve the
therapeutic outcome of pharmacological therapy, one aspect pertains
to the use of one or more current therapies in combination with
variants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21
peptide sequences and variants of fusions (chimeras) of FGF19
and/or FGF21 peptide sequences having one or more activities
associated with the treatment and/or prevention of PBC and
associated diseases, disorders and conditions. The most commonly
used and/or promising agents for combination therapy are set forth
hereafter, although it is to be understood that these agents are
illustrative, and not exclusionary.
[0283] PBC treatment most frequently involves the bile acid
ursodeoxycholic acid (Urosdiol, UDCA). UDCA therapy is helpful in
reducing the cholestasis and improving the liver function tests in
PBC patients; however, it does not demonstrably improve symptoms
and has a questionable impact on prognosis. UDCA has been shown to
reduce mortality, adverse events and the need for transplantation
in PBC. Although UDCA is considered the first-line therapy,
approximately one-third of patients may be non-responsive and
remain at risk of progressive liver disease and are candidates for
alternative or additive therapy.
[0284] There are several alternative and adjuvant therapies, some
of which are currently in clinical development, that can be used in
combination with variants of FGF19 peptide sequences, fusions of
FGF19 and/or FGF21 peptide sequences and variants of fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences provided herein
having one or more activities associated with the treatment and/or
prevention of PBC and associated diseases, disorders and
conditions.
[0285] Farnesoid-X-receptor agonists represent a promising class of
agents that may be used in combination therapy. One of the primary
functions of agonists of FXR, a nuclear receptor expressed at high
levels in the liver and intestine, is the suppression of
cholesterol 7a hydroxylase-1 (CYP7A1), the rate-limiting enzyme in
the synthesis of bile acids from cholesterol. Obeticholic acid
(OCA; Intercept Pharmaceuticals, NY) is a bile acid analog and FXR
agonist derived from the primary human bile acid chenodeoxycholic
acid, or CDCA. OCA is currently being evaluated for patients having
an inadequate therapeutic response to ursodiol or who are unable to
tolerate ursodiol.
[0286] Inhibitors of the apical sodium-dependent bile acid
transporter (ASBT) represent another class of agents that may be
used in combination with the variants of FGF19 peptide sequences,
fusions of FGF19 and/or FGF21 peptide sequences and variants of
fusions (chimeras) of FGF19 and/or FGF21 peptide sequences
described herein for the treatment and/or prevention of PBC and
associated diseases. ASBT, a member of the sodium/bile-salt
co-transport family coded by gene SLC10A2, is currently thought to
be the primary mechanism for bile acid reabsorption in the
intestine. Examples of ABST inhibitors include LUM001 and SC-435,
both of which are being developed by Lumena Pharmaceuticals (San
Diego, Calif.).
[0287] Bile acid sequestrants also find use in the treatment of
PBC. Cholestyramine and colestipol are the best known bile acid
sequestrants. However, their use is sometimes limited because they
are only available in powder form and are not tolerated by many
patients, often because of the poor texture and taste of the resin
powder. The bile acid sequestrant colesevelam is available in
tablet form and is often better tolerated. All bile acid
sequestrants are capable of binding other compounds, including the
fat-soluble vitamins A, D, E and K, and deficiencies of these
vitamins many necessitate supplementation. Importantly, the PBC
patient population inherently has poor lipid-dependent absorption
of vitamins A, D, E and K, and thus patients taking bile acid
sequestrants are at particular risk for deficiency of those
vitamins.
[0288] Agents associated with immune and inflammatory function are
candidates for combination therapy with the variants of FGF19
peptide sequences, fusions of FGF19 and/or FGF21 peptide sequences
and variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of PBC and associated diseases,
disorders and conditions.
[0289] The interleukin IL-12 is linked with autoimmunity. Data
indicate that the IL-12 signaling pathway plays a key role in the
effector mechanisms that lead to biliary destruction. Targeting the
p40 subunit of IL-12 has also been shown to ameliorate experimental
immune-mediated cholangiopathy. Thus, anti-IL-12 agents (e.g.,
monoclonal Ab inhibitors) provide a promising treatment.
Furthermore, because polymorphisms in CD80 have been identified as
conferring an increased susceptibility to PBC, blockade of
co-stimulation between T cells and antigen-presenting cells through
CD80 by use of an anti-CD80 agent could represent an important
therapeutic approach for the treatment of PBC. In addition,
improvement in IgM titre and an increase in intrahepatic regulatory
T-cell number using the anti-CD20 antibody rituximab (RITUXAN) have
shown promise.
[0290] The immune-mediated destruction of small-sized bile ducts in
PBC is predominantly cell-mediated, characterized by Th1 cells,
CD8+ T cells, NK cells and NKT cells which express CXCR3.
Therefore, neutralizing antibodies to CXCL10, a ligand for CXCR3,
may offer the possibility to interfere with one of the key
inflammatory processes and contribute to immune-mediated biliary
destruction in PBC. Similarly, blockade of co-stimulatory signals
between T cells expressing CD28 and antigen-presenting cells
expressing CD80 (e.g. cholangiocytes, antibody-secreting B cells)
might represent an important approach for the treatment of
autoimmune diseases.
[0291] The variants of FGF19 peptide sequences, fusions of FGF19
and/or FGF21 peptide sequences and variants of fusions (chimeras)
of FGF19 and/or FGF21 peptide sequences described herein can be
used alone or in combination with other agents for the treatment
and/or prevention of those bile acid-related or associated
disorders referenced herein that have an immune and/or inflammatory
component, including, but not limited to, PBC and associated
diseases, disorders and conditions. Examples of such other agents
include, for example, non-steroidal anti-inflammatory drugs
(NSAID); steroids; cytokine suppressive anti-inflammatory drug(s)
(CSAIDs); antibodies to, or antagonists of, other human cytokines
or growth factors (e.g., IL-2, IL-6, or PDGF); TNF antagonists
(e.g., agents such as REMICADE, p75TNFRIgG (ENBREL) or p55TNFR1gG
(LENERCEPT)); interferon-01a (AVONEX); interferon-01b (BETASERON);
and immune checkpoint inhibitors, including PD1 (associated agents
include the antibodies nivolumab and lambrolizumab), PDL1, BTLA,
CTLA4 (associated agents include the fully humanized CTLA4
monoclonal antibody ipilimumab (YERVOY), TIM3, LAGS, and A2aR.
[0292] Fibrates have been shown to improve various aspects of PBC,
including liver function tests, both as monotherapy and in
combination with UDCA non-responders. In certain embodiments, a
fibrate is a member selected from the group of bezafibrate
(BEZALIP), ciprofibrate (MODALIM), gemfibrozil (LOPID), clofibrate,
and fenofibrate (TRICOR). Fish oil has exhibited similar
benefits.
[0293] In PBC patients demonstrating certain characteristics of
hepatitis on biopsy, corticosteroids such as budesonide may improve
liver histology and biochemistry, particularly when used in
combination with UDCA. Colchicine has been shown to improve liver
function tests (e.g., AST and ALP) and represents another
alternative treatment for PBC.
[0294] Though not an exhaustive list, other drugs that have shown
promise include methotrexate as an immunomodulatory treatment,
azathioprine, cyclosporine, and certain agents used in
anti-retroviral therapy (e.g., combivir).
[0295] Various treatments exist for the sequelae associated with
PBC. For example, itching can be relieved by the bile acid
sequestrant cholestyramine, or alternatively naltrexone and
rifampicin. The fatigue associated with PBC may effectively be
treated with modafinil (Provigil; Teva (formerly Cephalon)) without
damaging the liver. As patients with PBC have increased risk of
developing osteoporosis and esophageal varices compared to the
general population (and others with liver disease), screening and
treatment of these complications is an important part of the
management of PBC. Variants of FGF19 peptide sequences, fusions of
FGF19 and/or FGF21 peptide sequences and variants of fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences having one or
more activities associated with the treatment and/or prevention of
PBC and associated diseases, disorders and conditions, as provided
herein, either alone or in combination with other agents, offer
novel, promising alternatives to the management of such
sequelae.
[0296] 4.5.1.2 NASH and NAFLD and Therapy with Agents Effective in
the Treatment or Prevention Thereof
[0297] Non-alcoholic steatohepatitis (NASH), considered part of a
spectrum of non-alcoholic fatty liver diseases (NAFLD), causes
inflammation and accumulation of fat and fibrous tissue in the
liver. Although the exact cause of NASH is unknown, risk factors
include central obesity, type-2 diabetes mellitus, insulin
resistance (IR) and dyslipidemia; combinations of the foregoing are
frequently described as the metabolic syndrome. In addition,
certain drugs have been linked to NASH, including tamoxifen,
amiodarone and steroids (e.g., prednisone and hydrocortisone).
Non-alcoholic fatty liver disease is the most common cause of
chronic liver disease in the United States, and the estimated
prevalence of NAFLD is 20-30% and for NASH it is estimated at
3.5-5%. (See, e.g., Abrams, G. A., et al., Hepatology, 2004.
40(2):475-83; Moreira, R. K., Arch Pathol Lab Med, 2007.
131(11):1728-34).
[0298] NASH frequently presents with no overt symptoms,
complicating its diagnosis. Liver function tests generally begin
the diagnostic process, with levels of AST (aspartate
aminotransferase) and ALT (alanine aminotransferase) elevated in
about 90% percent of individuals with NASH. Other blood tests are
often used for ruling out other causes of liver disease, such as
hepatitis. Imaging tests (e.g., ultrasound, CT scan, or Mill) may
reveal fat accumulation in the liver but frequently cannot
differentiate NASH from other causes of liver disease that have a
similar appearance. A liver biopsy is required to confirm NASH.
[0299] The prognosis for individuals suffering from NASH is
difficult to predict, although features in the liver biopsy can be
helpful. The most serious complication of NASH is cirrhosis, which
occurs when the liver becomes severely scarred. It has been
reported that between 8 and 26 percent of individuals with NASH
develop cirrhosis, and it is predicted that NASH will be the
leading indication for liver transplantation by 2020.
[0300] At the present time, treatment of NASH focuses primarily on
pharmacological and non-pharmacological management of those medical
conditions associated with it, including hyperlipidemia, diabetes
and obesity. Although not curative, pharmacological intervention of
NASH itself includes treatment with vitamin E, pioglitazone,
metformin, statins, omega-3 fatty acids, and ursodeoxycholic acid
(UDCA (ursodiol)). Other agents being evaluated, currently approved
for different indications, include losartan and telisartan,
exenatide, GLP-1 agonists, DPP IV inhibitors, and
carbamazepine.
[0301] In view of the deficiencies of the aforementioned current
therapies, therapy with agents having distinct mechanisms of action
offers a promising new avenue for the treatment and prevention of
NASH and NAFLD. Addressing such deficiencies is contemplated, for
example, by using the variants of FGF19 peptide sequences, fusions
of FGF19 and/or FGF21 peptide sequences and variants of fusions
(chimeras) of FGF19 and/or FGF21 peptide sequences as taught
herein. In certain embodiments, the peptides are used in
combination with other therapeutic agents and/or treatment
modalities. Also provided herein is the prophylactic and/or
therapeutic use of these variants of FGF19 peptide sequences,
fusions of FGF19 and/or FGF21 peptide sequences and variants of
fusions (chimeras) of FGF19 and/or FGF21 peptide sequences, either
alone or in combination with therapies developed in the future, for
the treatment or prevention of NASH and NAFLD.
[0302] 4.5.1.3 Therapy for the Treatment or Prevention of Other
Bile Acid-Related Disorders and Associated Diseases, Disorders and
Conditions
[0303] Also provided herein is the use of variants of FGF19 peptide
sequences, fusions of FGF19 and/or FGF21 peptide sequences and
variants of fusions (chimeras) of FGF19 and/or FGF21 peptide
sequences having one or more activities associated with the
treatment and/or prevention of other bile acid-related disorders
and associated diseases, disorders and conditions besides PBC. In
certain embodiments, the peptides are used in combination with
other therapeutic agents and/or treatment modalities.
[0304] By way of example, patients with bile acid diarrhea
secondary to Crohn's ileitis will be helped with glucocorticoid
treatment. Microscopic colitis is also helped by steroids. In
patients with a short-bowel syndrome (a bile acid deficiency occurs
in the proximal intestine that leads to impaired micellar
solubilization), cholylsarcosine (cholyl-N-methylglycine), a
synthetic bile acid analogue, has been shown to increase lipid
absorption.
[0305] Administration of the primary bile acid chenodeoxycholic
Acid (CDCA) has been shown to decrease biliary cholesterol
secretion and gradual dissolution of gallstones. Because CDCA is
slightly hepatotoxic, it was gradually replaced by UDCA. Despite
the efficacy and safety of UDCA administration for cholesterol
gallstone dissolution, it is not frequently used today because of
the success of laparoscopic cholecystectomy, which provides a rapid
cure for symptomatic disease. Medical therapy, in contrast,
requires months of therapy, does not always dissolve stones, and is
followed by gradual recurrence in some patients.
[0306] Bile acid replacement is used in inborn errors of bile acid
biosynthesis, usually with a mixture of CDCA or UDCA and cholic
acid, to suppress the synthesis of cytotoxic bile acid precursors
and restore the input of primary bile acids into the enterohepatic
circulation.
[0307] In addition to the agents and therapeutic modalities set
forth above, combination therapy with numerous additional agents
(and classes thereof) is also contemplated, including, but not
limited to, 1) insulin e.g., bolus and basal analogs), insulin
mimetics and agents that entail stimulation of insulin secretion,
including sulfonylureas (e.g., chlorpropamide, tolazamide,
acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and
meglitinides (e.g., repaglinide (PRANDIN) and nateglinide
(STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE)) and other
agents that act by promoting glucose utilization, reducing hepatic
glucose production and/or diminishing intestinal glucose output; 3)
alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and
other agents that slow down carbohydrate digestion and consequently
absorption from the gut and reduce postprandial hyperglycemia; 4)
thiazolidinediones (e.g., rosiglitazone (AVANDIA), troglitazone
(REZULIN), pioglitazone (ACTOS), glipizide, balaglitazone,
rivoglitazone, netoglitazone, troglitazone, englitazone,
ciglitazone, adaglitazone, darglitazone that enhance insulin action
(e.g., by insulin sensitization), thus promoting glucose
utilization in peripheral tissues; 5) glucagon-like-peptides
including DPP-IV inhibitors (e.g., vildagliptin (GALVUS) and
sitagliptin (JANUVIA)) and Glucagon-Like Peptide-1 (GLP-1) and
GLP-1 agonists and analogs (e.g., exenatide (BYETTA and ITCA 650
(an osmotic pump inserted subcutaneously that delivers an exenatide
analog over a 12-month period; Intarcia, Boston, Mass.)); 6) and
DPP-IV-resistant analogues (incretin mimetics), PPAR gamma
agonists, dual-acting PPAR agonists, pan-acting PPAR agonists,
PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXR
agonists, glycogen synthase kinase-3 inhibitors, immune modulators,
beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and
amylin analogues.
[0308] Other exemplary agents that can be used, in certain
embodiments, in combination with the peptides and methods provided
herein include dipeptidyl peptidase-4 (DPP-4) inhibitors,
bromocriptine formulations (e.g. and bile acid sequestrants (e.g.,
colesevelam), and SGLT-2 inhibitors. Appetite suppression drugs are
also well known and can be used in combination with the
compositions and methods provided herein. Supplementary therapies
can be administered prior to, contemporaneously with or following
methods and uses provided herein.
[0309] In one aspect, provided herein is a method for preventing or
treating a bile acid related disorder (BARD), or a symptom thereof,
in a subject comprising administering to the subject an effective
amount of a peptide, wherein the peptide has an amino acid sequence
comprising or consisting of:
TABLE-US-00040 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLRELYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKIIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70).
[0310] In one aspect, provided herein is a method for preventing or
treating a bile acid related disorder (BARD), or a symptom thereof,
in a subject comprising administering to the subject an effective
amount of a peptide, wherein the peptide has an amino acid sequence
comprising or consisting of:
TABLE-US-00041 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLRELYTSGPHGLSSCFLRIRADGVVDCARGQSAH
SLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIR
PDGYNVYRSEKIIRLPVSLSSAKQRQLYKNRGFLPLSEFLPMLPMVPEEP
EDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69).
[0311] In another aspect, provided herein is a method for
preventing or treating a BARD, or a symptom thereof, in a subject
comprising administering to the subject an effective amount of a
peptide, wherein the peptide comprises: 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.
[0312] Other peptides provided herein are also contemplated in the
methods provided herein.
[0313] In certain embodiments, the BARD, or symptom thereof, is
improved as compared to baseline. In some embodiments, baseline is
a pre-dose baseline.
[0314] In some embodiments, the BARD is non-alcoholic fatty liver
disease (NAFLD). In one embodiment, provided herein is a method of
preventing or treating NAFLD, or a symptom thereof, in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0315] In some embodiments, the method results in an improvement of
the NAFLD activity score (NAS).
[0316] In some embodiments, the BARD is hepatic fibrosis. In one
embodiment, provided herein is a method of preventing or treating
hepatic fibrosis, or a symptom thereof, in a subject, comprising
administering a peptide (e.g., M70, or M69) provided herein.
[0317] In some embodiments, the BARD is nonalcoholic
steatohepatitis (NASH). In one embodiment, provided herein is a
method of preventing or treating NASH or a symptom thereof, in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein. In some embodiments, the subject has
biopsy-confirmed NASH.
[0318] In some embodiments, the BARD is cholestatic liver disease.
In one embodiment, provided herein is a method of preventing or
treating cholestatic liver disease, or a symptom thereof, in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0319] In some embodiments, the cholestatic liver disease is
primary sclerosing cholangitis (PSC). In some embodiments, the
cholestatic liver disease is primary biliary cirrhosis (PBC). In
some embodiments, the cholestatic liver disease is intrahepatic
cholestatis of pregnancy. In some embodiments, the cholestatic
liver disease is alcoholic hepatitis. In some embodiments, the
cholestatic liver disease is drug-induced cholestatis.
[0320] In some embodiments, the methods provided herein result in a
decrease in liver steatosis. In one embodiment, provided herein is
a method of preventing or treating liver steatosis in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0321] In some embodiments, the methods provided herein result in a
decrease in liver inflammation. In one embodiment, provided herein
is a method of preventing or treating liver inflammation in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein. In certain embodiments, the liver inflammation is
lobular inflammation.
[0322] In some embodiments, the methods provided herein result in a
decrease in hepatocyte ballooning. In one embodiment, provided
herein is a method of decreasing hepatocyte ballooning in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0323] In some embodiments, the methods provided herein result in a
reduction of CYP7a1 levels in the subject. In one embodiment,
provided herein is a method of reducing CYP7a1 levels in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0324] In some embodiments, the methods provided herein result in a
reduction of serum bile acid levels in the subject. In one
embodiment, provided herein is a method of reducing serum bile acid
levels in a subject, comprising administering a peptide (e.g., M70,
or M69) provided herein.
[0325] In some embodiments, the methods provided herein result in a
reduction of triglycerides in the subject. In one embodiment,
provided herein is a method of reducing triglycerides in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0326] In some embodiments, the methods provided herein result in a
reduction in alkaline phosphatase (ALP) levels in the subject. In
one embodiment, provided herein is a method of reducing ALP levels
in a subject, comprising administering a peptide (e.g., M70, or
M69) provided herein. In some embodiments, the ALP levels are
reduced at least 10% in the subject. In some embodiments, the ALP
levels are reduced at least 15% in the subject.
[0327] In some embodiments, the methods provided herein result in a
reduction in alkaline aminotransferase (ALT) levels in the subject.
In one embodiment, provided herein is a method of reducing ALT in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein. In one embodiment, provided herein is a method of
reducing ALT levels in a subject, comprising administering a
peptide (e.g., M70, or M69) provided herein.
[0328] In some embodiments, the methods provided herein result in a
reduction in aspartate aminotransfease (AST) levels in the subject.
In one embodiment, provided herein is a method of reducing AST
levels in a subject, comprising administering a peptide (e.g., M70,
or M69) provided herein.
[0329] In some embodiments, the methods provided herein result in a
reduction in gamma-glutamyltransferase (GGT) levels in the subject.
In one embodiment, provided herein is a method of reducing GGT
levels in a subject, comprising administering a peptide (e.g., M70,
or M69) provided herein.
[0330] In some embodiments, the methods provided herein result in
an improvement in a biochemical marker of liver function. In one
embodiment, provided herein is a method of improving a biochemical
marker of liver function in a subject, comprising administering a
peptide (e.g., M70, or M69) provided herein. In some embodiments,
the biochemical marker of liver function is an enzyme. In some
embodiments, the enzyme is ALP. In some embodiments, the enzyme is
ALT. In some embodiments, the enzyme is AST. In some embodiments,
the enzyme is GGT.
[0331] In some embodiments, the methods provided herein result in a
reduction in cholesterol levels in the subject. In one embodiment,
provided herein is a method of reducing cholesterol levels in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0332] In some embodiments, the methods provided herein result in a
reduction in glucose levels in the subject. In one embodiment,
provided herein is a method of reducing glucose levels in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0333] In some embodiments, the methods provided herein result in
an improvement in insulin resistance in the subject. In one
embodiment, provided herein is a method of improving insulin
resistance in a subject, comprising administering a peptide (e.g.,
M70, or M69) provided herein.
[0334] In some embodiments, the methods provided herein result in
an improvement in insulin sensitivity in the subject. In one
embodiment, provided herein is a method of improving insulin
sensitivity in a subject, comprising administering a peptide (e.g.,
M70, or M69) provided herein. In some embodiments, the insulin
sensitivity is as measured by HOMA-IR.
[0335] In some embodiments, the methods provided herein result in a
reduction in body weight in the subject. In one embodiment,
provided herein is a method of reducing body weight in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0336] In some embodiments, the methods provided herein result in a
reduction in liver weight in the subject. In one embodiment,
provided herein is a method of reducing liver weight in a subject,
comprising administering a peptide (e.g., M70, or M69) provided
herein.
[0337] In some embodiments, the methods provided herein result in a
decrease in bilirubin levels in the subject. In one embodiment,
provided herein is a method of reducing bilirubin levels in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0338] In some embodiments, the methods provided herein result in a
decrease in a serum biomarker of early fibrosis in the subject. In
one embodiment, provided herein is a method of reducing the level
of a serum biomarker of early fibrosis in a subject, comprising
administering a peptide (e.g., M70, or M69) provided herein.
[0339] In some embodiments, the methods provided herein result in
the reduction of serum C4 levels in the subject. In one embodiment,
provided herein is a method of reducing serum C4 levels in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein. In some embodiments, the serum C4 levels are
decreased by at least 50%, at least 60%, at least 70%, at least
80%, or at least 90% in the subject. In some embodiments, the
reduction in serum C4 levels is a mean reduction in C4 levels. In
some embodiments, the mean reduction in serum C4 levels is at least
90%. In some embodiments, the serum C4 levels are decreased as
compared to the serum C4 levels in the subject prior to
administration of the peptide.
[0340] In some embodiments, the methods provided herein result in
an improvement in liver function in the subject. In one embodiment,
provided herein is a method of improving liver function in a
subject, comprising administering a peptide (e.g., M70, or M69)
provided herein.
[0341] In some embodiments, the methods provided herein result in
improving pruritus, or a symptom thereof, in the subject. In one
embodiment, provided herein is a method of preventing or treating
pruritus, or a symptom thereof, in a subject, comprising
administering a peptide (e.g., M70, or M69) provided herein. In one
embodiment, the method is a method of preventing pruritus, or a
symptom thereof, in a subject. In one embodiment, the method is a
method of treating pruritus, or a symptom thereof, in a subject. In
some embodiments, the pruritus symptom is itching. In some
embodiments, the pruritus symptom is impaired sleep. In some
embodiments, the pruritus symptom is depression.
[0342] In some embodiments, the peptide is administered at a dose
of 0.3 mg. In some embodiments, the peptide is administered at a
dose of 1 mg. In some embodiments, the peptide is administered at a
dose of 2 mg. In some embodiments, the peptide is administered at a
dose of 3 mg. In some embodiments, the peptide is administered at a
dose of 5 mg. In some embodiments, the peptide is administered at a
dose of 10 mg.
[0343] In some embodiments, the peptide is administered once a day.
In some embodiments, the peptide is administered twice a day.
[0344] In some embodiments, the peptide is administered
subcutaneously.
[0345] In some embodiments, the peptide is administered for 7 days
or longer. In some embodiments, the peptide is administered for 14
days or longer. In some embodiments, the peptide is administered
for 21 days or longer. In some embodiments, the peptide is
administered for 28 days or longer. In some embodiments, the
peptide is administered for 1 to 12 months. In some embodiments,
the peptide is administered for 12 months. In some embodiments, the
peptide is administered for more than 12 months.
[0346] In some embodiments, the peptide is administered in
combination with ursodeoxycholic acid (UDCA).
[0347] In some embodiments, the subject is overweight. In some
embodiments, the subject is obese. In some embodiments, the subject
has diabetes. In some embodiments, the subject does not have
diabetes. In some embodiments, the diabetes is type 2 diabetes.
[0348] 4.5.2 Methods of Preventing, Treating and Managing Metabolic
Disorders
[0349] Also provided herein are in vitro, ex vivo and in vivo
(e.g., on or in a subject) methods and uses. Such methods and uses
can be practiced with any of the peptide sequences set forth
herein. In various embodiments, the methods include administering a
peptide sequence, such as a FGF19 or FGF21 variant, fusion or
chimera disclosed herein (e.g., in the Sequence Listing or Tables
1-11), or a subsequence, a variant or modified form of a FGF19 or
FGF21 variant, fusion or chimera disclosed herein (e.g., the
Sequence Listing or Tables 1-11), to a subject in an amount
effective for treating a metabolic or associated disorder.
[0350] In certain embodiments, the peptide is administered in
combination with an additional therapeutic agent(s) and/or
treatment modalities (e.g., an agent useful in the treatment and/or
prevention of PBC). The additional therapeutic agent(s) can be
administered before, with, or following administration of the
peptides described herein.
[0351] Also provided herein are methods of preventing (e.g., in
subjects predisposed to having a particular disorder(s)), delaying,
slowing or inhibiting progression of, the onset of, or treating
(e.g., ameliorating) a metabolic or associated disorder relative to
an appropriate matched subject of comparable age, gender, race,
etc.). Thus, in various embodiments, a method provided herein for,
for example, modulating bile acid homeostasis or treating a
metabolic or associated disorder includes contacting or
administering one or more peptides provided herein (e.g., a variant
or fusion of FGF19 and/or FGF21 as set forth in the Sequence
Listing or Tables 1-11) in an amount effective to modulate bile
acid homeostasis or treat a metabolic or associated disorder. In
certain embodiments the method further comprises contacting or
administering at least one additional therapeutic agent or
treatment modality that is useful in the treatment or prevention of
a metabolic or associated disorder (e.g., PBC).
[0352] 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.
[0353] Subjects include those having a disorder, e.g., a metabolic
or associated disorder, or subjects that do not have a disorder but
may be at risk of developing the disorder.
[0354] Non-limiting exemplary disorders or conditions preventable,
treatable or manageable with the peptide formulations, methods and
uses thereof provided herein, include metabolic diseases and
disorders. Non limiting examples of diseases and disorders include:
metabolic syndrome; a lipid- or glucose-related disorder;
cholesterol or triglyceride metabolism; type 2 diabetes;
cholestasis, including, for example diseases of intrahepatic
cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and
drug induced cholestasis (e.g., estrogen)), and diseases of
extrahepatic cholestasis (e.g., bile cut compression from tumor,
bile duct blockade by gall stones); bile acid malabsorption and
other disorders involving the distal small intestine, including
ileal resection, inflammatory bowel diseases (e.g., Crohn's disease
and ulcerative colitis), disorders impairing absorption of bile
acids not otherwise characterized (idiopathic)) leading to diarrhea
(e.g., BAD) and GI symptoms, and GI, liver, and/or biliary cancers
(e.g., colon cancer and hepatocellular cancer); and/or bile acid
synthesis abnormalities, such as those contributing to NASH,
cirrhosis and portal hypertension. For treatment, peptide sequences
provided herein can be administered to subjects in need of
modulation of bile acid homeostasis or having a bile-acid related
or associated disorder. 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; dyslipidemias and their sequelae such
as, for example, atherosclerosis, coronary artery disease,
cerebrovascular disorders and the like.
[0355] Other conditions which may be associated with metabolic
syndrome, such as obesity and elevated body mass (including the
co-morbid conditions thereof such as, but not limited to,
nonalcoholic fatty liver disease (NAFLD), nonalcoholic
steatohepatitis (NASH), and polycystic ovarian syndrome (PCOS)),
and also include thromboses, hypercoagulable and prothrombotic
states (arterial and venous), hypertension (including portal
hypertension (defined as a hepatic venous pressure gradient (HVPG)
greater than 5 mm Hg), cardiovascular disease, stroke and heart
failure; Disorders or conditions in which inflammatory reactions
are involved, including atherosclerosis, chronic inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), asthma,
lupus erythematosus, arthritis, or other inflammatory rheumatic
disorders; Disorders of cell cycle or cell differentiation
processes such as adipose cell tumors, lipomatous carcinomas
including, for example, liposarcomas, solid tumors, and neoplasms;
Neurodegenerative diseases and/or demyelinating disorders of the
central and peripheral nervous systems and/or neurological diseases
involving neuroinflammatory processes and/or other peripheral
neuropathies, including Alzheimer's disease, multiple sclerosis,
Parkinson's disease, progressive multifocal leukoencephalopathy and
Guillian-Barre syndrome; Skin and dermatological disorders and/or
disorders of wound healing processes, including erythemato-squamous
dermatoses; and other disorders such as syndrome X, osteoarthritis,
and acute respiratory distress syndrome.
[0356] In one embodiment, a subject has 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, is
obese and/or has an undesirable body mass.
[0357] In particular aspects of the methods and uses, a peptide
sequence or chimeric peptide sequence provided herein 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.
[0358] 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.
[0359] Treatment of a metabolic or associated disorder (e.g.,
hyperglycemia) may have the benefit of alleviating or abolishing a
disorder secondary thereto. By way of example, a subject suffering
from hyperglycemia may also have depression or anxiety due to the
hyperglycemia; thus, treating the subject's hyperglycemia may also
indirectly treat the depression or anxiety. The use of the
therapies disclosed herein to target such secondary disorders is
also contemplated in certain embodiments.
[0360] In particular embodiments, the subject has or is at risk of
having hyperglycemia. In other particular embodiments, the subject
has or is at risk of having diabetes, such as Type 2 diabetes.
[0361] Subjects at risk of developing a metabolic or associated
disorder (such as the disorders described above) include, for
example, those who may have a family history or genetic
predisposition toward such disorder, as well those whose diet may
contribute to development of such disorders.
[0362] As disclosed herein, treatment methods include contacting or
administering a peptide as set forth herein (e.g., a variant or
fusion of FGF19 and/or FGF21 as set forth in the Sequence Listing
or Tables 1-11) in an amount effective to achieve a desired outcome
or result in a subject. A treatment that results in a desired
outcome or result includes decreasing, reducing or preventing the
severity or frequency of one or more symptoms of the condition in
the subject, e.g., an improvement in the subject's condition or a
"beneficial effect" or "therapeutic effect." Therefore, treatment
can decrease or reduce or prevent the severity or frequency of one
or more symptoms of the disorder, stabilize or inhibit progression
or worsening of the disorder, and in some instances, reverse the
disorder, transiently (e.g., for 1-6, 6-12, or 12-24 hours), for
medium term (e.g., 1-6, 6-12, 12-24 or 24-48 days) or long term
(e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greater than 24-48
weeks). Thus, in the case of a metabolic or associated disorder,
treatment can lower or reduce one or more symptoms or effects of
the metabolic or associated disorders described above.
[0363] In certain embodiments, the various methods provided herein
further include contacting or administering one or more additional
agents or therapeutic modalities useful in the treatment or
prevention of a metabolic or associated disorder, such as those
agents or therapeutic modalities described herein, in an amount
effective to achieve a desired outcome or result in a subject.
[0364] An "effective amount" or a "sufficient amount" for use
and/or for treating a subject refers to an amount that provides, in
single or multiple doses, alone, or in combination with one or more
other agents, treatments, protocols, or therapeutic regimens, a
detectable response of any duration of time (transient, medium or
long term), a desired outcome in or an objective or subjective
benefit to a subject of any measurable or detectable degree or for
any duration of time (e.g., for hours, days, months, years, in
remission or cured). Such amounts typically are effective to
ameliorate a disorder, or one, multiple or all adverse symptoms,
consequences or complications of the disorder, to a measurable
extent, although reducing or inhibiting a progression or worsening
of the disorder, is considered a satisfactory outcome.
[0365] 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
metabolic or associated disorder such as those described above, an
improvement can be a lowering or a reduction in one or more
symptoms or effects of the disorder.
[0366] 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.).
[0367] Thus, in the case of a disorder treatable by a peptide
sequence provided herein, either alone or in combination with an
additional agent, the amount of the peptide (and optionally the
additional agent)sufficient to ameliorate a disorder will depend on
the type, severity and extent, or duration of the disorder, the
therapeutic effect or outcome desired, and can be readily
ascertained by the skilled artisan. Appropriate amounts will also
depend upon the individual subject (e.g., the bioavailability
within the subject, gender, age, etc.). For example, a transient,
or partial, restoration of normal bile acid homeostasis in a
subject can reduce the dosage amount or frequency of the peptides
and agents described herein in order to treat the metabolic or
associated disorders described previously even though complete
freedom from treatment has not resulted. An effective amount can be
ascertained, for example, by measuring one or more relevant
physiological effects.
[0368] Methods and uses provided herein for treating a subject are
applicable for prophylaxis to prevent or reduce the likelihood of a
disorder in a subject, such as a metabolic or associated disorder.
Accordingly, methods and uses provided herein for treating a
subject having, or at risk of developing, a metabolic or associated
disorder can be practiced prior to, substantially contemporaneously
with, or following administration or application of another agent
useful for the treatment or prevention of a metabolic or associated
disorder, and/or can be supplemented with other forms of therapy.
Supplementary therapies include other glucose lowering treatments,
such as insulin, an insulin sensitivity enhancer and other drug
treatments, a change in diet (low sugar, fats, etc.), weight loss
surgery--(reducing stomach volume by gastric bypass, gastrectomy),
gastric banding, gastric balloon, gastric sleeve, etc. For example,
a method or use provided herein for treating a hyperglycemic or
insulin resistance disorder can be used in combination with drugs
or other pharmaceutical compositions that lower glucose or increase
insulin sensitivity in a subject.
[0369] In one embodiment, a method or use includes contacting or
administering to a subject one or more variant or fusion FGF19
and/or FGF21 peptide sequences in an amount effective for
preventing a metabolic or associated disorder. In one embodiment, a
method or use 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 a metabolic or associated
disorder. In one embodiment, a method or use includes contacting or
administering to a subject one or more variant or fusion FGF19
and/or FGF21 peptide sequences in an amount effective for managing
a metabolic or associated disorder.
4.6 Nucleic Acid Molecules
[0370] Also provided are nucleic acid molecules encoding peptide
sequences provided herein, including subsequences, sequence
variants and modified forms of the sequences listed in the Sequence
Listing (and in PCT Pub. No. WO 2013/006486 and US Pub. No.
2013/0023474, as well as PCT Publ. No. WO 2014/085365) or Tables
1-11, and vectors that include nucleic acid encoding the peptides
used in the methods described herein. Accordingly, "nucleic acids"
include those that encode the exemplified peptide sequences
disclosed herein, as well as those encoding functional
subsequences, sequence variants and modified forms of the
exemplified peptide sequences, so long as the foregoing retain at
least detectable or measureable activity or function useful in the
treatment or prevention of a bile acid-related or associated
disorder (e.g., PBC).
[0371] Nucleic acid, which can also be referred to herein as a
gene, polynucleotide, nucleotide sequence, primer, oligonucleotide
or probe, refers to natural or modified purine- and
pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotides or mixed
polyribo-polydeoxyribo nucleotides and .alpha.-anomeric forms
thereof. The two or more purine- and pyrimidine-containing polymers
are typically linked by a phosphoester bond or analog thereof. The
terms can be used interchangeably to refer to all forms of nucleic
acid, including deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). The nucleic acids can be single strand, double, or triplex,
linear or circular. Nucleic acids include genomic DNA and cDNA. RNA
nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA or
antisense. Nucleic acids include naturally occurring, synthetic, as
well as nucleotide analogs and derivatives.
[0372] As a result of the degeneracy of the genetic code, the
nucleic acid molecules provided herein include sequences degenerate
with respect to nucleic acid molecules encoding the peptide
sequences useful in the methods provided herein. Thus, degenerate
nucleic acid sequences encoding peptide sequences, including
subsequences, variants and modified forms of the peptide sequences
exemplified herein (e.g., in the Sequence Listing or Tables 1-11),
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.
[0373] Nucleic acid can be produced using any of a variety of known
standard cloning and chemical synthesis methods, and can be altered
intentionally by site-directed mutagenesis or other recombinant
techniques known to one skilled in the art. Purity of
polynucleotides can be determined through, for example, sequencing,
gel electrophoresis, and UV spectrometry.
[0374] 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.
[0375] 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.
[0376] Expression control elements include elements that activate
transcription constitutively, that are inducible (i.e., require an
external signal or stimuli for activation), or derepressible (i.e.,
require a signal to turn transcription off; when the signal is no
longer present, transcription is activated or "derepressed"). Also
included in the expression cassettes provided herein are control
elements sufficient to render gene expression controllable for
specific cell types or tissues (i.e., tissue-specific control
elements). Typically, such elements are located upstream or
downstream (i.e., 5' or 3') of the coding sequence. Promoters are
generally positioned 5' of the coding sequence. Promoters, produced
by recombinant DNA or synthetic techniques, can be used to provide
for transcription of the polynucleotides provided herein. A
"promoter" typically means a minimal sequence element sufficient to
direct transcription.
[0377] 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, or producing peptide sequences, for example.
[0378] As used herein, the term "transgene" means a polynucleotide
that has been introduced into a cell or organism by artifice. For
example, in a cell having a transgene, the transgene has been
introduced by genetic manipulation or "transformation" of the cell.
A cell or progeny thereof into which the transgene has been
introduced is referred to as a "transformed cell" or
"transformant." Typically, the transgene is included in progeny of
the transformant or becomes a part of the organism that develops
from the cell. Transgenes may be inserted into the chromosomal DNA
or maintained as a self-replicating plasmid, YAC, minichromosome,
or the like.
[0379] Bacterial system promoters include T7 and inducible
promoters such as pL of bacteriophage plac, ptrp, ptac (ptrp-lac
hybrid promoter) and tetracycline-responsive promoters. Insect cell
system promoters include constitutive or inducible promoters (e.g.,
ecdysone). Mammalian cell constitutive promoters include SV40, RSV,
bovine papilloma virus (BPV) and other virus promoters, or
inducible promoters derived from the genome of mammalian cells
(e.g., metallothionein IIA promoter; heat shock promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the
inducible mouse mammary tumor virus long terminal repeat).
Alternatively, a retroviral genome can be genetically modified for
introducing and directing expression of a peptide sequence in
appropriate host cells.
[0380] 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.
[0381] 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).
[0382] Expression vectors also can contain a selectable marker
conferring resistance to a selective pressure or identifiable
marker (e.g., beta-galactosidase), thereby allowing cells having
the vector to be selected for, grown and expanded. Alternatively, a
selectable marker can be on a second vector that is co-transfected
into a host cell with a first vector containing a nucleic acid
encoding a peptide sequence. Selection systems include, but are not
limited to, herpes simplex virus thymidine kinase gene (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad.
Sci. USA 48:2026 (1962)), and adenine phosphoribosyltransferase
(Lowy et al., Cell 22:817 (1980)) genes that can be employed in
tk-, hgprt- or aprt-cells, respectively. Additionally,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, which
confers resistance to mycophenolic acid (Mulligan et al., Proc.
Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers
resistance to aminoglycoside G-418 (Colberre-Garapin et al., J.
Mol. Biol. 150:1(1981)); puromycin; and hygromycin gene, which
confers resistance to hygromycin (Santerre et al., Gene 30:147
(1984)). Additional selectable genes include trpB, which allows
cells to utilize indole in place of tryptophan; hisD, which allows
cells to utilize histinol in place of histidine (Hartman et al.,
Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine
decarboxylase), which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue (1987) In: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory).
4.7 Cell Lines and Animal Models
[0383] In certain embodiments, also provided is a transformed
cell(s) (in vitro, ex vivo and in vivo) and host cells that produce
a variant or fusion of FGF19 and/or FGF21 as set forth herein,
where expression of the variant or fusion of FGF19 and/or FGF21 is
conferred by a nucleic acid encoding the variant or fusion of FGF19
and/or FGF21. As used herein, a "transformed" or "host" cell is a
cell into which a nucleic acid is introduced that can be propagated
and/or transcribed for expression of an encoded peptide sequence.
The term also includes any progeny or subclones of the host cell.
Transformed and host cells that express peptide sequences provided
herein typically include a nucleic acid that encodes the peptide
sequence. In one embodiment, a transformed or host cell is a
prokaryotic cell. In another embodiment, a transformed or host cell
is a eukaryotic cell. In various aspects, the eukaryotic cell is a
yeast or mammalian (e.g., human, primate, etc.) cell.
[0384] 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.
[0385] 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.
[0386] 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.
[0387] 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.
[0388] 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.
[0389] 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, nucleic acids for 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, can be introduced for stable expression in
mice.
[0390] Mice strains that develop or are susceptible to developing a
particular disease (e.g., diabetes, degenerative disorders, cancer,
etc.) are also useful for introducing therapeutic proteins as
described herein in order to study the effect of therapeutic
protein expression in the disease-susceptible mouse. Transgenic and
genetic animal models that are susceptible to particular disease or
physiological conditions, such as streptozotocin (STZ)-induced
diabetic (STZ) mice, are appropriate targets for expressing
variants of FGF19 and/or FGF21, fusions/chimeric sequences (or
variant) thereof, such as a chimeric peptide sequence including all
or a portion of FGF19, or including all or a portion of FGF21, as
set forth herein. Thus, in certain embodiments, there are provided
non-human transgenic animals that produce a variant of FGF19 and/or
FGF21, or a fusion/chimeric sequence (or variant) thereof, such as
a chimeric peptide sequence including all or a portion of FGF19, or
including all or a portion of FGF21, the production of which is not
naturally occurring in the animal which is conferred by a transgene
present in somatic or germ cells of the animal.
[0391] 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.
[0392] Peptide sequences, nucleic acids encoding peptide sequences,
vectors and transformed host cells expressing peptide sequences
include isolated and purified forms. The term "isolated," when used
as a modifier of a composition provided herein, means that the
composition is separated, substantially, completely, or at least in
part, from one or more components in an environment. Generally,
compositions that exist in nature, when isolated, are substantially
free of one or more materials with which they normally associate
with in nature, for example, one or more protein, nucleic acid,
lipid, carbohydrate or cell membrane. The term "isolated" does not
exclude alternative physical forms of the composition, such as
variants, modifications or derivatized forms, fusions and chimeras,
multimers/oligomers, etc., or forms expressed in host cells. The
term "isolated" also does not exclude forms (e.g., pharmaceutical
compositions, combination compositions, etc.) in which there are
combinations therein, any one of which is produced by the hand of
man. An "isolated" composition can also be "purified" when free of
some, a substantial number of, or most or all of one or more other
materials, such as a contaminant or an undesired substance or
material.
[0393] As used herein, the term "recombinant," when used as a
modifier of peptide sequences, nucleic acids encoding peptide
sequences, etc., means that the compositions have been manipulated
(i.e., engineered) in a fashion that generally does not occur in
nature (e.g., in vitro). A particular example of a recombinant
peptide would be where a peptide sequence provided herein is
expressed by a cell transfected with a nucleic acid encoding the
peptide sequence. A particular example of a recombinant nucleic
acid would be a nucleic acid (e.g., genomic or cDNA) encoding a
peptide sequence cloned into a plasmid, with or without 5', 3' or
intron regions that the gene is normally contiguous within the
genome of the organism. Another example of a recombinant peptide or
nucleic acid is a hybrid or fusion sequence, such as a chimeric
peptide sequence comprising a portion of FGF19 and a portion of
FGF21.
[0394] In accordance with the methods provided herein, there are
provided 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 Tables 1-11 and the Sequence
Listing, and the FGF19/FGF21 fusions and chimeras listed in Tables
1-11 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 a bile acid homeostasis modulating or
anti-diabetic, or glucose lowering, drug or therapeutic agent.
Combinations, such as one or more peptide sequences in a
pharmaceutically acceptable carrier or excipient, with one or more
of a bile acid homeostasis modulating or a treatment for a bile
acid-related or associated disorder, or anti-diabetic, or glucose
lowering drug or therapeutic agent are also provided. Such
combinations of a peptide sequence provided herein with another
drug or agent, such as a bile acid homeostasis modulating or acid
related disorder treating, or 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.
[0395] 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, non-encapsulated nanoparticles, 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.
Methods of preparing liposomes are described in, for example, U.S.
Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods of
encapsulated-free controlled release using nanoparticles are
described, for example, in Pakulska et al., Science Advances 2(5):
e1600519 (2016). Methods for preparation of the above-mentioned
formulations will be apparent to those skilled in the art.
[0396] The peptides provided herein including subsequences,
variants and modified forms of the exemplified peptide sequences
(including the FGF19 and FGF21 variants and subsequences listed in
Tables 1-11 and the Sequence Listing, and the FGF19/FGF21 fusions
and chimeras listed in Tables 1-11 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.
[0397] In case of conflict, the specification, including
definitions, will control. As used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide sequence" or "a treatment,"
includes a plurality of such sequences, treatments, and so forth.
It is further noted that the claims can be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology such as
"solely," "only" and the like in connection with the recitation of
claim elements, or use of a "negative" limitation.
[0398] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges can
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0399] As used herein, numerical values are often presented in a
range format throughout this document. The use of a range format is
merely for convenience and brevity and should not be construed as
an inflexible limitation on the scope of the invention unless the
context clearly indicates otherwise. Accordingly, the use of a
range expressly includes all possible subranges, all individual
numerical values within that range, and all numerical values or
numerical ranges including integers within such ranges and
fractions of the values or the integers within ranges, unless the
context clearly indicates otherwise. This construction applies
regardless of the breadth of the range and in all contexts
throughout this patent document. Thus, for example, reference to a
range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%,
91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range
of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc.,
as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%,
92.3%, 92.4%, 92.5%, etc., and so forth. In addition, reference to
a range of 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60,
60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,
140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225,
225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, etc. In a further example, reference to a range
of 25-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-25,000, or
5000-50,000 includes any numerical value or range within or
encompassing such values, e.g., 25, 26, 27, 28, 29 . . . 250, 251,
252, 253, 254 . . . 500, 501, 502, 503, 504 . . . , etc. The use of
a series of ranges includes combinations of the upper and lower
ranges to provide another range. This construction applies
regardless of the breadth of the range and in all contexts
throughout this patent document. Thus, for example, reference to a
series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75,
75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50,
5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150,
and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth.
[0400] 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-00042 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)
[0401] 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.
[0402] Particular embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Upon reading the foregoing description,
variations of the disclosed embodiments may become apparent to
individuals working in the art, and it is expected that those
skilled artisans may employ such variations as appropriate.
Accordingly, it is intended that the invention be practiced
otherwise than as specifically described herein, and that the
invention includes all modifications and equivalents of the subject
matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described
elements in all possible variations thereof is encompassed by the
invention unless otherwise indicated herein or otherwise clearly
contradicted by context.
[0403] All publications, patent applications, accession numbers,
and other references cited in this specification are herein
incorporated by reference in its entirety as if each individual
publication or patent application were specifically and
individually indicated to be incorporated by reference. The
publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of
prior invention. Further, the dates of publication provided can be
different from the actual publication dates which can need to be
independently confirmed.
[0404] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, the descriptions in the
Experimental section are intended to illustrate but not limit the
scope of invention described in the claims.
5. EXPERIMENTAL
5.1 Example 1
[0405] The following is a description of various methods and
materials used in the studies herein.
[0406] Animals.
[0407] 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. Mice 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. All animal studies were
approved by the NGM Institutional Animal Care and Use
Committee.
[0408] DNA and Amino Acid Sequences.
[0409] 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).
[0410] PCR.
[0411] FGF19 ORF was amplified with polymerase chain reaction (PCR)
using recombinant DNA (cDNA) prepared from human small intestinal
tissue. PCR reagents kits with Phusion.RTM. high-fidelity DNA
polymerase were purchased from New England BioLabs (F-530L,
Ipswich, Mass.). The following primers were used: forward PCR
primer:
TABLE-US-00043 (SEQ ID NO: 136) 5'
CCGACTAGTCACCatgcggagcgggtgtgtgg
and reverse PCR primer:
TABLE-US-00044 (SEQ ID NO: 137) 5'
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.
[0412] Cyp7a1 Repression Assay in Primary Human Hepatocytes.
[0413] Primary human hepatocytes were plated on collagen coated
plates (Becton Dickinson Biosciences) in Williams E media
(Invitrogen) supplemented with 100 nM dexamethasone (Sigma) and
0.25 mg/ml MatriGel.TM. (Becton Dickinson Biosciences). Cells were
treated with FGF19 or variants at 37.degree. C. for 6 hours. Cyp7a1
expression was evaluated in triplicate by quantitative RT-PCR
(TaqMane ABI PRISM 7700, Applied Biosystems) and normalized to
GAPDH expression.
[0414] Cyp7a1 In Vivo Repression Assay.
[0415] Nine-week-old male db/db mice (Jackson Laboratories) were
injected intraperitoneally with recombinant proteins FGF19 or FGF21
at 0.1 mg/kg, 1 mg/kg, and 10 mg/kg. Animals were euthanized 5
hours post-injection. Liver was harvested and homogenized in
TRIzol.RTM. reagent (Invitrogen). Total RNA was extracted and
treated with DNase (Ambion) followed by quantitative RT-PCR
analysis and normalized to GAPDH expression.
[0416] Production and Purification of AAV.
[0417] 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.TM. plasmids (Agilent Technologies) and AAV2/9
plasmid (Gao et al., J. Virol. 78:6381 (2004)). Forty-eight (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.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.
[0418] 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.
[0419] Viral stock was diluted with PBS to desirable GC/ml. Viral
working solution (200 .mu.l) was delivered into mice via tail vein
injection.
[0420] Hepatocellular Carcinoma (HCC) Assay.
[0421] Liver specimens were harvested from db/db mice 24 weeks
after AAV injection. HCC scores were 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.
[0422] Serum FGF19/FGF21/Variants Exposure Level Assay.
[0423] Whole blood (about 50 .mu.l/mouse) 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.). FGF19, FGF21, and variant exposure
levels in serum was determined using EIA kits (Biovendor) by
following the manufacturer's instructions.
[0424] FGFR4 binding and activity assays. Solid phase ELISA
(binding) and ERK phosphorylation assay can be performed using
purified recombinant proteins. FGFR binding assay can be conducted
using solid phase ELISA. Briefly, a 96-well plate can be coated
with 2 .mu.g/ml anti-hFc antibody and can be incubated with 1
.mu.g/ml FGFR1-hFc or FGFR4-hFc. Binding to FGF19 variants in the
presence of 1 .mu.g/ml soluble f3-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.
5.2 Example 2
[0425] In order to confirm that FGF19 variants such as those set
forth herein repress cyp7a1 expression, inhibition of cyp7a1
expression by wild-type FGF19 was determined following
administration of various concentrations. The effects of FGF21 were
assessed in a comparable manner.
[0426] Briefly, at time 0, db/db mice were dosed intraperitoneally
with either recombinant FGF19 (0.1 mg/kg; 1 mg/kg; 10 mg/kg) or
recombinant FGF21 (0.1 mg/kg; 1 mg/kg; 10 mg/kg). Five hours after
dosing, livers were harvested, RNA was extracted, and cyp7a1
expression was determined by real-time PCR (QPCR) using GADPH as a
normalization control. In each group of mice, n=3, and cyp7a1
expression values for the various FGF19 and FGF21 concentrations
were compared to mice dosed with PBS vehicle control.
[0427] As set forth in FIG. 1, FGF19 dramatically decreased cyp7a1
expression in a concentration-dependent manner. Although
administration of FGF21 caused a reduction of cyp7a1 expression,
the effect was demonstrably less than that observed with FGF19.
[0428] The effect of variant M70 on cyp7a1 expression in human
primary hepatocytes was compared to that of FGF19. As noted in FIG.
2, variant M70 repressed cyp7a1 expression in an amount comparable
to that of FGF19.
5.3 Example 3
[0429] Using the assays described above, repression of cyp7a1 in
primary human hepatocytes was determined for a number of FGF19
variants. As indicated in FIG. 3-FIG. 5, several variants (e.g.,
M1, M2, etc.) exhibited strong cyp7a1 repression.
[0430] To evaluate effects of some additional FGF19 variants on
Cyp7a1 repression, the in vitro cell-based assay (primary human
hepatocyte) and the in vivo assay (protein dosing in db/db mice)
were utilized in which the variants were compared with
saline-treated controls. FIG. 5 sets forth the results (IC.sub.50
and Cyp7a1(%)) in tabular form. While most FGF19 variants that were
evaluated exhibited Cyp7a1-inhibiting activity, a few variants
(e.g., M90, M96, M98, M5 and M32) no longer repressed Cyp7a1.
[0431] FGF19 variants that retain Cyp7a1 repression activity can be
further evaluated in the HCC assay (or other relevant assay or
model) described above to identify variants that might be useful
for modulating bile acid metabolism and/or for treating bile
acid-related diseases (e.g., bile acid diarrhea and primary biliary
cirrhosis) without causing induction of HCC. The figures set forth
data for variants that were evaluated in the HCC assay.
5.4 Example 4
[0432] 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.
[0433] The Tables summarize 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-00045 TABLE 2 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 Eleva- tion HCC Forma- tion FGF19 RPLAFSDAGPHVHYGWGDPI
99 (aa 1-20) RLRHLYTSG 185 + + FGF21 HPIPDSSPLLQ--FGGQV 100 (aa
1-16) RQRYLYTDD 186 - - M5 R-HPIPDSSPLLQ--FGGQV 5 (aa 1-17)
RLRHLYTSG 185 - - M74 R-----------DAGPHVHYGWGDPI 74 (aa 1-10)
RLRHLYTSG 185 + + M75 R---------------VHYGWGDPI 75 (aa 1-10)
RLRHLYTSG 185 - - M76 R--------------------GDPI 76 (aa 1-5)
RLRHLYTSG 185 - - M77 R------------------------ 77 (aa 1) RLRHLYTSG
185 - - M78 R-----------AGPHVHYGWGDPI 78 (aa 1-14) RLRHLYTSG 185 +
+ M79 R------------GPHVHYGWGDPI 79 (aa 1-13) RLRHLYTSG 185 + + M80
R-------------PHVHYGWGDPI 80 (aa 1-12) RLRHLYTSG 185 - - M81
R--------------HVHYGWGDPI 81 (aa 1-11) RLRHLYTSG 185 - -
TABLE-US-00046 TABLE 3 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 Forma- tion 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-00047 TABLE 4 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/H1432A(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-00048 M88 (H31A/S141A): (SEQ ID NO: 88)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGELPLAHFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M89
(H31A/H142A): (SEQ ID NO: 89)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGELPLSAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M90
(K127A/R129A): (SEQ ID NO: 90)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGELPLSHFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M91
(K127A/S141A): (SEQ ID NO: 91)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGELPLAHFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M92
(K127A/H142A): (SEQ ID NO: 92)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGELPLSAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M93
(R129A/S141A): (SEQ ID NO: 93)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQAQLYKN
RGELPLAHFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M94
(S141A/H142A): (SEQ ID NO: 94)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKN
RGELPLAAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M95
(K127A/H142A): (SEQ ID NO: 95)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKN
RGELPLSAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M96
(K127A/R129A/S141A): (SEQ ID NO: 96)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGELPLAHFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M97
(K127A/R129A/H142A): (SEQ ID NO: 97)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGELPLSAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK M98
(K127A/R129A/S141A/H142A): (SEQ ID NO: 98)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKN
RGELPLAAFLPMLPMVPEEPEDLRGHLESDMESSPLETDSMDPFGLVTGLEAVRSPSFEK.
5.5 Example 5
[0434] The following is a data summary of additional FGF19 variant
peptides analyzed for glucose lowering activity and lipid elevating
activity.
[0435] Table 5 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-00049 TABLE 5 Additional Variants and Fine Mapping of the
N-terminal Domain (see SEQ ID NOs: 99, 100, and 5 to 40) SEQ ID NO
SEQ of N-term- 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 - + M11
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--FGIYQV 32 (aa 1-18) RLRHLYTSG 185 - - 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-00050 TABLE 5a (see SEQ ID NOs: 99, 100, 5, 9, 8, 12, 10,
13, 15, 14, 43, 6 and 7) ##STR00004## SEQ ID NO. Glucose Lowering
Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG
99 (aa 129) + + + 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-00051 TABLE 5b (see SEQ ID NOs: 99, 5 and 31 to 40)
##STR00005## 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-00052 TABLE 5c (see SEQ ID NOs: 99, 100, 5, 52, 54, to 68,
4, 69, 70 and 53) ##STR00006## 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 50 (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) + + -
[0436] Table 6 illustrates the peptide sequences of additional
variants.
TABLE-US-00053 TABLE 6 Additional Variants (SEQ ID NOs: 41, 42 and
44-46) M41: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR
GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF
EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEP
PGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 41) M42:
HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSA
HSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEI
RPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGIL
APQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 42) M44:
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAAD
QSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFR
ELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAL
PEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 44) M45:
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPE
SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLL
EDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPMVP
EEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 45) M46:
RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAAD
QSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFR
ELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAL
PEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASPMVPEEPEDLRGHLE
SDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 46)
[0437] Table 7 illustrates the peptide sequences of 3 FGF19
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-00054 TABLE 7 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).
5.6 Example 6
[0438] 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.
[0439] Mice were injected with FGF19 or FGF21 in AAV vector. Body
weight was recorded 4 weeks after injection.
TABLE-US-00055 TABLE 8 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-00056 TABLE 9 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
5.7 Example 7
[0440] The following is a study showing that variant M5 and variant
M69 peptides reduce blood glucose.
[0441] 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. The results of
variant M5 and variant M69 showed similar glucose lowering effects
as wild type FGF19 (data not shown).
5.8 Example 8
[0442] This example sets forth several variant polypeptides and
particular characteristics thereof, including the variants' effect
on glucose lowering, lipid profile parameters, and HCC
formation.
[0443] In particular, Table 10 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-00057 TABLE 10 ##STR00009## Core Sheet- 8/Loop8/Sheet-9
region Glucose Lower- ing Body Weight Re- duction HDL Eleva- tion
Tri- glyceride Elevation HCC Formation 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 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:
5) SEQ ID NO: 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: 6) SEQ ID NO: 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: 5) SEQ ID
NO: 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:
6) SEQ ID NO: 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: 50) SEQ ID NO: 50)
[0444] As the data in Table 10 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.
5.9 Example 9
[0445] 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.
[0446] The data in Table 10 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-00058 (SEQ ID NO: 193)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139); (SEQ ID NO: 194)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140); (SEQ ID NO: 195)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141); and (SEQ ID NO:
196) RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARG
QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEE
EILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160).
[0447] Only the following sequence domains for each of the
aforementioned variants are listed in Table 10: 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-00059 TABLE 11 ##STR00010## Core Glucose Lower- ing Body
Weight Re- duction HCL Ele- vation Tri- glyceride Elevation HCC
Forma- tion 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 SEQ ID NO: 100) NO: 100) M3
RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + + + + +/- (aa 1-20
of (aa 21-29 of (aa 102-112 of SEQ SEQ ID NO: 3) SEQ ID NO: 3) ID
NO: 3) M139 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILPDGYNVY// + - + +
+ (aa 1-20 of (aa 21-29 of (aa 102-112 of SEQ ID NO193) 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 SEQ ID NO: 194) SEQ ID ID NO: 194) NO: 194) M141
RPLAFSDAGHVHYGWGDPI 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)
[0448] Referring to Table 11, 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).
[0449] 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-00060 M1 (SEQ ID NO: 1)
RPLAFSDASPHVHYGWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M2 (SEQ ID NO: 2)
RPLAFSDSSPLVHYGWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV
ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS
SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA
VRSPSFEK M3 (SEQ ID NO: 3)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK M5 (SEQ ID NO: 5)
REIPIPDSSPLLQFGGQVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M5-R (SEQ ID NO: 160)
HPIPDSSPLLQFGGQVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
M48 (SEQ ID NO: 48)
RDSSPLLQFGGQVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M49 (SEQ ID NO: 49)
RPLAFSDSSPLLQFGGQVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL
RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA
KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR
SPSFEK M50 (SEQ ID NO: 50)
REIPIPDSSPLLQFGDQVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M51 (SEQ ID NO: 51)
REIPIPDSSPLLQFGGNVRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M52 (SEQ ID NO: 52)
RDSSPLLQWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI
KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M53 (SEQ ID NO: 192)
MDSSPLLQWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVA
IKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQ
LYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K
M69 (SEQ ID NO: 69)
RDSSPLVHYGWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT
VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQ
RQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK
M70 (SEQ ID NO: 70)
MRDSSPLVHYGWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR
TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK
QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS
PSFEK M71 (SEQ ID NO: 71)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLEIFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M72 (SEQ
ID NO: 72)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLEIFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M73 (SEQ
ID NO: 73)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGV
IQILGVKTSRFLCQRPDGALYGSLEIFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPH
RDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPE
NCKTLLTDIDRTHTEKPVWDGITGE M75 (SEQ ID NO: 75)
RVHYGWGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKG
VHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLY
KNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M76
(SEQ ID NO: 76)
RGDPIRLREILYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVR
YLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFL
PLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK FGF19 (SEQ
ID NO: 99)
RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA
VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL
SSAKQRQLYKNRGFLPLSEIFLPMLPMVPEEPEDLRGEILESDMFSSPLETDSMDPFGLVTGLE
AVRSPSFEK.
5.10 Example 10
[0450] This example shows that administration of M70 in human
patients results in suppression of 7a-hydroxy-4-cholsten-3-one
(C4), a marker of bile acid synthesis.
[0451] Study Subjects:
[0452] Healthy adults in the age range 18-65 years and with normal
body weight (body mass index, BMI 20-35) were enrolled in the
study. The study protocol was approved by the Human Research Ethics
Committee in Australia, and written informed consent was obtained
from each subject. For inclusion in the study each subject had to
be in good health determined by no clinically significant findings
from medical history, physical exam, 12 lead ECG, clinical
laboratory findings, and vital signs at screening. Subjects with
history or clinical manifestation of any significant metabolic,
allergic, dermatological, hepatic, renal, hematological, pulmonary,
cardiovascular, GI, neurological, or psychiatric disorder were
excluded from enrollment.
[0453] Study Design:
[0454] The study was a randomized, double-blind, placebo-controlled
design.
[0455] Prescreening of subjects was performed 7-30 days prior to
entry, and baseline evaluations were performed before treatment.
Each subject was given subcutaneous injection of M70 at doses 3
mg/day in a single bolus dose daily for 7 days. Blood samples were
collected into heparinized tubes through an indwelling catheter.
Blood samples taken on Day 1 and Day 7 at 4.5 hrs or 24 hrs after
administration of M70 or placebo were analyzed. Serum levels of
7a-hydroxy-4-cholesten-3-one (C4) were used to monitor CYP7A1
enzymatic activity (bile acid synthesis). They were analyzed from
individual serum samples after sample extraction followed by
high-pressure liquid chromatography (HPLC) as described previously
(Galman et al. (2003) J Lipid Res. 2003; 44(4):859-66).
[0456] Results:
[0457] The data provided in FIG. 6 show that on days 1 and 7, at
both 4.5 hours and 24 hours post-dose, serum levels of C4 were
significantly suppressed in the patients, as compared to patients
receiving a placebo.
5.11 Example 11
[0458] This example shows activation of mouse FGFR4-.beta.-klotho
signaling by FGF19, M3, and M70 in a rat myoblast cell line
[0459] Methods:
[0460] An ELK luciferase assay was performed in L6 cells
transiently transfected with mouse FGFR4, b-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.
[0461] 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.
[0462] Following transfection, concentration response of
ligand-dependent luciferase expression was analyzed in whole-cell
lysates in the presence of luciferin substrate.
[0463] Results:
[0464] 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 12 and FIG. 7).
TABLE-US-00061 TABLE 12 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
[0465] 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.
5.12 Example 12
[0466] This example shows that M70 selectively activates signaling
through the KLB/FGFR4 receptor complex in a manner that
beneficially does not cause HCC in mice, as shown in two different
models of oncogenic potential.
[0467] Study Subjects:
[0468] An FDA-accepted model of accelerated tumorigenesis, known as
the rasH2 transgenic model, as well as the db/db animal model.
[0469] Study Design and Results:
[0470] M70 expressed at exposures roughly 1,000 times greater than
normal levels of FGF19 in human blood did not cause HCC after
exposure for one year. By contrast, human FGF19, utilized as a
positive control in the mouse experiment, did cause HCC.
[0471] Co-administration of M70 and FGF19 via gene delivery in the
db/db animal model obviated the expected FGF19-driven HCC,
suggesting that M70 blocked the ability of FGF19 to occupy the
relevant receptor and signal in such a way as to cause HCC.
5.13 Example 13
[0472] This example discusses the results of a Phase 1 randomized,
double blind, placebo controlled, single ascending dose (SAD) and
multiple ascending dose (MAD) study to evaluate the safety,
tolerability and pharmacokinetics of M70 in healthy adult
participants. An overview of the study is provided in Table 13.
TABLE-US-00062 TABLE 13 Phase 1 Study Design to Evaluate the
Safety, Tolerability and Pharmacokinetics of M70 in Healthy Adult
Participants. Study Population Primary Outcome Measure Primary
Outcome Results Selected Secondary Outcomes Healthy subjects Safety
and tolerability No safety or tolerability PK supports qd dosing
signals identified Statistically significant No serious adverse
events reduction in C4 at all doses reported tested (0.3, 1 and
Majority of adverse 3 mg) vs. pre-dose levels events were mild
(MAD); p<0.001 Statistically significant reduction in
triglycerides with doses >1 mg (MAD); p<0.05 Statistically
significant increase in total cholesterol (MAD); p<0.05
[0473] As shown in FIG. 8, the Phase 1 trial with M70 demonstrated
a favorable safety profile with signs of biological activity
consistent with FGF19-like activity related to FGFR1c and FGFR4
signaling, supports its application in NASH and bile acid related
disorders (BARDs).
[0474] Study Design:
[0475] In this blinded, placebo-controlled, Phase 1 trial,
overweight or obese but otherwise healthy adults were randomized to
receive M70 or placebo as a daily subcutaneous injection in
escalating doses.
[0476] Results:
[0477] As shown in FIG. 8, a rapid and dose-proportional reduction
of serum C4 concentrations indicated that M70 has a statistically
significant effect on bile acid synthesis at the 0.3 mg, 1 mg and 3
mg doses. A mean reduction of approximately 94% in serum C4
concentrations was noted after the sixth dose at 3 mg when compared
with pre-dose levels. This rapid reduction in C4 supports the
potential biological activity of M70 as an inhibitor of bile acid
synthesis through CYP7a1. Two outlier data points are not shown in
FIG. 8, but were included in the statistic analysis (placebo, Day
7: 45.1 ng/ml; 0.3 mg NGM282 at baseline: 62.1 ng/ml).
[0478] Laboratory analysis of blood samples collected from subjects
receiving M70 in the Phase 1 MAD trial showed administration of the
drug was associated with statistically significant reductions in
triglycerides at doses of 1 mg and greater (p<0.05) and a
statistically significant decrease in total cholesterol (p<0.05)
(data not shown).
[0479] In both the SAD and MAD trials, M70 was well tolerated and
exhibited linear pharmacokinetics with no immunogenicity. There
were no serious adverse events. The most frequently observed
adverse events were diarrhea, vomiting and injection site
reactions. Also, there were no clinically significant laboratory
abnormalities documented in M70-treated subjects, as determined by
the Safety Data Monitoring Committee for the study, and there were
no anti-drug antibodies, or ADAs, observed.
5.14 Example 14
[0480] This example discusses the results of preclinical testing,
which supports the role of M70 for the treatment of NASH.
[0481] Normally the liver contains some fat. However, if more than
5-10% of the liver's weight is fat, it is referred to as a fatty
liver, or steatosis. The spectrum of NAFLD ranges from simple
steatosis to NASH, which can ultimately progress to end-stage liver
disease.
[0482] Bile acid synthesis and serum bile acid levels are
correlated with NAFLD and progression of disease to NASH, as
evidenced by elevations of CYP7a1 and increased serum bile acid
levels observed in NAFLD and NASH patients, respectively.
Accordingly, by reducing triglycerides and blocking bile acid
synthesis through the CYP7a1 pathway, M70 can disrupt the cascade
that leads from NAFLD to NASH, and through fibrosis and cirrhosis
to either transplant or death.
[0483] Study Design:
[0484] A mouse model of NASH, known as STAM.TM. was used to study
the beneficial effect of M70 in treatment of NASH. This model is
characterized by steatosis, lobular inflammation and hepatocyte
ballooning consistent with NASH pathology in humans. Mice in which
M70 was continuously expressed had statistically significant
decreases in total body weight, liver weight and liver-to-body
weight ratio reflective of a decrease in total liver fat content
(p<0.001 relative to control).
[0485] Results:
[0486] M70 expression demonstrated statistically significant
improvements in all components of the NAFLD Activity Score (NAS),
resulting in a total NAS score of 1.5 compared to 5.33 for control,
as shown in the chart below. The NAS is a histological feature
scoring system that is widely used to grade the activity of fatty
liver disease and the total score represents the sum of the scores
for steatosis, lobular inflammation and ballooning. Generally, a
score of 5 or greater is considered to be diagnostic of NASH. These
results are summarized below.
TABLE-US-00063 TABLE 14 Treatment Effect on NAS by M70 Treatment
Effect on NAS NAS M70 Control M70 vs. NAS Component Score (n = 6)
(n = 6) Control Steatosis 0 6 1 P = 0.0117 1 0 4 2 0 1 3 0 0
Lobular Inflammation 0 3 0 P = 0.0041 1 2 0 2 1 4 3 0 2 Hepatocyte
Ballooning 0 1 0 P = 0.0009 1 5 0 2 0 6 Total NAS (mean .+-. SD)
1.5 .+-. 1.0 5.33 .+-. 1.5 P = 0.0005
[0487] Additional preclinical work in a mouse bile duct ligation
model of liver fibrosis has demonstrated that expression of M70
effectively prevents mice from developing hepatic fibrosis, as
indicated by histology as well as gene expression analysis of
several markers of fibrosis and inflammation. These preclinical
data, combined with the Phase 1 MAD study data, further supports
the role for M70 providing benefits in patients with NASH.
5.15 Example 15
[0488] This example shows the role of M70 in the treatment of
cholestatic liver disease and other BARDs.
[0489] Cholestatic liver disease is a form of BARD defined as an
impairment of bile flow from the liver and is often characterized
by fatigue, pruritus and, in its more advanced form, jaundice.
Elevated serum bile acids are a hallmark of many cholestatic liver
diseases including PSC, PBC, intrahepatic cholestasis of pregnancy,
alcoholic hepatitis and drug-induced cholestasis. Impairment of
bile acid flow from the liver leads to cholestasis, hepatocellular
injury and progressive liver disease that may ultimately result in
liver failure.
[0490] Bile acids are believed to play a role in causing pruritus,
and elevated serum levels of certain forms of bile acid have been
correlated to higher rates of pruritus. Severe pruritus, which
patients often describe as intense, constant, unrelievable itching
under the skin at any place on the body, may present at all stages
of cholestatic liver disease and is the most debilitating symptom
afflicting cholestatic disease patients. Patients often resort to
destructive scratching behaviors that can cause bleeding and
scarring, and the condition can lead to a marked decrease in
quality of life, impaired sleep, depression and, potentially,
suicidal thoughts or actions. Caregivers also suffer from impaired
sleep and anxiety as they struggle to help manage this debilitating
symptom. In some patients, the emotional and physical effects of
pruritus alone can justify liver transplantation.
[0491] The potent bile acid regulation effect of M70, and the fact
that it is not a derivative of a bile acid, support its role as a
treatment for certain cholestatic liver diseases such as PSC, PBC
and other BARDs. A large body of in vivo preclinical data testing
the efficacy of M70 in a bile duct ligation (BDL) model, an
alpha-naphthylisothiocyanate (ANIT) model, and an Mdr2 knockout
model showed statistically significant reduction of serum bile acid
(p<0.001) and improvements in biochemical markers of liver
damage. In addition, as described above in Example 13, the Phase 1
data demonstrated that M70 administration statistically significant
reductions in serum C4 levels (p<0.001), indicating biological
activity consistent with FGF19 suppression of CYP7a1 in the liver
and reduction in serum bile acid levels. In a Phase 2a trial in
PBC, subjects demonstrated statistically significant reductions in
ALP, GGT, ALT and AST (p<0.05) without generally exacerbating
pruritus. All these observations support the view that M70 offers a
safe and effective, non-invasive pharmacological approach to reduce
serum bile acid and decrease the damaging effects of high bile acid
levels in the liver and the debilitating pruritus often associated
with cholestatic liver diseases. Accordingly, these results support
that M70 can be effective in treating liver cholestatic diseases,
such as PSC and other orphan BARDs.
5.16 Example 16
[0492] This example shows that M70 improved liver function in
preclinical studies.
[0493] M70 potently represses in vitro CYP7a1 expression in primary
human hepatocytes, or liver cells, and in vivo CYP7a1 expression in
mice. In addition, an average reduction of 81% in serum C4
concentrations was observed in cynomolgus monkeys treated for six
days with FGF19 (1 mg/kg subcutaneous daily injection) relative to
control. Furthermore, preclinical studies using two in vivo models
of cholestasis showed that inhibiting de novo bile acid synthesis
through the CYP7a1 pathway with M70 showed statistically
significant improvements in biochemical markers of liver function
in mice.
[0494] Study Design and Results:
[0495] The first model, bile duct ligation (BDL), uses a surgical
method to transect the common bile duct and prevent bile flow out
of the liver and induce a state of cholestasis. Mice that were
subjected to BDL and received M70 showed a statistically
significant reduction of serum bile acids (p<0.001) and
improvements in biochemical markers of liver damage, such as
alkaline phosphatase (ALP), alkaline aminotransferase (ALT),
aspartate aminotransfease (AST) and gamma-glutamyltransferase
(GGT), following BDL surgery.
[0496] The results shown in FIG. 9 compare the results from the
control group (the mice were subjected to BDL, but did not receive
any treatment), a group receiving M70, a group receiving INT-747
(Intercept Pharmaceuticals, Inc.; an FXR agonist ligand and novel
bile acid analogue shown to be efficacious in treating humans with
PBC in Phase 3 studies), and a group receiving bezafibrate (a drug
that is not approved for the treatment of PBC but is nevertheless
sometimes prescribed off-label by some physicians).
[0497] Notably, as shown in FIG. 9, M70 reduced circulating bile
acid levels and improved liver function in the BDL animal
model.
[0498] Mice treated with ANIT, represent an animal model in which
the cholestatic state is pharmacologically induced by chemical
treatment that leads to damage of the cells that line the bile
ducts.
[0499] As shown in FIG. 10, ANIT-treated mice showed a
statistically significant elevation in serum bile acids (p<0.01)
and an impaired liver function, similar to profiles of human
patients suffering from cholestatic disease. As with the BDL model,
inhibiting de novo bile acid synthesis through the classical
pathway with M70 resulted in statistically significant improvements
in biochemical markers of liver function (p<0.001) in the ANIT
model.
[0500] The Mdr2 knockout mouse model of chronic cholestasis and
liver inflammation resembles many aspects of human PSC. In a study
in which M70 was continuously expressed in Mdr2 knockout mice for
24 weeks, reduced serum levels of total bile acid, normalized liver
enzymes such as ALP, ALT and AST, and reduced liver weight was
observed.
[0501] Overall, these data further confirmed that M70 is a
non-tumorigenic FGF19 variant that can effectively treat PSC and
PBC and other manifestations of BARDs. Results from a range of
nonclinical safety studies indicated that M70 is safe and
well-tolerated and support the dosing range and duration of
treatment in clinical trials. Thus, M70 can be a safe and effective
pharmacological approach to reducing serum bile acids and
decreasing the damaging effects of high bile acid levels in the
liver and, potentially, the debilitating pruritus often associated
with cholestatic liver diseases.
5.17 Example 17
[0502] This example describes a Phase 2, randomized, double blind,
placebo controlled, parallel group, multiple center study to
evaluate the safety, tolerability and pharmacodynamic activity of
M70 in combination with ursodeoxycholic acid (UDCA) administered
for 28 days in patients with PBC, and shows the role of M70 in
treating human patients.
[0503] PBC is a chronic cholestatic liver disease in which the bile
ducts become inflamed and are slowly destroyed by an apparent
autoimmune reaction, driving bile acid build-up in the liver and,
eventually, leading to irreversible scarring. Although a large
proportion of patients are asymptomatic at diagnosis, common
symptoms of pruritus and fatigue can develop as the disease
progresses. The one approved treatment in the United States, UDCA
has been shown to slow disease progression in some patients, but
only approximately one-third of patients with PBC fully respond to
treatment.
[0504] Study design and results: A Phase 2a PBC trial was designed
to investigate the effects of M70 in combination with UDCA after 28
days of treatment, compared to control. Eligible subjects were
randomized to control or one of two treatment groups, including a
high dose (3.0 mg) or a low dose (0.3 mg) of M70.
TABLE-US-00064 TABLE 15 Clinical Trial Design on PBC treatment by
M70 Primary Primary Selected Study Outcome Outcome Secondary Safety
and Population Measure Results Outcomes Tolerability PBC subjects
on Change in ALP Statistically Liver enzymes: No statistically UDCA
for at least (absolute significant ALP statistically significant 12
months with an international units per reductions with significant
evidence of incomplete liter, or IU/L, %) both doses reduction in
drug-induced response from baseline at 0.3 mg: -49 IU/L ALT, AST
and pruritus Day 28 (-15.8%) GGT at both Majority of 3 mg: -69 IU/L
dose levels vs. adverse events (-19.2%) placebo (p<0.05) were
mild or Serum C4: moderate reduction One serious observed with 3
adverse event mg dose reported (not Cholesterol: no drug related)
statistically significant change
[0505] All subjects completed the 28-day treatment phase of the
study and were eligible to participate in a 52-week extension
trial, also referred to as the Phase 2b trial in PBC subjects. The
Phase 2a trial achieved statistical significance in the primary
endpoint (change in ALP from baseline, as noted below) at both
doses. There were improvements in a number of secondary endpoints
(change in the biochemical markers, ALT, AST, GGT, C4, fasting
serum bile levels and pruritus and fatigue), including: (1)
statistically significant percentage reduction in ALP from baseline
to Day 28 with both M70 doses (0.3 mg=-15.8%, p-value=0.009; 3.0
mg=-19.2%, p-value=0.003); (2) marked reductions in other markers
of liver injury, including ALT (0.3 mg=-17.5 IU/L; 3.0 mg=-26.7
IU/L), AST (0.3 mg=-10.9 IU/L; 3.0 mg=-15.3 IU/L) and GGT (0.3
mg=-28.2 IU/L; 3.0 mg=-50.9 IU/L); (3) no statistically significant
change in pruritus in either M70 treatment arm; and (4) acceptable
safety and tolerability profile with no drug-related safety
signals. Most adverse events were mild, with a single serious
adverse event that was deemed not related to treatment. The most
frequent adverse events were mild headache and mild lower GI
symptoms. The lower GI symptoms were observed in 21% of the 0.3 mg
and 43% of the 3 mg cohorts, compared to 13% of the control group.
Mild injection site reactions were also observed more frequently
with M70.
[0506] The Phase 2b trial was designed as a 52-week extension to
enable subjects from the Phase 2a 28-day PBC study to get access to
M70 for an extended period and thus allow collection of data on the
longer term safety profile and disease impact of M70. An analysis
of available data was performed for those subjects that
transitioned from the Phase 2a study and reached 12 weeks of
treatment. A reduction of ALP from baseline was observed in all
groups at that time point, with the lowest dose (0.3 mg) cohort
achieving a statistically significant reduction (p=0.004). In the
Phase 2b PBC trial, M70 has thus far exhibited a safety and
tolerability profile consistent with that seen in the Phase 2a PBC
trial.
[0507] While UDCA is the only treatment approved for PBC in the
United States, there are several treatments in development.
INT-747, an FXR agonist ligand and novel bile acid analogue, is one
such treatment. FXR is a nuclear receptor involved in regulating
the expression of numerous genes, including the gene that produces
the FGF19 hormone. Although INT-747's Phase 3 trial demonstrated a
statistically significant effect on ALP reduction (p<0.0001),
the drug nearly doubled the rate of pruritus in PBC subjects as
compared to control (68% and 38% at the 10 mg and placebo doses,
respectively), perhaps as a consequence of introducing a bile acid
analog into the livers of subjects suffering from excessive bile
acid accumulation.
5.18 Example 18
[0508] This example discussed the role of M70 for the treatment of
primary sclerosing cholangitis (PSC). PSC is a chronic cholestatic
liver disease, characterized by progressive inflammation, fibrosis
and obstruction of the bile ducts leading to cholestasis and, in
most cases, liver failure and an increased risk of liver cancer.
Though cholestatic symptoms will eventually present, patients can
remain asymptomatic and undiagnosed for several years. The median
life expectancy after diagnosis with PSC is 12 to 18 years without
liver transplantation and, even in the case of liver transplanted
patients, PSC returns in 30% to 50% of patients within ten years.
PSC is often associated with ulcerative colitis and also appears to
have overlap with other forms of autoimmune disease, including
autoimmune hepatitis and autoimmune pancreatitis. The patient
population is estimated to be between 50,000 and 132,000 in the
United States and Europe, with a 2:1 incidence in men versus women
and a particularly high incidence in northern Europe. There are no
approved therapeutics for the treatment of PSC, but liver
transplantation is the most frequent treatment approach in
end-stage PSC, making it the fifth leading indication for liver
transplant in the United States. Many PSC patients suffer from the
same pruritus symptoms of PBC and for which there are currently no
drug treatments available.
[0509] The bile acid synthesis-inhibiting properties of M70 can
help slow the progression of PSC by reducing the pool of bile acid
in the obstructed bile ducts and thereby lessening the impact on
liver fibrosis.
[0510] Study design: The study of M70 in PSC subjects will explore
the activity of the compound in approximately 60 subjects in a
12-week, randomized, placebo-controlled, double-blind, multi-center
trial. The subjects will be confirmed PSC patients as assessed by
elevated ALP and cholangiography or liver biopsy with no evidence
of cirrhosis or advanced liver disease. The primary endpoint will
be change in ALP from baseline at 12 weeks of treatment. The study
is designed to investigate the effects of M70 on changes from
baseline in other biochemical markers associated with PSC, such as
ALT, GGT and bilirubin, serum bile acid, C4, pruritus and
inflammatory bowel disease symptoms, following daily dosing over 12
weeks.
5.19 Example 19
[0511] This example describes a Phase 2 randomized, double blind,
placebo controlled, parallel group, multiple center study to
evaluate the safety, tolerability and activity of M70 administered
for 28 days to participants with type 2 diabetes
[0512] Study Design:
[0513] A four-week, randomized, double-blind, multi-center trial
was conducted to evaluate M70 in subjects with type 2 diabetes. As
a consequence of the contribution of obesity and insulin resistance
to both conditions, there is a substantial overlap in the
prevalence of type 2 diabetes and NASH patients.
[0514] The type 2 diabetes trial was also designed to measure
several of the metabolic parameters that are believed to play a
role in the disease progression of NAFLD and NASH, including
indicators of insulin sensitivity, triglyceride levels and liver
enzyme levels. Three doses of M70 were tested in subjects with type
2 diabetes inadequately controlled by metformin to assess changes
from baseline in biochemical markers associated with type 2
diabetes, such as fasting plasma glucose and stimulated
glucose/insulin.
[0515] The primary endpoint measured by this trial was the change
in fasting plasma glucose after 28 days of treatment. Although this
endpoint was not different in the M70 subjects as compared to the
control arm, there were trends towards improvement in insulin
sensitivity, as measured by HOMA-IR and a statistically significant
weight loss observed in the 10 mg group, which lost an average of
2.6 kilograms over the 28 days of treatment (p<0.041). Moreover,
there was a statistically significant reduction in triglycerides
with the 2 mg (p=0.009) and 10 mg (p=0.007) doses and
dose-dependent reductions in ALT, or alanine transaminase, and AST,
or aspartate transaminase, consistent with improvements in liver
health. The trial further established that M70 improves both
metabolic and liver health in a patient population that closely
resembles NASH patients.
TABLE-US-00065 TABLE 16 Clinical Trial Design on Type 2 Diabetes
treatment by M70 Primary Outcome Primary Outcome Selected Secondary
Safety and Study Population Measure Results Outcomes Tolerability
Type 2 diabetes subjects Change in fasting No statistically
HOMA-IR: No serious inadequately controlled plasma glucose (FPG)
significant reduction statistically adverse events by metformin
from baseline at in FPG significant reported Day 28 reduction at
Majority of 10 mg dose adverse events (p = 0.001) were mild or Body
weight: moderate statistically significant reduction at 10 mg, dose
(p = 0.019) Serum triglycerides: statistically significant
reduction at 2 and 10 mg doses (p = 0.009 and p = 0.007,
respectively) Liver enzymes: reduction in ALT and AST Cholesterol:
statistically significant increase (p<0.05)
[0516] Overall, M70 was well tolerated at each dose. There were no
serious adverse events reported. These preclinical and clinical
data suggest that M70 offers a potentially novel approach in the
treatment of NASH by reducing body weight and triglyceride levels
and improving insulin sensitivity to combat the metabolic drivers
of the disease, while also reducing bile acid synthesis to combat
the liver damage caused by pooling of toxic bile acid.
6. SEQUENCE LISTING
[0517] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing in ASCII text
format submitted via EFS-Web. The CRF copy of the Sequence Listing,
entitled 13370-114-999_SEQLIST.txt, which was created on Jun. 4,
2020 and is 255,629 bytes in size, 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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