U.S. patent application number 13/680986 was filed with the patent office on 2013-06-06 for chimeric polypeptides and uses thereof.
This patent application is currently assigned to AMGEN INC.. The applicant listed for this patent is AMGEN INC.. Invention is credited to Yang Li, Zhulun Wang, Xinle Wu.
Application Number | 20130143796 13/680986 |
Document ID | / |
Family ID | 42541288 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130143796 |
Kind Code |
A1 |
Li; Yang ; et al. |
June 6, 2013 |
CHIMERIC POLYPEPTIDES AND USES THEREOF
Abstract
The disclosure provides nucleic acid molecules encoding chimeric
polypeptides, chimeric polypeptides, pharmaceutical compositions
comprising chimeric polypeptides, and methods for treating
metabolic disorders such as diabetes and obesity using such nucleic
acids, polypeptides, or pharmaceutical compositions.
Inventors: |
Li; Yang; (Mountain View,
CA) ; Wu; Xinle; (Belmont, CA) ; Wang;
Zhulun; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMGEN INC.; |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Family ID: |
42541288 |
Appl. No.: |
13/680986 |
Filed: |
November 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12817084 |
Jun 16, 2010 |
8324160 |
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13680986 |
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61265548 |
Dec 1, 2009 |
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61187767 |
Jun 17, 2009 |
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Current U.S.
Class: |
514/4.8 ;
435/252.33; 435/320.1; 514/6.9; 514/9.1; 530/387.3; 530/387.9;
530/399; 536/23.5 |
Current CPC
Class: |
A61P 3/04 20180101; C07K
14/50 20130101; C07K 16/22 20130101; C07K 2319/00 20130101; A61P
3/10 20180101 |
Class at
Publication: |
514/4.8 ;
435/252.33; 435/320.1; 514/6.9; 514/9.1; 530/387.3; 530/387.9;
530/399; 536/23.5 |
International
Class: |
C07K 14/50 20060101
C07K014/50 |
Claims
1. A chimeric polypeptide comprising a wild type mature FGF19
polypeptide scaffold comprising SEQ ID NO:4, further comprising a
modification that decreases FGFR4-mediated signaling activity.
2. The chimeric polypeptide of claim 1, wherein the modification
comprises substituting one or more of the residues WGDPI at
positions 16-20 of the FGF19 polypeptide scaffold with (a) no amino
acid; or (b) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence.
3. The chimeric polypeptide of claim 2, wherein the tryptophan
residue of the WGDPI sequence is deleted.
4. The chimeric polypeptide of claim 2, wherein the residues WGDPI
are substituted with 1-5 contiguous residues present in a wild type
FGF21 or a wild type FGF23 amino acid sequence.
5. The chimeric polypeptide of claim 4, wherein the 1-5 contiguous
residues are present in a wild type FGF21 amino acid sequence.
6. The chimeric polypeptide of claim 5, wherein the 1-5 contiguous
residues are GQV.
7. The chimeric polypeptide of claim 1, wherein the modification
comprises substituting one or more of the residues SGPHGLSS at
positions 28-35 of the FGF19 polypeptide scaffold with (a) no amino
acid; or (b) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence.
8. The chimeric polypeptide of claim 7, wherein the residues
SGPHGLSS are substituted with 1-8 contiguous residues present in a
wild type FGF21 or a wild type FGF23 amino acid sequence.
9. The chimeric polypeptide of claim 8, wherein the 1-8 contiguous
residues are present in a wild type FGF21 amino acid sequence.
10. The chimeric polypeptide of claim 9, wherein the 1-8 contiguous
residues are DDAQQTE.
11. The chimeric polypeptide of claim 1, wherein the modification
comprises substituting one or more of the residues
SSAKQRQLYKNRGFLPL at positions 124-140 of the FGF19 polypeptide
scaffold with (a) no amino acid; or (b) an amino acid other than
the amino acid located at the position in the wild type amino acid
sequence.
12. The chimeric polypeptide of claim 11, wherein the residues
SSAKQRQLYKNRGFLPL are substituted with 1-17 contiguous residues
present in either a wild type FGF21 or a wild type FGF23 amino acid
sequence.
13. The chimeric polypeptide of claim 12, wherein the 1-17
contiguous residues are present in a wild type FGF21 amino acid
sequence.
14. The chimeric polypeptide of claim 13, wherein the 1-17
contiguous residues are PGNKSPHRDPAPRGP.
15. A chimeric polypeptide that exhibits decreased FGFR4-mediated
signaling activity comprising a wild type FGF19 polypeptide
scaffold comprising SEQ ID NO:4, wherein one or more of the
residues WGDPI at positions 16-20 of SEQ ID NO:4 has been
substituted with (a) no amino acid; or (b) an amino acid other than
the amino acid located at the position in the wild type amino acid
sequence; and one or both of: (i) one or more of the residues
SGPHGLSS at positions 28-35 of SEQ ID NO:4 has been substituted
with (1) no amino acid; or (2) an amino acid other than the amino
acid located at the position in the wild type amino acid sequence;
and (ii) one or more of the residues SSAKQRQLYKNRGFLPL at positions
124-140 of SEQ ID NO:4 has been substituted with (1) no amino acid;
or (2) an amino acid other than the amino acid located at the
position in the wild type amino acid.
16. The chimeric polypeptide of claim 15, wherein the tryptophan
residue of the WGDPI sequence is deleted.
17. The chimeric polypeptide of claim 15, wherein the residues
WGDPI are substituted with 1-5 contiguous residues present in a
wild type FGF21 or a wild type FGF23 amino acid sequence.
18. The chimeric polypeptide of claim 17, wherein the 1-5
contiguous residues are present in wild type FGF21 amino acid
sequence.
19. The chimeric polypeptide of claim 17, wherein the 1-5
contiguous residues are GQV.
20. The chimeric polypeptide of claim 15, wherein the residues
SGPHGLSS are substituted with 1-8 contiguous residues present in a
wild type FGF21 or a wild type FGF23 amino acid sequence.
21. The chimeric polypeptide of claim 20, wherein the 1-8
contiguous residues are present in a wild type FGF21 amino acid
sequence.
22. The chimeric polypeptide of claim 21, wherein the 1-8
contiguous residues are DDAQQTE.
23. The chimeric polypeptide of claim 15, wherein the residues
SSAKQRQLYKNRGFLPL are substituted with 1-17 contiguous residues
present in a wild type FGF21 or wild type FGF23 amino acid
sequence.
24. The chimeric polypeptide of claim 23, wherein the 1-17
contiguous residues are present in a wild type FGF21 amino acid
sequence.
25. The chimeric polypeptide of claim 24, wherein the 1-17
contiguous residues are PGNKSPHRDPAPRGP.
26. The chimeric polypeptide of claim 15, wherein the residues
WGDPI at positions 16-20 of SEQ ID NO:4 are substituted with GQV;
and one or both of: (a) the residues SGPHGLSS at positions 28-35 of
SEQ ID NO:4 are substituted with DDAQQTE; and (b) the residues
SSAKQRQLYKNRGFLPL at positions 124-140 of SEQ ID NO:4 are
substituted with PGNKSPHRDPAPRGP.
27. A nucleic acid molecule encoding the chimeric polypeptide of
claim 1 or 15.
28. A vector comprising the nucleic acid molecule of claim 27.
29. A host cell comprising the nucleic acid molecule of claim
27.
30. A pharmaceutical composition comprising the chimeric
polypeptide of claim 1 or 15 and a pharmaceutically acceptable
carrier.
31. A method of treating a metabolic disease selected from the
group consisting of diabetes and obesity comprising administering
to a human patient in need thereof the pharmaceutical composition
of claim 30.
32. An antigen binding protein that specifically binds to a
chimeric polypeptide of claim 1 or 15.
33. A chimeric fusion polypeptide comprising the chimeric
polypeptide of claim 1 or 15 fused to a heterogenous moiety.
34. The chimeric fusion polypeptide of claim 33, wherein the
heterogenous moiety is selected from the group consisting of an Fc
region of an IgG molecule and a PEG molecule.
35. The chimeric polypeptide of claim 1 or 15, wherein SEQ ID NO:4
is truncated on the N terminus by 1-15 amino acids, the C terminus
by 1-15 amino acids, or on both the N terminus by 1-15 amino acids
and the C terminus by 1-15 amino acids.
36. The chimeric polypeptide of claim 1 or 15, which, except for
the modification that decreases FGFR4-mediated signaling activity,
comprises a polypeptide scaffold that is 95% or more identical to
SEQ ID NO:4.
Description
[0001] This application claims the benefit of U.S. Non-Provisional
application Ser. No. 12/817,084, filed Jun. 16, 2010 and U.S.
Provisional Appln. No. 61/265,548 filed Dec. 1, 2009 and U.S.
Provisional Appln. No. 61/187,767 filed Jun. 17, 2009, all of which
are incorporated by reference herein.
REFERENCE TO THE SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled A-1498-US-DIV_Sequence_listing_ST25.txt, created May
25, 2010 which is 121 KB in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to nucleic acid molecules encoding
chimeric polypeptides, chimeric polypeptides, pharmaceutical
compositions comprising chimeric polypeptides and methods for
treating a variety of metabolic disorders using such nucleic acids,
polypeptides or pharmaceutical compositions.
BACKGROUND OF THE INVENTION
[0004] FGF19, FGF21, and FGF23 form a unique subfamily of
fibroblast growth factors (FGFs). Unlike other FGFs, all three have
been shown to function as endocrine hormones in the regulation of
various metabolic processes (Fukumoto, (2008). Endocr. J.
55:23-31). For example, FGF23 originates in bone and regulates
phosphate homeostasis in kidney (Fukumoto & Yamashita, (2007)
Bone 40:1190-1195), FGF21 is expressed predominantly in liver but
can signal in adipose tissue (Ogawa et al., (2007) Proc. Natl.
Acad. Sci. U.S.A. 104:7432-7437), and FGF19 is secreted from ileum
and functions as an enterohepatic signal for the regulation of bile
acid metabolism (Inagaki et al. (2005) Cell. Metab. 2:217-225).
[0005] FGF19 and FGF21 appear to share many similarities with
reported effects on the regulation of glucose, lipid, and energy
metabolism. Both FGF19 and FGF21 transgenic mice are resistant to
diet induced obesity, have lower body fat mass, and improved
insulin sensitivity, glucose disposal, and plasma lipid parameters
(Tomlinson et al., (2002) Endocrinology 143:1741-1747; Fu et al.,
(2004) Endocrinology 145:2594-2603; Kharitonenkov et al. (2005) J
Clin Invest 115:1627-1635; Xu et al., (2008) Diabetes 58:250-59).
Injection of recombinant FGF19 or FGF21 proteins in diabetic mouse
models resulted in the reduction of serum glucose and insulin
levels, improvements in glucose tolerance and liver steatosis, and
reduction in body weight (Kharitonenkov et al., (2005) J. Clin.
Invest. 115:1627-1635; Xu et al. (2008) Diabetes 58:250-59). In
addition, FGF21 has also been shown to improve glucose, insulin and
lipid profiles with reduced body weight in diabetic rhesus monkeys
(Kharitonenkov et al., (2007) Endocrinology 148:774-781). Taken
together, these observations signify the potential utility of these
molecules as novel therapies for the treatment of diabetes and
obesity.
[0006] Although this subfamily displays unique features as compared
to other FGF molecules (Kurosu & Kuro-o, (2008) Curr. Opin.
Nephrol. Hypertens. 17:368-372 (2008); Wu et al., (2008) J. Biol.
Chem. 283(48):33304-9), FGF19, hepatocellular carcinoma (HCC)
formation was observed in transgenic mice overexpressing FGF19 in
skeletal muscle (Nicholes et al., (2002) Am. J. Pathol.
160:2295-2307). This has been a consideration in developing FGF19
as a therapy for diabetes, obesity and other metabolic
disorders.
[0007] A chimeric polypeptide that exhibits potential for
therapeutic use, while at the same time does not exhibit
undesirable properties, such as mitogenicity, that would compromise
the use of the polypeptide as a therapeutic, is therefore
desirable.
SUMMARY OF THE INVENTION
[0008] A chimeric polypeptide comprising a wild type mature FGF19
polypeptide scaffold comprising SEQ ID NO:4, further comprising a
modification that decreases FGFR4-mediated signaling activity is
provided.
[0009] In one embodiment, one or more of the residues WGDPI at
positions 16-20 of the FGF19 polypeptide scaffold has been
substituted with (a) no amino acid; or (b) an amino acid other than
the amino acid located at the position in the wild type amino acid
sequence. The tryptophan residue of the WGDPI sequence can be
deleted. Additionally, the residues WGDPI can be substituted with
1-5 contiguous residues present in either a wild type FGF21 or a
wild type FGF23 amino acid sequence. The 1-5 contiguous residues
can be present in a wild type FGF21 amino acid sequence, for
example the 1-5 contiguous residues are GQV.
[0010] In a further embodiment one or more of the residues SGPHGLSS
at positions 28-35 of the FGF19 polypeptide scaffold has been
substituted with either (a) no amino acid; or (b) an amino acid
other than the amino acid located at the position in the wild type
amino acid sequence. The residues SGPHGLSS can be substituted with
1-8 contiguous residues present in either a wild type FGF21 or a
wild type FGF23 amino acid sequence. The 1-8 contiguous residues
can be present in a wild type FGF21 amino acid sequence, for
example the 1-8 contiguous residues can be DDAQQTE.
[0011] In another embodiment one or more of the residues
SSAKQRQLYKNRGFLPL at positions 124-140 of the FGF19 polypeptide
scaffold can be been substituted with either (a) no amino acid; or
(b) an amino acid other than the amino acid located at the position
in the wild type amino acid sequence. The residues
SSAKQRQLYKNRGFLPL can be substituted with 1-17 contiguous residues
present in either a wild type FGF21 or a wild type FGF23 amino acid
sequence. When the 1-17 contiguous residues are present in a wild
type FGF21 amino acid sequence the 1-17 contiguous residues can be
PGNKSPHRDPAPRGP.
[0012] Also provided is a chimeric polypeptide comprising a wild
type FGF19 polypeptide scaffold comprising SEQ ID NO:4, wherein one
or more of the residues WGDPI at positions 16-20 of SEQ ID NO:4 has
been substituted with either (a) no amino acid; or (b) an amino
acid other than the amino acid located at the position in the wild
type amino acid sequence, and one or both of: (i) one or more of
the residues SGPHGLSS at positions 28-35 of SEQ ID NO:4 has been
substituted with either (1) no amino acid; or (2) an amino acid
other than the amino acid located at the position in the wild type
amino acid sequence; and (ii) one or more of the residues
SSAKQRQLYKNRGFLPL at positions 124-140 of SEQ ID NO:4 has been
substituted with either (1) no amino acid; or (2) an amino acid
other than the amino acid located at the position in the wild type
amino acid.
[0013] The residues WGDPI can be substituted with 1-5 contiguous
residues present in either FGF21 or FGF23. The 1-5 contiguous
residues can be present in wild type FGF21 amino acid sequence. The
1-5 contiguous residues can be GQV. Further, the tryptophan residue
of the WGDPI sequence can be deleted.
[0014] The residues SGPHGLSS can be substituted with 1-8 contiguous
residues present in either a wild type FGF21 or a wild type FGF23
amino acid sequence. The 1-8 contiguous residues can be present in
a wild type FGF21 amino acid sequence. The 1-8 contiguous residues
are DDAQQTE.
[0015] The residues SSAKQRQLYKNRGFLPL can be substituted with 1-17
contiguous residues present in either a wild type FGF21 or wild
type FGF23 amino acid sequence. The 1-17 contiguous residues can be
present in a wild type FGF21 amino acid sequence. The 1-17
contiguous residues can be PGNKSPHRDPAPRGP.
[0016] In one particular embodiment, the residues WGDPI at
positions 16-20 of SEQ ID NO:4 are substituted with GQV, and one or
both of: (a) the residues SGPHGLSS at positions 28-35 of SEQ ID
NO:4 are substituted with DDAQQTE; and (b) the residues
SSAKQRQLYKNRGFLPL at positions 124-140 of SEQ ID NO:4 are
substituted with PGNKSPHRDPAPRGP.
[0017] In other embodiments of a chimeric polypeptide provided
herein, the polypeptide scaffold of SEQ ID NO:4 is truncated on the
N terminus by 1-5 amino acids, on the C terminus by 1-15 amino
acids or on both N terminus by 1-15 amino acids and on the C
terminus by 1-15 amino acids. In yet another embodiment of a
chimeric polypeptide except for the modification that decreases
FGFR4-mediated signaling activity, the chimeric polypeptide
comprises a polypeptide scaffold that is 95% or more identical to
SEQ ID NO:4.
[0018] Nucleic acid molecules encoding the chimeric polypeptides
are also disclosed, as well as vectors and host cells comprising
the nucleic acid molecules.
[0019] Pharmaceutical compositions comprising the chimeric
polypeptides and a pharmaceutically acceptable carrier are also
disclosed. In another aspect, methods of treating diabetes and
obesity comprising administering to a human patient in need thereof
such pharmaceutical compositions are also disclosed.
[0020] Antibodies that specifically binds to the disclosed chimeric
polypeptides are also disclosed. A kit for detecting the presence
of the disclosed chimeric polypeptides comprising such antibodies
are also disclosed.
[0021] Chimeric fusion polypeptides comprising the chimeric
polypeptides fused to a heterogenous moiety, such as a Fc region of
an IgG molecule or a PEG molecule are also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows the incorporation of BrdU by FGF19 and FGF21;
FIG. 1A shows liver sections from FGF19 and FGF21 treated animals
and FIG. 1B is a bar graph depicting BrdU label incorporation in
each test group.
[0023] FIG. 2 is a series of Western blots depicting FGF21 or
FGF19-mediated activation of FGFR1c (FIG. 2A), FGFR2c (FIG. 2B),
FGFR3c (FIG. 2C), and FGFR4 (FIG. 2D).
[0024] FIG. 3 depicts the structure and activity a C-terminally
truncated form of FGF19; FIG. 3A is a diagram graphically depicting
the structure of the C-terminally truncated form of FGF19; FIG. 3B
is a series of Western blots depicting FGF19 or truncated
FGF19-mediated activation of FGFR4 or FGFR1c; FIG. 3C is a bar
graph depicting BrdU incorporation by FGF19 or the truncated form
of FGF19.
[0025] FIG. 4 depicts the structure of several FGF19/FGF21 chimeric
polypeptides and the effect of each on FGFR4-mediated activity and
BrdU incorporation; FIG. 4A is a graphical depiction of the FGF
19/21-1, FGF 19/21-2, FGF 19/21-3, FGF 19/21-4 and FGF19/21-5
chimeric proteins, with FGF19 shown in white and FGF21 shown as
gray; FIG. 4B is a series of Western blots showing the effect of
the FGF19/21-1, FGF19/21-2, FGF 19/21-3, FGF 19/21-4 and FGF
19/21-5 chimeric polypeptides on FGFR1c (top panel) and FGFR4
(lower panel) mediated activity; FIG. 4C is a plot showing the
effect of the FGF 19/21-1, FGF 19/21-2, FGF 19/21-3, FGF 19/21-4
and FGF 19/21-5 chimeric polypeptides on glucose uptake; FIG. 4D is
a bar graph depicting incorporation of BrdU by the FGF19/21-1,
FGF19/21-2, FGF19/21-3, FGF19/21-4 and FGF19/21-5 chimeric
polypeptides.
[0026] FIG. 5 depicts the structure and activity of the
FGF21/19.sup.38-42 chimeric polypeptide in which residues 42-44 of
FGF21 were replaced by residues 38-42 of FGF19; FIG. 5A is a
graphical depiction of the chimeric polypeptide with FGF21 shown in
gray and FGF19 in white; FIG. 5B is a series of Western blots
showing FGFR1c and FGFR4-mediated activity of the chimeric
FGF21/19.sup.38-42 polypeptide; FIG. 5C is a bar graph depicting
incorporation of BrdU by the chimeric FGF21/19.sup.38-42
polypeptide.
[0027] FIG. 6 depicts the structure and activity of two chimeric
polypeptides, the FGF21/19.sup.38-42 chimeric polypeptide in which
either residues 42-44 of FGF21 were replaced by residues 38-42 of
FGF19, and the FGF19/21.sup.42-44 chimeric polypeptide in which
residues 38-42 of FGF19 were replaced with residues 42-44 of FGF21;
FIG. 6A is a graphical depiction of the chimeric polypeptides and
indicates FGFR4 activity and BrdU incorporation; FIG. 6B depicts
regions of FGF19 (SEQ ID NO:47) and FGF21 (SEQ ID NO:48),
subsequences of which (SEQ ID NO:49 for FGF19) were exchanged in
two chimeric polypeptides, FGF21/19.sup.38-42 and
FGF19/21.sup.42-44, and analogous regions of FGF23 (SEQ ID NO:50)
are also shown; FIG. 6C is a series of Western blots showing FGFR1c
and FGFR4-mediated activity of the FGF21/19.sup.38-42 and
FGF19/21.sup.42-44chimeric polypeptides; FIG. 6D is a series of
plots showing the results of a solid-phase binding assay measuring
the interaction between FGFR4 and FGF21/19 .sup.38-42 or
FGF19/21.sup.42-44 in the presence and absence of heparin; FIG. 6E
is bar graph depicting the results of a semiquantitative analysis
of BrdU immunostaining of livers from female FVB mice treated for 6
days with PBS, 2 mg/kg/day recombinant FGF19, FGF21/19.sup.38-42 or
FGF 19/21.sup.42-44.
[0028] FIG. 7 depicts the .beta.1-.beta.2 loop region (SEQ ID
NO:51) and the loop itself (underlined, SEQ ID NO:52), and
.beta.10-.beta.12 segment region (SEQ ID NO:57) and the segment
itself (underlined, SEQ ID NO:58) in FGF19 and analogous sequences
in FGF21 (.beta.1-.beta.2 loop region, SEQ ID NO:53, loop
underlined, SEQ ID NO:54; .beta.10-.beta.12 segment region, SEQ ID
NO:57, segment underlined, SEQ ID NO:60) and FGF23 (.beta.1-.beta.2
loop region SEQ ID NO:55, loop underlined SEQ ID NO:56;
.beta.10-.beta.12 segment region, SEQ ID NO:61, segment underlined,
SEQ ID NO:62).
[0029] FIG. 8 depicts the structure and activity of the three
chimeric polypeptides, FGF19-1, FGF19-2 and FGF19-3, in which
residues 50-57 of FGF19 were replaced with residues 52-58 of FGF21
(FGF19-1), residues 146-162 of FGF19 were replaced with residues
147-161 of FGF21 (FGF-2), or residues 50-57 of FGF19 were replaced
with residues 52-58 of FGF21 and residues 146-162 of FGF19 were
replaced with residues 147-161 of FGF21 (FGF19-3); FIG. 8A is a
graphical depiction of the three chimeric polypeptides FGF19-1,
FGF19-2 and FGF19-3; FIG. 8B is a series of plots showing the
glucose lowering effect of the FGF19-1, FGF19-2 and FGF19-3
constructs in the presence and absence of heparin; FIG. 8C is a
series of plots showing the glucose lowering effect of the FGF19-1,
FGF19-2 and FGF19-3 constructs in the presence and absence of
.beta.Klotho; FIG. 8D is a series of Western blots showing FGFR1c
and FGFR4-mediated activity of the FGF19-1, FGF19-2 and FGF19-3
chimeric polypeptides in the presence and absence of .beta.Klotho;
FIG. 8E is a bar graph showing BrdU incorporation mediated by FGF19
and FGF19-1.
[0030] FIG. 9 depicts the structure and activity of the three
chimeric polypeptides FGF19-4, FGF19-5 and FGF19-6 in which
residues 38-42 of FGF19 were replaced with residues 42-44 of FGF21
and residues 50-57 of FGF19 were replaced with residues 52-58 of
FGF21 (FGF19-4), residues 38-42 of FGF19 were replaced with
residues 42-44 of FGF21 and residues 146-162 of FGF19 were replaced
with residues 147-161 of FGF21 (FGF19-5), and residues 38-42 of
FGF19 were replaced with residues 42-44 of FGF21, residues 50-57 of
FGF19 were replaced with residues 52-58 of FGF21, and residues
146-162 of FGF19 were replaced with residues 147-161 of FGF21
(FGF19-6); FIG. 9A is a graphical depiction of the three chimeric
polypeptides FGF19-4, FGF19-5 and FGF19-6; FIG. 9B is a series of
Western blots showing FGFR1c and FGFR4-mediated activity of the
FGF19-4, FGF19-5 and FGF19-6 chimeric polypeptides in the presence
and absence of .beta.Klotho; FIG. 9C is a bar graph showing BrdU
incorporation mediated by FGF19 and FGF19-4, FGF19-5 and
FGF19-6.
[0031] FIG. 10 is a series of plots depicting the results of
several assays performed on FGF19, FGF19-1 and FGF19-4; FIG. 10A
depicts the effect of FGF19 and the FGF19-4 chimeric polypeptide on
glucose uptake in a 3T3L1 cell-based assay; FIG. 10B shows the
effect of FGF19 and FGF19-4 on plasma glucose in a ob/ob mouse
model; FIG. 10C depicts the effect of FGF19 and the FGF19-1
chimeric polypeptide on glucose uptate in a 3T3L1 cell-based assay;
FIG. 10D shows the effect of FGF19 and FGF19-1 on plasma glucose in
a ob/ob mouse model.
[0032] FIG. 11 is a plot depicting the pharmacokinetic properties
of various FGF19/21 chimeric proteins.
[0033] FIG. 12 is a table showing the binding response of various
FGF19 mutants having one or more mutations or deletions in the
WGDPI region to FGFR1c and FGFR4.
[0034] FIG. 13 is a series of bar graphs showing FGFR1c-induced
activity of several FGF19 mutants having one or more mutations or
deletions in the WGDPI region; each construct was tested at
concentrations of 0, 2.5, 16 and 100 nM.
[0035] FIG. 14 is a series of bar graphs showing FGFR1c-mediated
activity of several FGF19 mutants having one or more mutations or
deletions in the WGDPI region; each construct was tested at
concentrations of 0, 2.5, 7.4, 33, 67 and 200 nM.
[0036] FIG. 15 is a series of bar graphs showing FGFR4-mediated
activity of several FGF19 mutants having one or more mutations or
deletions in the WGDPI region; each construct was tested at
concentrations of 0, 2.5, 16 and 100 nM.
[0037] FIG. 16 is a series of bar graphs showing FGFR4-mediated
activity of several FGF19 mutants having one or more mutations or
deletions in the WGDPI region; each construct was tested at
concentrations of 0, 2.5, 7.4, 22, 67 and 200 nM.
DETAILED DESCRIPTION OF THE INVENTION
[0038] It has been suggested that constitutive hepatocellular
proliferation is a prerequisite for transformation (Fausto, (1999)
Seminars in Liver Disease 19:243-252). Accordingly, it is noted
that dramatic increases in the proliferation of pericentral
hepatocytes, as measured by enhanced BrdU labeling, was observed as
early as 2 to 4 months of age in FGF19 transgenic animals as well
as in normal mice subjected to six daily injections of recombinant
FGF19 (Nicholes et al., (2002) Am. J. Pathol. 160:2295-2307). Cell
lineage analysis of FGF19 induced tumors suggest that dysplastic
and neoplastic hepatocytes originated from around the central
veins, coincident with the increased pericentral proliferation
observed by BrdU labeling (Nicholes et al., (2002) Am. J. Pathol.
160:2295-2307). Thus the relatively shorter BrdU labeling assay
could serve as a marker to study mitogenic potential of these
molecules in vivo. FGF21 has been shown to lack potential for cell
proliferation in vitro (Kharitonenkov et al., (2005) J. Clin.
Invest. 115:1627-1635).
[0039] The receptors for the FGF19, FGF21, FGF23 subfamily have
been elucidated in recent years. Both FGF19 and FGF21 utilize
.beta.Klotho, a single transmembrane protein, as a co-receptor
required for signaling mediated through FGFRs 1c, 2c, and 3c
(Kurosu & Kuro-o, (2009) Mol. Cell. Endocrinol. 299:72-78).
Because FGFR1c and 2c are the predominant receptors expressed in
adipose tissue, induction of ERK phosphorylation and increased
glucose uptake in adipocytes in vitro and in vivo upon treatment
with either FGF19 or FGF21 are likely mediated through these
receptors complexed with .beta.Klotho in adipocytes (Kurosu et al.,
(2007) J. Biol. Chem. 282:26687-26695). Despite these similarities,
a major difference between FGF19 and FGF21 exists with respect to
FGFR4. Although both FGF19 and FGF21 appear to be able to bind to
.beta.Klotho/FGFR4 complexes, only FGF19 signals efficiently
through FGFR4 (Kurosu et al., (2007) J. Biol. Chem.
282:26687-26695). Consistent with these in vitro observations,
FGF19, but not FGF21, activates liver ERK phosphorylation, which is
likely mediated through FGFR4, the predominant receptor expressed
in liver (Kurosu et al., (2007) J. Biol. Chem. 282:26687-26695).
Additionally, it has been suggested that FGFR4 could contribute to
hepatocellular carcinoma progression, and increased production of
alpha-fetoprotein, a hepatocellular carcinoma biomarker, has been
observed with FGF19 stimulated liver cancer cell lines (Ho et al.,
(2009) J. Hepatol. 50:118-127).
[0040] As disclosed herein, a C-terminally truncated FGF19 and a
series of FGF19 and FGF21 chimeric proteins was prepared and it was
possible to identify three regions in FGF19 that are responsible
for FGFR4 activation. A correlation between FGFR4 activation and
hepatocellular proliferation, as indicated by BrdU incorporation,
is disclosed. These results provide a direct link between liver
FGFR4 activity and hepatocyte proliferation in vivo. Furthermore,
it is disclosed that in contrast to FGF19, FGF21 does not activate
FGFR4 and does not induce hepatocyte proliferation in vivo, making
the various forms of FGF19 disclosed herein a unique potential
therapeutic approach for the treatment metabolic diseases, such as
obesity, diabetes and dyslipidemia.
[0041] Recombinant nucleic acid methods used herein, including in
the Examples, are generally those set forth in Sambrook et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1989) or Current Protocols in Molecular Biology
(Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons
1994), all of which are incorporated herein by reference for any
purpose.
I. DEFINITIONS
[0042] As used herein, the terms "a" and "an" mean one or more
unless specifically indicated otherwise.
[0043] As used herein, the term "chimeric polypeptide" refers to a
polypeptide scaffold in which at least one amino acid of one or
more regions comprising between 1 to 20 contiguous amino acids has
been replaced with either no amino acid or an amino acid that is
not found at the replaced amino acid's position in the region in a
wild type polypeptide scaffold. In one example of a chimeric
polypeptide, residues 38-42 of an FGF19 polypeptide scaffold are
replaced with three residues from an FGF21 polypeptide, for example
GQV, which is found at positions 42-44 of FGF21 or three residues
from a FGF23 polypeptide, for example WGG which is found at
positions 36-38 of FGF23. In another example the tryptophan residue
at position 38 of a FGF19 polypeptide scaffold is replaced by an
amino acid other than a tryptophan.
[0044] As used herein, the term "isolated nucleic acid molecule"
refers to a nucleic acid molecule of the present disclosure that
(1) has been separated from at least about 50, 60, 70, 80, 90, 95
or more percent of proteins, lipids, carbohydrates, or other
materials with which it is naturally found when total nucleic acid
is isolated from the source cells, (2) is not linked to all or a
portion of a polynucleotide to which the "isolated nucleic acid
molecule" is linked in nature, (3) is operably linked to a
polynucleotide which it is not linked to in nature, or (4) does not
occur in nature as part of a larger polynucleotide sequence.
Preferably, the isolated nucleic acid molecule of the present
invention is substantially free from any other contaminating
nucleic acid molecules or other contaminants that are found in its
natural environment that would interfere with its use in
polypeptide production or its therapeutic, diagnostic, prophylactic
or research use.
[0045] The terms "polynucleotide" and "nucleic acid" are generally
used interchangeably herein and refer to a polymeric molecule
having a backbone that supports bases capable of hydrogen bonding
to typical polynucleotides, where the polymer backbone presents the
bases in a manner to permit such hydrogen bonding in a sequence
specific fashion between the polymeric molecule and a typical
polynucleotide (e.g., single-stranded DNA). Common bases include
inosine, adenosine, guanosine, cytosine, uracil and thymidine.
[0046] As used herein, the term "isolated polypeptide" refers to a
polypeptide of the present invention that (1) has been separated
from at least about 50, 60, 70, 80, 90, 95 or more percent of
polynucleotides, lipids, carbohydrates, or other materials with
which it is naturally found when isolated from the source cell, (2)
is not linked (by covalent or noncovalent interaction) to all or a
portion of a polypeptide to which the "isolated polypeptide" is
linked in nature, (3) is operably linked (by covalent or
noncovalent interaction) to a polypeptide with which it is not
linked in nature, or (4) does not occur in nature. Preferably, the
isolated polypeptide is substantially free from any other
contaminating polypeptides or other contaminants that are found in
its natural environment that would interfere with its therapeutic,
diagnostic, prophylactic or research use.
[0047] The terms "polypeptide" and "protein" are used
interchangeably and refer to a compound made up of a single chain
of amino acid residues linked by peptide bonds. A polypeptide or
protein can, but need not, comprise non-naturally occurring amino
acids and amino acid derivatives. A non-limiting lists of examples
of non-naturally occurring amino acids that can be inserted into a
protein or polypeptide (including chimeric polypeptides disclosed
herein) include .beta.-amino acids, homoamino acids, cyclic amino
acids and amino acids with derivatized side chains. Examples
include (in the L-form or D-form; abbreviated as in parentheses):
para-acetyl-phenylalanine, para-azido-phenylalanine,
para-bromo-phenylalanine, para-iodo-phenylalanine and
para-ethynyl-phenylalanine, citrulline (Cit), homocitrulline
(hCit), N.alpha.-methylcitrulline (NMeCit),
N.alpha.-methylhomocitrulline (N.alpha.-MeHoCit), ornithine (Orn),
N.alpha.-Methylornithine (N.alpha.-MeOrn or NMeOrn), sarcosine
(Sar), homolysine (hLys or hK), homoarginine (hArg or hR),
homoglutamine (hQ), N.alpha.-methylarginine (NMeR),
N.alpha.-methylleucine (N.alpha.-MeL or NMeL), N-methylhomolysine
(NMeHoK), N.alpha.-methylglutamine (NMeQ), norleucine (Nle),
norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),
Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine
(1-Nal), 3-(2-naphthyl)alanine (2-Nal),
1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI),
para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or
4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine
(abbreviated "K(N.epsilon.-glycyl)" or "K(glycyl)" or "K(gly)"),
nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or
Amino-Phe), benzylphenylalanine (benzylphe),
.gamma.-carboxyglutamic acid (.gamma.-carboxyglu), hydroxyproline
(hydroxypro), p-carboxyl-phenylalanine (Cpa), .alpha.-aminoadipic
acid (Aad), N.alpha.-methyl valine (NMeVal), N-.alpha.-methyl
leucine (NMeLeu), N.alpha.-methylnorleucine (NMeNle),
cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine
(acetylarg), .alpha.,.beta.-diaminopropionoic acid (Dpr),
.alpha.,.gamma.-diaminobutyric acid (Dab), diaminopropionic acid
(Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe),
.beta.,.beta.-diphenyl-alanine (BiPhA), aminobutyric acid (Abu),
4-phenyl-phenylalanine (or biphenylalanine; 4Bip),
.alpha.-amino-isobutyric acid (Aib), beta-alanine,
beta-aminopropionic acid, piperidinic acid, aminocaprioic acid,
aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic
acid, N-ethylglycine, N-ethylaspargine, hydroxylysine,
allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine,
N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp),
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other
similar amino acids, and derivatized forms of any of those
specifically listed.
[0048] As used herein, the term "vector" is used to refer to any
molecule (e.g., nucleic acid, plasmid, or virus) used to transfer
coding information to a host cell.
[0049] As used herein, the term "expression vector" refers to a
vector that is suitable for transformation of a host cell and
contains nucleic acid sequences that direct and/or control the
expression of inserted heterologous nucleic acid sequences.
Expression includes, but is not limited to, processes such as
transcription, translation, and RNA splicing, if introns are
present.
[0050] As used herein, the term "host cell" is used to refer to a
cell which has been transformed, or is capable of being transformed
with a nucleic acid sequence and then of expressing a selected gene
of interest. The term includes the progeny of the parent cell,
whether or not the progeny is identical in morphology or in genetic
make-up to the original parent, so long as the selected gene is
present.
[0051] As used herein, the term "naturally occurring" when used in
connection with biological materials such as nucleic acid
molecules, polypeptides, host cells, and the like, refers to
materials which are found in nature and are not manipulated by man.
Similarly, "non-naturally occurring" as used herein refers to a
material that is not found in nature or that has been structurally
modified or synthesized by man. When used in connection with
nucleotides, the term "naturally occurring" refers to the bases
adenine (A), cytosine (C), guanine (G), thymine (T), and uracil
(U). When used in connection with amino acids, the term "naturally
occurring" refers to the 20 amino acids alanine (A), cysteine (C),
aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine
(G), histidine (H), isoleucine (I), lysine (K), leucine (L),
methionine (M), asparagine (N), proline (P), glutamine (Q),
arginine (R), serine (S), threonine (T), valine (V), tryptophan
(W), and tyrosine (Y).
[0052] As used herein, the term "FGF19 polypeptide" refers to a
polypeptide expressed in any species, including humans. For
purposes of this disclosure, the term "FGF19 polypeptide" can be
used interchangeably to refer to any full-length FGF19 polypeptide,
e.g., SEQ ID NO:2, which consists of 216 amino acid residues and
which is encoded by the nucleotide sequence of SEQ ID NO: 1, and
any mature form of the polypeptide, e.g., SEQ ID NO:4, which
consists of 194 amino acid residues and which is encoded by the
nucleotide sequence of SEQ ID NO:3, and in which the 22 amino acid
residues at the amino-terminal end of the full-length FGF19
polypeptide (i.e., those residues which constitute the signal
peptide) have been removed. A bacterially expressed form of a
mature FGF19 polypeptide can be produced from the nucleotide of SEQ
ID NO:5 and have the amino acid sequence of SEQ ID NO:6, and which
will comprise an N-terminal methionine residue. A "FGF19
polypeptide" can be encoded by SEQ ID NOs:1, 3 and 5, for example,
as well as any polynucleotide sequence that, due to the degeneracy
of the genetic code, has a polynucleotide sequence that is altered
by one or more bases from the polynucleotide sequences of SEQ ID
NOs:1, 3 and 5, as well as allelic variants of SEQ ID NOs:1, 3 and
5. The term "FGF19 polypeptide" also encompasses
naturally-occurring FGF19 variants. A "FGF19" polypeptide can but
need not incorporate one or more non-naturally occurring amino
acids.
[0053] As used herein, the term "FGF21 polypeptide" refers to a
polypeptide expressed in any species, including humans. For
purposes of this disclosure, the term "FGF21 polypeptide" can be
used interchangeably to refer to any full-length FGF21 polypeptide,
e.g., SEQ ID NO:8, which consists of 209 amino acid residues and
which is encoded by the nucleotide sequence of SEQ ID NO:7; any
mature form of the polypeptide, e.g., SEQ ID NO:10, which consists
of 181 amino acid residues and which is encoded by the nucleotide
sequence of SEQ ID NO:9, and in which the 28 amino acid residues at
the amino-terminal end of the full-length FGF21 polypeptide (i.e.,
those residues which constitute the signal peptide) have been
removed. A bacterially expressed form of a mature FGF21 polypeptide
can be produced from the nucleotide of SEQ ID NO:11 and have the
amino acid sequence of SEQ ID NO:12 and will comprise an N-terminal
methionine residue. A "FGF21 polypeptide" can be encoded by SEQ ID
NOs:7, 9 and 11, for example, as well as any polynucleotide
sequence that, due to the degeneracy of the genetic code, has a
polynucleotide sequence that is altered by one or more bases from
the polynucleotide sequence of SEQ ID NOs:7, 9 and 11, as well as
allelic variants of SEQ ID NOs:7, 9 and 11. The term "FGF21
polypeptide" also encompasses naturally-occurring variants. A
"FGF21" polypeptide can but need not incorporate one or more
non-naturally occurring amino acids.
[0054] As used herein, the term "FGF23 polypeptide" refers to a
polypeptide expressed in any species, including humans. For
purposes of this disclosure, the term "FGF23 polypeptide" can be
used interchangeably to refer to any full-length FGF23 polypeptide,
e.g., SEQ ID NO:14, which consists of 251 amino acid residues and
which is encoded by the nucleotide sequence of SEQ ID NO:13; any
mature form of the polypeptide, e.g., SEQ ID NO:16, which consists
of 227 amino acid residues and which is encoded by the nucleotide
sequence of SEQ ID NO:15, and in which the 24 amino acid residues
at the amino-terminal end of the full-length FGF23 polypeptide
(i.e., those residues which constitute the signal peptide) have
been removed. A bacterially expressed form of a mature FGF23
polypeptide can be produced from the nucleotide of SEQ ID NO:18 and
have the amino acid sequence of SEQ ID NO:17, and will comprise an
N-terminal methionine residue. A "FGF23 polypeptide" can be encoded
by SEQ ID NOs: 13, 15 and 17, for example, as well as any
polynucleotide sequence that, due to the degeneracy of the genetic
code, has a polynucleotide sequence that is altered by one or more
bases from the polynucleotide sequence of SEQ ID NOs:13, 15 and 17,
as well as allelic variants of SEQ ID NOs:13, 15 and 17. The term
"FGF23 polypeptide" also encompasses naturally-occurring variants.
A "FGF23" polypeptide can but need not incorporate one or more
non-naturally occurring amino acids.
[0055] As used herein, the terms "effective amount" and
"therapeutically effective amount" each refer to the amount of a
chimeric polypeptide disclosed herein used to support an observable
level of one or more biological activities of the wild-type
polypeptide scaffold, such as the ability to lower blood glucose,
insulin, triglyceride, or cholesterol levels; reduce body weight;
or improve glucose tolerance, energy expenditure, or insulin
sensitivity in a human or non-human subject.
[0056] As used herein, the term "pharmaceutically acceptable
carrier" or "physiologically acceptable carrier" as used herein
refers to one or more formulation materials suitable for
accomplishing or enhancing the delivery of a chimeric polypeptide
disclosed herein.
[0057] As used herein, the terms "biological activity" and
"biologically active" when used in connection with a polypeptide
scaffold or a chimeric polypeptide of the instant disclosure mean
that the polypeptide scaffold or chimeric polypeptide possesses an
activity of the polypeptide scaffold, such as the ability to lower
blood glucose, insulin, triglyceride, or cholesterol; to reduce
body weight; or to improve glucose tolerance, energy expenditure,
or enhance insulin sensitivity when assayed in an appropriate assay
such as those provided herein, regardless of the type or number of
modifications that have been introduced into the chimeric
polypeptide. Chimeric polypeptides possessing a somewhat decreased
level of activity relative to the polypeptide scaffold can
nonetheless be considered to be biologically active chimeric
polypeptides. One particular example of a biological activity is
the ability to increase glucose uptake in 3T3L1 cells by 1.2 fold
or higher over basal levels in an in vitro glucose uptake assay as
shown in Example 10.
[0058] As used herein, the term "polypeptide scaffold" means a
polypeptide which has been modified to form a chimeric polypeptide
as described herein. Examples of polypeptide scaffolds that can
form the basis of a chimeric polypeptide include wild type FGF19,
FGF21 and FGF23 polypeptide sequences.
[0059] As used herein, the term "conservative amino acid
substitution" means a substitution of a native amino acid residue
(i.e., a residue found in a given position of a wild-type
polypeptide scaffold sequence) with a nonnative residue (i.e., a
residue that is not found in a given position of the wild type
polypeptide scaffold sequence) such that there is little or no
effect on the polarity or charge of the amino acid residue at that
position. Conservative amino acid substitutions also encompass
non-naturally occurring amino acid residues that are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics, and other
reversed or inverted forms of amino acid moieties.
[0060] Naturally occurring residues can be divided into classes
based on common side chain properties:
[0061] (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0062] (2) neutral hydrophilic: Cys, Ser, Thr;
[0063] (3) acidic: Asp, Glu;
[0064] (4) basic: Asn, Gln, His, Lys, Arg;
[0065] (5) residues that influence chain orientation: Gly, Pro;
and
[0066] (6) aromatic: Trp, Tyr, Phe.
[0067] Conservative substitutions can involve the exchange of a
member of one of these classes for another member of the same
class. Non-conservative substitutions can involve the exchange of a
member of one of these classes for a member from another class.
[0068] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. An exemplary (but not
limiting) list of amino acid substitutions is set forth in Table
1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Residue
Exemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln
Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln,
Lys, Arg Ile Leu, Val, Met, Ala, Phe Leu Ile, Val, Met, Ala, Phe
Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro
Ala Ser Thr, Ala, Cys Thr Ser Tip Tyr, Phe Tyr Trp, Phe, Thr, Ser
Val Ile, Met, Leu, Phe, Ala
II. CHIMERIC POLYPEPTIDES
[0069] In one aspect of the present disclosure, a series of
chimeric polypeptides are described. These chimeric polypeptides
are based on a wild type FGF19, FGF21 or FGF23 polypeptide scaffold
wherein one or more of the residues in a contiguous region of 1-20
amino acids of the polypeptide scaffold, e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids,
have been replaced with (a) no amino acid (a deletion); or (b) an
amino acid other than the amino acid located at the position in the
wild type sequence (i.e., a substitution or a mutation).
[0070] In addition to having a modulating effect on particular
biological activities of a polypeptide scaffold, the chimeric
polypeptides described herein can have other retained or enhanced
biological activities of the polypeptide scaffold, such as the
ability to lower blood glucose in vivo. For example, the chimeric
polypeptides disclosed herein can exhibit an enhanced or decreased
degree of FGFR4 activation and yet still retain any glucose
lowering effects inherent to the polypeptide scaffold, possibly
making such a molecule more therapeutically attractive than the
unmodified polypeptide scaffold. In the case of a FGF19 polypeptide
scaffold, for example, a chimeric polypeptide built on the FGF19
scaffold can have decreased ability to activate FGFR4 and/or
decreased hepatocyte mitogenicity, while at the same time still
showing the glucose lowering effects inherent to FGF19. In another
example, a chimeric polypeptide built on a FGF21 scaffold can have
an increased ability to activate FGFR4 and/or enhanced hepatocyte
mitogenicity, while at the same time showing the glucose lowering
effects inherent in FGF21. Thus, in some embodiments the chimeric
polypeptides disclosed herein, comprise molecules in which
properties deemed desirable in a given circumstance have been
maintained or augmented, while properties deemed undesirable in the
same circumstance have been decreased or eliminated.
[0071] In any of the disclosed chimeric polypeptides, a
substitution can comprise any naturally or non-naturally occurring
amino acid. Such a substitution can be a conservative substitution,
as described herein, or non-conservative. In some cases, in which
it is desired to disrupt a particular effect it may be desirable to
make a non-conservative substitution at a given position or insert
a non-naturally occurring amino acid. In other cases, a
conservative substitution may mitigate or enhance a particular
effect to a desired degree. In still other cases, a substitution of
no amino acid at one or more positions (a deletion) may mitigate or
enhance a particular effect to a desired degree.
[0072] Over the span of a contiguous region of a polypeptide
scaffold in which two or more amino acid residues are being
replaced with amino acids or no amino acid, any combination can be
employed without restriction. That is, a two or more amino acids in
a contiguous region can be replaced with no amino acid, a naturally
occurring amino acid, a non-naturally occurring amino acid, a
conservative substitution, a non-conservative substitution or any
combination thereof. In fact, in some cases an omission at a given
position in a region of a polypeptide scaffold coupled with a
conservative and/or non-conservative substitution at one or more
other positions in the same region of the scaffold may mitigate or
enhance a particular effect to a desired degree, allowing a level
of control over the activity of a chimeric polypeptide.
[0073] The present disclosure, therefore, encompasses not only
chimeric polypeptides that fully impart a desired effect or
property, but also that that partially impart a desired effect or
property. By way of example, the present disclosure encompasses
both chimeric polypeptides that completely eliminate an effect such
as the mitogenicity or FGFR4 activation normally associated with a
FGF19 polypeptide scaffold, and chimeric polypeptides that
partially eliminate the same effects, which can be desirable in
some circumstances. Also encompassed are chimeric polypeptides that
enhance an effect such as mitogenicity or FGFR4 activation, which
can be desirable in some circumstances.
[0074] The biological activity of the chimeric polypeptides
disclosed herein can be assayed in any assay appropriate to the
metric desired. For example, a binding assay such as an in vitro or
in vivo ERK or FRS2 assay can be employed to examine FGFR activity,
target gene expression analysis can be performed in vitro or in
vivo, glucose uptake can be studied in adipocytes, and glucose
lowering ability, as shown herein in Examples 2.3 and 10, effects
on body weight, plasma lipid profiles, energy expenditures, can be
studied in other in vitro or in vivo functional assays. Various in
vivo assays can also be employed to study the biological activity
of polypeptide scaffolds and chimeric polypeptides, including
histopathological analysis to examine BrdU incorporation in the
livers of test animals. This assay is demonstrated in Example 2.4
and exemplary results are shown in FIG. 1A.
[0075] II.A. Chimeric Polypeptides That Do Not Signal Through
FGFR4
[0076] The present disclosure relates to chimeric polypeptides that
do not signal through FGFR4. In one embodiment, an isolated
chimeric polypeptide comprises the amino acid sequence of a wild
type FGF19 (e.g., SEQ ID NOs:2, 4 or 6) polypeptide scaffold, which
has been modified such that it does not signal through FGFR4. As
shown in the Examples presented herein, three regions or a subset
thereof, are sufficient for FGFR4 signaling in a FGF19 polypeptide
scaffold, namely residues 38-42, 50-57 and 146-162 of SEQ ID NO:2
(residues 16-20, 28-35 and 124-140 of SEQ ID NO:4 and residues
17-21, 29-36 and 125-141 of SEQ ID NO:6). Thus, a chimeric
polypeptide based on a FGF19 polypeptide scaffold that signals
through FGFR1c but not FGFR4 will have at least one of these
regions modified. In one example, at least one of these regions of
the FGF19 polypeptide scaffold can be modified by replacing at
least one amino acid in the region with either no amino acid or an
amino acid not found at the position in the region of the scaffold.
Thus, in this example at least one residue of residues 38-42 of the
FG19 scaffold will be replaced with either no amino acid or an
amino acid not found at the position in the scaffold. In one
particular embodiment, the tryptophan residue at position 38 is
mutated to a residue other than tryptophan or is deleted (see
Example 12).
[0077] Similarly, if it is desired to impart the ability to signal
through FGFR4 but not FGFR1c, residues 38-42, 50-57 and 146-162 of
SEQ ID NO:2 (residues 16-20, 28-35 and 124-140 of SEQ ID NO:4 and
residues 17-21, 29-36 and 125-141 of SEQ ID NO:6) can be used to
replace the analogous regions of a non-FGF19 polypeptide scaffold,
such as a FGF21 or FGF23 polypeptide scaffold.
[0078] It is noted that although the disclosed regions of FGF19 are
sufficient for FGFR4-mediated signaling, additional regions of
FGF19 may also contribute to FGFR4-mediated signaling. The present
disclosure, therefore, contemplates that additional residues and/or
regions of a scaffold polypeptide may play a role in FGFR4-mediated
signaling, in conjunction with the disclosed regions. Accordingly,
chimeric polypeptides comprising substitutions and/or deletions at
those regions form an embodiment of the present disclosure.
[0079] II.B. Chimeric Polypeptides Lacking or Incorporating FGF19
Residues 38-42
[0080] In one aspect, the present disclosure relates to chimeric
polypeptides that are based on an FGF21 polypeptide scaffold. In
one embodiment, such a chimeric polypeptide comprises the amino
acid sequence of a wild type FGF21 (e.g., SEQ ID NOs:8, 10 and 12)
polypeptide scaffold, wherein one or more of the residues of GQV at
positions 42-44 of SEQ ID NO:8 (positions 14-16 of SEQ ID NO:10 and
15-17 of SEQ ID NO:12) has been replaced with (a) no amino acid (a
deletion); or (b) an amino acid other than the amino acid located
at the position in the wild type sequence. In another example, the
residues at positions 42-44 of a wild type FGF21 polypeptide (i.e.,
GQV) are replaced by residues at position 38-42 of a wild type
FGF19 sequence (i.e., WGDPI) (SEQ ID NO:49). In this particular
chimeric polypeptide, it is noted that three residues in a region
of a FGF21 polypeptide scaffold are replaced by five residues from
a FGF19 polypeptide, highlighting the option of replacing a
particular residue at a particular position in a polypeptide
scaffold with more than one amino acid, i.e., replacing three amino
acids in a region with five amino acids.
[0081] The present disclosure also relates to chimeric polypeptides
that are based on a FGF19 polypeptide scaffold. In one embodiment,
such a chimeric polypeptide comprises the amino acid sequence of a
wild type FGF19 (SEQ ID NOs:2, 4 or 6) polypeptide scaffold,
wherein one or more of the residues of WGDPI (SEQ ID NO:49) at
positions 38-42 of SEQ ID NO:2 (positions 16-20 of SEQ ID NO:4 and
17-21 of SEQ ID NO:6) has been replaced with (a) no amino acid (a
deletion); or (b) an amino acid other than the amino acid located
at the position in the wild type sequence. In another example, the
residues of FGF21 at positions 42-44 of SEQ ID NO:8 (positions
14-16 of SEQ ID NO:10 and 15-17 of SEQ ID NO:12), i.e., the
residues GQV, are inserted at position 38-42 of SEQ ID NO:2
(positions 16-20 of SEQ ID NO:4 and 17-21 of SEQ ID NO:6). In this
particular chimeric polypeptide, it is noted that five residues of
a FGF19 polypeptide scaffold are being replaced by three residues
from a FGF21 polypeptide, highlighting the option of replacing a
particular residue at a particular position in a polypeptide
scaffold with no amino acid (i.e., making a deletion). Such
chimeric polypeptides can exhibit the properties of reduced FGF4
activation and/or hepatocyte mitogenicity.
[0082] In another embodiment, a chimeric polypeptide comprises the
amino acid sequence of a wild type FGF23 (SEQ ID NOs:14, 16 or 18)
polypeptide scaffold, wherein one or more of the residues of WGG at
positions 36-38 of SEQ ID NO:14 (positions 12-14 in SEQ ID NO:16
and 13-15 in SEQ ID NO:18) has been replaced with at (a) no amino
acid (a deletion); or (b) an amino acid other than the amino acid
located at the position in the wild type sequence. In another
example, the residues of FGF23 at positions 36-38 of SEQ ID NO:14
(positions 12-14 of SEQ ID NO:16 and 13-15 in SEQ ID NO:18), e.g.,
the residues WGG, are replaced by the FGF19 residues at position
38-42 of SEQ ID NO:2 (positions 16-20 of SEQ ID NO:4 and 17-21 of
SEQ ID NO:6), e.g., WGDPI (SEQ ID NO:49). In this particular
chimeric polypeptide, it is noted that three residues of a FGF23
polypeptide scaffold are being replaced by five residues from a
FGF19 polypeptide, highlighting the option of replacing a
particular residue at a particular position in a region of a
polypeptide scaffold with more than one amino acid, i.e., replacing
three amino acids in a region with five amino acids.
[0083] As with all of the chimeric polypeptides of the present
invention, the particular amino acids to be replaced in a FGF19,
FGF21 or FGF23 polypeptide scaffold can be substituted with either
no amino acid or with any amino acid other than the residue that
appears at that position in the wild type sequence. For example,
FGF19 residues 38-42 of SEQ ID NO:2 (positions 16-20 of SEQ ID NO:4
and 17-21 of SEQ ID NO:6) comprise the five residue sequence WGDPI,
and can be substituted with any sequence other than WGDPI and can
also comprise less than five residues or more than five residues. A
more specific example is the replacement of the five residues of
FGF19, namely WGDPI, with the three residues normally found in
FGF21 at positions 42-44 of SEQ ID NO:8 (positions 14-16 of SEQ ID
NO:10 and 15-17 of SEQ ID NO:12), namely GQV.
[0084] II.C. Chimeric Polypeptides Lacking or Incorporating the
FGF19 .beta.1-.beta.2 Loop
[0085] The .beta.1-.beta.2 loop of FGF19 is thought to contribute
to heparin binding activity, and analogous regions are found in
FGF21 and FGF23. A chimeric polypeptide comprising this loop region
is expected contribute to the activation of FGFR4, which is not
normally activated by FGF21 or FGF23, for example, and can
contribute to hepatocyte mitogenicity, again, a property not
normally observed in FGF21 or FGF23. Similarly, a chimeric
polypeptide lacking this loop region, in conjunction with other
modifications, is expected to lack the ability to signal through
FGFR4.
[0086] In one aspect, the present disclosure relates to chimeric
polypeptides that are based on an FGF21 scaffold. In one aspect, a
chimeric polypeptide comprises the amino acid sequence of a wild
type FGF21 (SEQ ID NOs: 8, 10 or 12) polypeptide scaffold wherein
one or more of the residues DDAQQTE (SEQ ID NO:54) at positions
52-58 of SEQ ID NO:8 (positions 24-30 in SEQ ID NO:10 and 25-31 in
SEQ ID NO:12) has been replaced with (a) no amino acid (a
deletion); or (b) an amino acid other than the amino acid located
at the position in the wild type sequence.
[0087] The present disclosure relates, in one aspect, to chimeric
polypeptides that are based on an FGF23 scaffold. In one aspect, a
chimeric polypeptide comprises the amino acid sequence of a wild
type FGF23 (SEQ ID NOs:14, 16 or 18) polypeptide scaffold wherein
one or more of the residues ATARNS (SEQ ID NO:56) at positions
45-50 of SEQ ID NO:14 (positions 21-26 of SEQ ID NO:16 and 22-27 of
SEQ ID NO:18) has been replaced with at least one of (a) no amino
acid (a deletion); or (b) an amino acid other than the amino acid
located at the position in the wild type sequence.
[0088] The present disclosure also relates to chimeric polypeptides
that are based on a FGF19 scaffold. In one embodiment such a
chimeric polypeptide comprises the amino acid sequence of a wild
type FGF19 (SEQ ID NOs:2, 4 or 6) polypeptide scaffold wherein one
or more of the residues SGPHGLSS (SEQ ID NO:52) at positions 50-57
of SEQ ID NO:2 (positions 28-35 of SEQ ID NO:4 and positions 29-36
of SEQ ID NO:6) has been replaced with at least one of (a) no amino
acid (a deletion); or (b) an amino acid other than the amino acid
located at the position in the wild type sequence. In one example,
the FGF21 residues at positions 52-58 of SEQ ID NO:8 (positions
24-30 of SEQ ID NO:10 and 25-31 of SEQ ID NO:12), e.g., the
residues DDAQQTE, (SEQ ID NO:54) are inserted at position 50-57 of
a wild type FGF19 sequence. In this particular chimeric
polypeptide, it is noted that eight residues of a FGF19 polypeptide
are being replaced by seven residues from a FGF21 polypeptide,
highlighting the option of replacing a particular residue at a
particular position in a polypeptide scaffold with no amino acid.
Such chimeric polypeptides can exhibit the properties of reduced
FGF4 activation and/or hepatocyte mitogenicity in and of itself, or
such a substitution can form one element in a combination chimeric
polypeptide, as described herein.
[0089] As with all of the chimeric polypeptides of the present
invention, the particular amino acids to be replaced can be
substituted with either no amino acid or with any amino acid other
than the residue that appears at that position in the wild type
sequence. For example, FGF19 residues 50-57 of SEQ ID NO:2
(positions 28-35 of SEQ ID NO:4 and positions 29-36 of SEQ ID NO:6)
is the eight residue sequence SGPHGLSS, and could be substituted
with any sequence other than SGPHGLSS and could also comprise less
than eight residues. A more specific example is the replacement of
these residues with the seven FGF21 residues found at positions
52-58 of SEQ ID NO:8 (positions 24-30 of SEQ ID NO:10 and 25-31 of
SEQ ID NO:12), namely DDAQQTE (SEQ ID NO:54).
[0090] II.D. Chimeric Polypeptides Lacking or Incorporating the
FGF19 .beta.10-.beta.12 Segment
[0091] The .beta.10-.beta.12 segment of FGF19 is thought to
contribute to heparin binding activity and is a region found
analogously in FGF19, FGF21 and FGF23. Such a chimeric polypeptide
can contribute to the activation of FGFR4, which is not normally
activated by FGF21, as well as contribute to hepatocyte
mitogenicity, again, a property not normally observed in FGF21. In
various embodiments, the present disclosure relates to chimeric
polypeptides that are based on FGF19, FGF21 and FGF23
scaffolds.
[0092] In one aspect, a chimeric polypeptide comprising the amino
acid sequence of a wild type FGF21 polypeptide scaffold (e.g., SEQ
ID NOs:10, 12 or 14) wherein one or more of the residues
PGNKSPHRDPAPRGP (SEQ ID NO:60) at positions 147-161 of SEQ ID NO:8
(positions 119-133 in SEQ ID NO:10 and 120-134 in SEQ ID NO:12) has
been replaced with at least one of (a) no amino acid (a deletion);
or (b) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence.
[0093] In one aspect, a chimeric polypeptide comprising the amino
acid sequence of a wild type FGF23 polypeptide scaffold (e.g., SEQ
ID NOs:14, 16 or 18) wherein one or more of the residues
GRAKRAFLPGMNPPPY (SEQ ID NO:62) at positions 139-154 of SEQ ID
NO:14 (positions 115-130 in SEQ ID NO:16 and 116-131 in SEQ ID
NO:18) has been replaced with (a) no amino acid (a deletion); or
(b) an amino acid other than the amino acid located at the position
in the wild type amino acid sequence.
[0094] The present disclosure also relates to chimeric polypeptides
that are based on a FGF19 scaffold. In one embodiment of such a
chimeric polypeptide comprises the amino acid sequence of a wild
type FGF19 polypeptide scaffold (e.g., SEQ ID NOs:2, 4 or 6)
wherein one or more of the residues SSAKQRQLYKNRGFLPL (SEQ ID
NO:58) at positions 146-163 of SEQ ID NO:2 (positions 124-140 in
SEQ ID NO:4 and 125-141 in SEQ ID NO:6) has been replaced with (a)
no amino acid (a deletion); or (b) an amino acid other than the
amino acid located at the position in the wild type amino acid
sequence. In one example, the FGF21 residues at positions 147-161
of SEQ ID NO:8 (corresponding to positions 119-133 of SEQ ID NO:10
and 120-134 of SEQ ID NO:12), e.g., PGNKSPHRDPAPRGP (SEQ ID NO:60),
are inserted at position 146-162 of FGF19 sequence SEQ ID NO:2
(corresponding to positions 124-140 of SEQ ID NO:4 and 125-141 of
SEQ ID NO:6). In this particular chimeric polypeptide, it is noted
that 17 residues of a FGF19 polypeptide are being replaced by 15
residues from a FGF21 polypeptide, highlighting the option of
replacing a particular residue at a particular position in a
polypeptide scaffold with no amino acid. Such chimeric polypeptides
are expected to exhibit the properties of reduced FGFR4 activation
and/or hepatocyte mitogenicity in and of itself, or such a
substitution can form one element in a combination chimeric
polypeptide, as described herein.
[0095] As with all of the chimeric polypeptides of the present
invention, the particular amino acids to be replaced can be
substituted with either no amino acid or with any amino acid other
than the residue that appears at that position in the wild type
sequence. For example, FGF19 residues 146-162 of SEQ ID NO:2
(corresponding to positions 124-140 of SEQ ID NO:4 and 125-141 of
SEQ ID NO:6) is the 17 residue sequence SSAKQRQLYKNRGFLPL (SEQ ID
NO:58), and could be substituted with any sequence other than
SSAKQRQLYKNRGFLPL and could also comprise less than 17 residues. A
more specific example is the replacement of these residues with the
15 FGF21 residues found at positions 147-161 of SEQ ID NO:8
(corresponding to positions 119-133 of SEQ ID NO:10 and 120-134 of
SEQ ID NO:12), namely PGNKSPHRDPAPRGP (SEQ ID NO:60).
[0096] II.E. Chimeric Combination Polypeptides
[0097] The present disclosure also relates to chimeric combination
polypeptides. A chimeric combination polypeptide is a chimeric
polypeptide in which two or more regions of a polypeptide scaffold
have been replaced at each position of each region with either no
amino acid or an amino acid not normally found at the position in
the wild type polypeptide scaffold. Chimeric combination
polypeptides can therefore be engineered to demonstrate enhanced or
reduced properties normally associated with the polypeptide
scaffold, or properties not normally associated with the
polypeptide scaffold. In various embodiments, a chimeric
combination polypeptide can have the property of exhibiting
decreased FGFR4-mediated signaling.
[0098] By way of example, a chimeric combination polypeptide can be
built on a FGF19 scaffold. The amino acids in two or more
particular regions of a FGF19 scaffold, such as the region
comprising positions 38-42 and/or the region comprising positions
50-57 and/or the region comprising positions 146-162 of SEQ ID NO:2
(corresponding to positions 16-20, 28-35 and 124-140 of SEQ ID NO:4
and 17-21, 29-36 and 125-141 of SEQ ID NO:6), can each be replaced
by either residues not found at each of positions 38-42 and/or
50-57 and/or 146-162 in SEQ ID NO:2, (positions 16-20, 28-35 and
124-140 of SEQ ID NO:4, or positions 17-21, 29-36 and 125-141 of
SEQ ID NO:6), or by no amino acid. In one particular embodiment, a
chimeric combination polypeptide can have amino acids from regions
of FGF21 substituted for the wild type regions of the FGF19
polypeptide scaffold. One possible sequence of this chimeric
polypeptide comprises an FGF19 scaffold in which (a) one or more of
the residues WGDPI at positions 38-42 of SEQ ID NO:2 has been
substituted with (i) no amino acid (a deletion); or (ii) an amino
acid other than the amino acid located at the position in the wild
type amino acid sequence; (b) one or more of the residues SGPHGLSS
at positions 50-57 of SEQ ID NO:2 (corresponding to positions 28-35
of SEQ ID NO:4 and positions 29-36 of SEQ ID NO:6) has been
substituted with (i) no amino acid (a deletion); or (ii) an amino
acid other than the amino acid located at the position in the wild
type amino acid sequence; and (c) one or more of the residues
SSAKQRQLYKNRGFLPL at positions 146-163 of SEQ ID NO:2
(corresponding to positions 124-140 of SEQ ID NO:4 and 125-141 of
SEQ ID NO:6) has been substituted with (i) no amino acid; or (ii)
an amino acid other than the amino acid located at the position in
the wild type amino acid.
[0099] As disclosed herein, by replacing at least one amino acid of
the 38-42 region of a FGF19 polypeptide scaffold of SEQ ID NO:2
(corresponding to positions 14-20 of SEQ ID NO:4 and 15-21 of SEQ
ID NO:6) with either no amino acid or a non wild type residue at
least one of these positions, the ability of FGF19 to activate
FGFR4 is diminished and hepatocyte mitogenicity is also diminished.
By replacing at least one of the 50-57 region of a FGF19
polypeptide scaffold of SEQ ID NO:2 (corresponding to positions
28-35 of SEQ ID NO:4 and 29-36 of SEQ ID NO:6), which comprises the
heparin binding .beta.1-.beta.2 loop and/or at least one amino acid
of the 146-162 region of a FGF19 polypeptide scaffold of SEQ ID
NO:2 (corresponding to positions 124-140 of SEQ ID NO:4 and 125-141
of SEQ ID NO:6), which comprises the .beta.10-.beta.12 segment,
with either no amino acid or a non-wild type residue at least one
of these positions, FGFR4 activation and hepatocyte mitogenicity of
FGF19 can be still further diminished or eliminated. It is
significant to note, however, that a chimeric combination
polypeptide comprising substitutions at one or more regions can
still maintain other biological activities, such as the ability to
lower blood glucose levels or decrease body weight. Thus, a
chimeric combination polypeptide can be engineered to achieve
various goals and to exhibit a desired activity profile by
substituting the amino acids of two or more regions of a
polypeptide scaffold with amino acids not found at each of the
positions of the regions in the wild type polypeptide scaffold or
with no amino acid (a deletion).
[0100] In another example, a chimeric combination polypeptide can
be built on a FGF21 scaffold. The amino acids in two or more
particular regions of a FGF21 scaffold (e.g., SEQ ID NOs:8, 10 or
12), such as the region comprising positions 42-44 and/or the
region comprising positions 52-58 and/or the region comprising
positions 147-161 of SEQ ID NO:8 (corresponding to positions 14-16,
24-30 and 119-133 of SEQ ID NO:10 and 15-17, 25-31 and 120-134 of
SEQ ID NO:12), can each be replaced with residues not found at each
of positions 42-44 and/or 52-58 and/or 147-161 of SEQ ID NO:8
(corresponding to positions 14-16, 24-30 and 119-133 of SEQ ID
NO:10 and 15-17, 25-31 and 120-134 of SEQ ID NO:12), or with no
amino acid. In one particular embodiment of such a chimeric
combination polypeptide could have amino acids from regions of
FGF19 substituted for the wild type regions of the FGF21
polypeptide scaffold. One possible sequence of such a chimeric
polypeptide comprises a polypeptide scaffold in which (a) one or
more of the residues GQV at positions 42-44 of SEQ ID NO:8
(corresponding to positions 14-16 of SEQ ID NO:10 and 15-17 of SEQ
ID NO:12) has been substituted with (i) no amino acid (a deletion);
or (ii) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence; (b) one or more of
the residues DDAQQTE at positions 52-58 of SEQ ID NO:8
(corresponding to positions 24-30 in SEQ ID NO:10 and 25-31 of SEQ
ID NO:12) has been substituted with (ii) no amino acid (a
deletion); or (ii) an amino acid other than the amino acid located
at the position in the wild type amino acid sequence; and (c) one
or more of the residues PGNKSPHRDPAPRGP at positions 147-161 of SEQ
ID NO:8 (corresponding to positions 119-133 in SEQ ID NO:10 and
120-134 in SEQ ID NO:12) has been substituted with (i) no amino
acid (a deletion); or (ii) an amino acid other than the amino acid
located at the position in the wild type amino acid.
[0101] As described herein, by replacing at least one amino acid of
the 42-44 region of a FGF21 polypeptide scaffold of SEQ ID NO:8
(and corresponding residues in SEQ ID NOs:10 and 12) with either no
amino acid or a non wild type residue at one or more of these
positions, the ability of FGF21 to activate FGFR4 is imparted and
hepatocyte mitogenicity is also imparted. By replacing at least one
of the 52-58 region of a FGF21 polypeptide scaffold of SEQ ID NO:8
(and corresponding residues of SEQ ID NOs:10 and 12), which
comprises the heparin binding 131-132 loop, and/or one or more
amino acids of the 147-161 region of a FGF19 polypeptide scaffold
of SEQ ID NO:8 (and corresponding residues of SEQ ID NOs:10 and
12), which comprises the 1310-1312 segment, with no amino acid or a
non-wild type residue at one or more of these positions, FGFR4
activation and hepatocyte mitogenicity of FGF19 can be further
augmented.
[0102] In yet another example, a chimeric combination polypeptide
can be built on a FGF23 scaffold. The amino acids in two or more
particular regions of a FGF23 scaffold (e.g., SEQ ID NOs:14, 16 or
18), such as the region comprising positions 36-38 and/or the
region comprising positions 45-50 and/or the region comprising
positions 139-154 of SEQ ID NO:14 (corresponding to positions
12-14, 21-26 and 115-130 of SEQ ID NO:16 and 13-15, 22-27 and
116-131 of SEQ ID NO:18), can each be replaced by residues not
found at each of positions 36-38 and/or 45-50 and/or 139-154 in SEQ
ID NO:14 (corresponding to positions 12-14, 21-26 and 115-130 of
SEQ ID NO:16 and 13-15, 22-27 and 116-131 of SEQ ID NO:18), or by
no amino acid. In one particular embodiment of such a chimeric
combination polypeptide can have amino acids from regions of FGF19
substituted for the wild type regions of the FGF23 polypeptide
scaffold. One possible sequence of such a chimeric polypeptide
comprises a polypeptide scaffold in which (a) one or more of the
residues WGG at positions 36-38 of SEQ ID NO:14 (corresponding to
positions 12-14 in SEQ ID NO:16 and 13-15 in SEQ ID NO:18) has been
substituted with (i) no amino acid; or (ii) an amino acid other
than the amino acid located at the position in the wild type amino
acid sequence; (b) one or more of the residues ATARNS at positions
45-50 of SEQ ID NO:14 (corresponding to positions 21-26 in SEQ ID
NO:16 and 22-27 in SEQ ID NO:18) has been substituted with at least
one of (i) no amino acid; or (ii) an amino acid other than the
amino acid located at the position in the wild type amino acid
sequence; and (c) one or more of the residues GRAKRAFLPGMNPPPY at
positions 139-154 of SEQ ID NO:14 (corresponding to positions
115-130 of SEQ ID NO:16 and 116-131 of SEQ ID NO:18) has been
substituted with at least one of (i) no amino acid; or (ii) an
amino acid other than the amino acid located at the position in the
wild type amino acid.
[0103] As described herein, by replacing at least one amino acid of
the 36-38 region of a FGF23 polypeptide scaffold of SEQ ID NO:14
(corresponding to positions 12-14 in SEQ ID NO:16 and 13-15 in SEQ
ID NO:18) with either no amino acid or a non wild type residue at
least one of these positions, the ability of FGF23 to activate
FGFR4 is imparted and hepatocyte mitogenicity is also imparted. By
replacing one or more of the 45-50 region of a FGF23 polypeptide
scaffold of SEQ ID NO:14 (corresponding to positions 21-26 in SEQ
ID NO:16 and 22-27 in SEQ ID NO:18), which comprises the heparin
binding .beta.1-.beta.2 loop and/or at least one amino acid of the
139-154 region of a FGF23 polypeptide sequence of SEQ ID NO:14
(corresponding to positions 115-130 of SEQ ID NO:16 and 116-131 of
SEQ ID NO:18), which comprises the .beta.10-.beta.12 segment, with
either no amino acid or a non-wild type residue at one or more of
these positions, FGFR4 activation and hepatocyte mitogenicity of
FGF23 can be further augmented.
III. TRUNCATED CHIMERIC POLYPEPTIDES
[0104] N and C-terminally truncated forms of the chimeric
polypeptides described herein form another aspect of the present
disclosure. As used herein, the term "truncated chimeric
polypeptide" refers to a chimeric polypeptide in which one or more
amino acid residues have been removed from the amino-terminal (or
N-terminal) end of the chimeric polypeptide, amino acid residues
have been removed from the carboxyl-terminal (or C-terminal) end of
the chimeric polypeptide, or one or more amino acid residues have
been removed from both the amino-terminal and carboxyl-terminal
ends of the chimeric polypeptide. A truncated chimeric polypeptide
can be truncated, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 20 or more residues from the C-terminal end,
N-terminal end or both C- and N-terminal ends of the
polypeptide.
[0105] The activity of N-terminally truncated chimeric polypeptides
and C-terminally truncated chimeric polypeptides can be assayed as
described herein, for example by employing an in vitro ERK assay as
described in Examples 2.1 and 2.2. Specific details of the in vitro
assays that can be used to examine the activity of truncated
chimeric polypeptides can be found in Example 2 and herein.
[0106] The activity of the truncated chimeric polypeptides
disclosed herein can also be assessed in an in vivo assay as can be
done for any of the chimeric polypeptides disclosed herein, for
example in a BrdU incorporation assay as shown in Example 2.4, or
by examining the effects of the truncated chimeric polypeptide on
one or more metabolic parameters in a diseased model (e.g., ob/ob
or DIO mice), or by examining the effects of a truncated chimeric
polypeptide on tissues functions, such as ERK phosphorylation
levels. Generally, to assess the in vivo activity of a truncated
chimeric polypeptide, the truncated chimeric polypeptide can be
administered to a test animal intraperitoneally. After a desired
incubation period (e.g., 5, 10, 15, 20, 30, 40, 50 or 60 or more
minutes), a blood sample can be drawn, and blood glucose levels can
be measured and/or tissues can be harvested for subsequent
analysis. Specific details of the in vivo assays that can be used
to examine the activity of truncated chimeric polypeptides can be
found in Example 10. In another assay, a test animal can be
administered a BrdU label, subsequently sacrificed and the liver
examined for BrdU incorporation and/or for observable morphological
changes.
[0107] As with all chimeric polypeptides of the present disclosure,
truncated chimeric polypeptides can optionally comprise an
amino-terminal methionine residue, which can be introduced by
directed mutation or as a result of a bacterial expression
process.
[0108] The truncated chimeric polypeptides disclosed herein, and
indeed all of the chimeric polypeptides disclosed herein, can be
prepared as described in Example 1. Those of ordinary skill in the
art, familiar with standard molecular biology techniques, can
employ that knowledge, coupled with the instant disclosure, to make
and use the truncated chimeric polypeptides described herein.
Standard techniques can be used for recombinant DNA,
oligonucleotide synthesis, tissue culture, and transformation
(e.g., electroporation, lipofection). See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, supra, which is
incorporated herein by reference for any purpose. Enzymatic
reactions and purification techniques can be performed according to
manufacturer's specifications, as commonly accomplished in the art,
or as described herein. Unless specific definitions are provided,
the nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques can be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0109] The truncated chimeric polypeptides of the present invention
can also be fused to another entity, which can impart additional
properties to the truncated chimeric polypeptide. In one embodiment
of the present invention, a truncated chimeric polypeptide can be
fused to an Fc sequence as described herein. Such a fusion can be
generated using known molecular biological methods and/or the
guidance provided herein. The benefits of such fusion polypeptides,
as well as methods for making such fusion polypeptides, are
discussed in more detail herein.
IV. CHIMERIC POLYPEPTIDE VARIANTS
[0110] In another aspect, chimeric polypeptide variants are
provided. A chimeric polypeptide variant comprises an amino acid
sequence that exhibits enhanced or decreased FGFR4-mediated
signaling and is at least about 85 percent identical to the amino
acid sequence of FGF19 (e.g., SEQ ID NO: 4), but wherein the
specific residues comprising the modification(s) that enhance or
decrease FGF19's FGFR4-mediated signaling activity have not been
further modified. In other words, with the exception of residues in
the chimeric polypeptide that have been modified in order to confer
enhanced or decreased FGFR4-mediated signaling, about 15 percent of
all other amino acid residues in the chimeric polypeptide sequence
can be modified. In the particular example of a chimeric
polypeptide exhibiting decreased FGFR4-mediated activity in which
one or more of the residues WGDPI at positions 16-20 of the FGF19
polypeptide scaffold has been substituted with (a) no amino acid;
or (b) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence (SEQ ID NO:4), up to
15 percent of all amino acid residues other than the residues
changed at position 16-20 of the FGF19 polypeptide scaffold could
be modified.
[0111] In still other embodiments, a chimeric polypeptide comprises
an amino acid sequence that exhibits enhanced or decreased
FGFR4-mediated signaling and that is at least about 90 percent, or
about 95, 96, 97, 98, or 99 percent identical to the amino acid
sequence of the polypeptide scaffold (e.g., SEQ ID NO: 4), but
wherein the specific residues conferring the chimeric polypeptide's
enhanced or decreased FGFR4-mediated signaling properties have not
been further modified.
[0112] Also provided are nucleic acids encoding such chimeric
polypeptide variants. Thus, a nucleic acid molecule encoding an
amino acid sequence that exhibits enhanced or decreased
FGFR4-mediated signaling and is at least about 85 percent identical
to the amino acid sequence of FGF19 (e.g., SEQ ID NO: 4), but
wherein the specific residues comprising the modification(s) that
enhance or decrease FGF19's FGFR4-mediated signaling activity have
not been further modified, is provided. In other words, with the
exception of nucleotides that encode residues in the chimeric
polypeptide that have been modified in order to confer decreased
FGFR4-mediated signaling or other properties, nucleotides encoding
about 15 percent of all other amino acids in the chimeric
polypeptide can be modified. Again using the case of a chimeric
polypeptide showing decreased FGFR4-mediated signaling in which one
or more of the residues WGDPI at positions 16-20 of the FGF19
polypeptide scaffold has been substituted with (a) no amino acid;
or (b) an amino acid other than the amino acid located at the
position in the wild type amino acid sequence as an example,
nucleotides encoding up to 15 percent of all amino acids other than
the nucleotides encoding residues at positions 16-20 of the FGF19
polypeptide scaffold could be modified.
[0113] Also provided is a nucleic acid molecule encoding a chimeric
polypeptide showing decreased FGFR4-mediated signaling and
comprising an amino acid sequence that is at least about 90
percent, or about 95, 96, 97, 98, or 99 percent identical to the
amino acid sequence of SEQ ID NO: 4, but wherein the specific
residues comprising the modification(s) that decrease FGF19's
FGFR4-mediated signaling activity have not been further
modified.
V. CHIMERIC FUSION POLYPEPTIDES
[0114] Chimeric fusion polypeptides form another aspect of the
present disclosure. As used herein, the term "chimeric fusion
polypeptide" or "chimeric fusion protein" refers to a fusion of an
amino acid sequence comprising one or more amino acid residues
(including longer sequences such as a heterologous protein or
peptide) at the N-terminus or C-terminus of any of the chimeric
polypeptides disclosed herein.
[0115] Heterologous peptides and polypeptides include, but are not
limited to, an epitope to allow for the detection and/or isolation
of a chimeric polypeptide; a transmembrane receptor protein or a
portion thereof, such as an extracellular domain or a transmembrane
and intracellular domain; a ligand or a portion thereof which binds
to a transmembrane receptor protein; an enzyme or portion thereof
which is catalytically active; a polypeptide or peptide which
promotes oligomerization, such as a leucine zipper domain; a
polypeptide or peptide which increases stability, such as an
immunoglobulin constant region (an "Fc" domain); a functional or
non-functional antibody, or a heavy or light chain thereof; and a
polypeptide which has an activity, such as a therapeutic activity,
different from the chimeric polypeptides of the present invention.
Also encompassed by the present invention are chimeric polypeptides
fused to human serum albumin (HSA).
[0116] Chimeric fusion polypeptides can be made by fusing
heterologous sequences at either the N-terminus or at the
C-terminus of a chimeric polypeptide. As described herein, a
heterologous sequence can be an amino acid sequence or a non-amino
acid-containing polymer. Heterologous sequences can be fused either
directly to the chimeric polypeptide or via a linker or adapter
molecule. A linker or adapter molecule can be one or more amino
acid residues (or -mers), e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9
residues (or -mers), preferably from 10 to 50 amino acid residues
(or -mers), e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, or 50 residues (or -mers), and more preferably from
15 to 35 amino acid residues (or -mers). Examples of linkers
include the peptides of SEQ ID NOs:63-70. A linker or adapter
molecule can also be designed with a cleavage site for a DNA
restriction endonuclease or for a protease to allow for the
separation of the fused moieties.
[0117] V.A. Fc Fusions
[0118] In one embodiment of the present invention, a chimeric
polypeptide is fused to one or more domains of an Fc region of
human IgG. Antibodies comprise two functionally independent parts,
a variable domain known as "Fab," that binds an antigen, and a
constant domain known as "Fc," that is involved in effector
functions such as complement activation and attack by phagocytic
cells. An Fc has a long serum half-life, whereas a Fab is
short-lived (Capon et al., (1989) Nature 337: 525-31). When joined
together with a therapeutic protein, an Fc domain can provide
longer half-life or incorporate such functions as Fc receptor
binding, protein A binding, complement fixation, and perhaps even
placental transfer (Capon et al., 1989).
[0119] The resulting chimeric fusion polypeptide can be purified,
for example, by the use of a Protein A affinity column. Peptides
and proteins fused to an Fc region have been found to exhibit a
substantially greater half-life in vivo than the unfused
counterpart. Also, a fusion to an Fc region allows for
dimerization/multimerization of the fusion polypeptide. The Fc
region can be a naturally occurring Fc region, or can be altered to
improve certain qualities, such as therapeutic qualities,
circulation time, or reduced aggregation.
[0120] Useful modifications of protein therapeutic agents by fusion
with the "Fc" domain of an antibody are discussed in detail in
International Publication No. WO 00/024782, which is hereby
incorporated by reference in its entirety. This document discusses
linkage to a "vehicle" such as polyethylene glycol (PEG), dextran,
or an Fc region.
[0121] V.B. Fusion Protein Linkers
[0122] When forming a chimeric fusion polypeptide of the present
disclosure, a linker can, but need not, be employed. When present,
the linker's chemical structure may not always be critical, since
it serves primarily as a spacer. The linker can be made up of amino
acids linked together by peptide bonds. In some embodiments of the
present invention, the linker is made up of from 1 to 20 amino
acids linked by peptide bonds, wherein the amino acids are selected
from the 20 naturally occurring amino acids. In various
embodiments, the 1 to 20 amino acids are selected from the amino
acids glycine, serine, alanine, proline, asparagine, glutamine, and
lysine. In some embodiments, a linker is made up of a majority of
amino acids that are sterically unhindered, such as glycine and
alanine In some embodiments, linkers are polyglycines (such as
(Gly).sub.4 (SEQ ID NO:63) and (Gly).sub.5 (SEQ ID NO:64)),
polyalanines, combinations of glycine and alanine (such as
poly(Gly-Ala)), or combinations of glycine and serine (such as
poly(Gly-Ser)). Other suitable linkers include:
(Gly).sub.5-Ser-(Gly).sub.3-Ser-(Gly).sub.4-Ser (SEQ ID NO:65),
(Gly).sub.4-Ser-(Gly).sub.4-Ser-(Gly).sub.4-Ser (SEQ ID NO:66),
(Gly).sub.3-Lys-(Gly).sub.4 (SEQ ID NO:67),
(Gly).sub.3-Asn-Gly-Ser-(Gly).sub.2 (SEQ ID NO:68),
(Gly).sub.3-Cys-(Gly).sub.4 (SEQ ID NO:69), and Gly-Pro-Asn-Gly-Gly
(SEQ ID NO:70). Linkers of any length or composition can be
employed in the formation of a chimeric fusion polypeptide.
[0123] The linkers described herein are exemplary, and linkers that
are much longer and which include other residues are contemplated
by the present invention. Non-peptide linkers are also contemplated
by the present invention. For example, alkyl linkers such as
--NH--(CH.sub.2).sub.s--C(O)--, wherein s=2 to 20, could be used.
These alkyl linkers can further be substituted by any
non-sterically hindering group, including, but not limited to, a
lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN,
NH.sub.2, or phenyl. An exemplary non-peptide linker is a
polyethylene glycol linker, wherein the linker has a molecular
weight of 100 to 5000 kD, for example, 100 to 500 kD.
VI. CHEMICALLY MODIFIED CHIMERIC POLYPEPTIDES
[0124] Chemically modified forms of the chimeric polypeptides
described herein, including their truncated forms, can be prepared
by one skilled in the art, using the present disclosure coupled
with techniques known in the art. Such chemically modified chimeric
polypeptides are altered such that the chemically modified chimeric
polypeptide is different from the unmodified chimeric polypeptide,
either in the type or location of the molecules naturally attached
to the chimeric polypeptide. Chemically modified chimeric
polypeptides can include molecules formed by the deletion of one or
more naturally-attached chemical groups.
[0125] In one embodiment, chimeric polypeptides of the present
invention can be modified by the covalent attachment of one or more
polymers. For example, the polymer selected is often water-soluble
so that the protein to which it is attached does not precipitate in
an aqueous environment, such as a physiological environment.
Included within the scope of suitable polymers is a mixture of
polymers. Preferably, for therapeutic use of the end-product
preparation, the polymer will be pharmaceutically acceptable.
Non-water soluble polymers conjugated to the chimeric polypeptides
of the present disclosure also form an aspect of the invention.
[0126] Exemplary polymers each can be of any molecular weight and
can be branched or unbranched. The polymers each typically have an
average molecular weight of between about 2 kDa to about 100 kDa
(the term "about" indicating that in preparations of a
water-soluble polymer, some molecules will weigh more and some less
than the stated molecular weight). The average molecular weight of
each polymer is preferably between about 5 kDa and about 50 kDa,
more preferably between about 12 kDa and about 40 kDa, and most
preferably between about 20 kDa and about 35 kDa.
[0127] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C.sub.1-C.sub.10), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran of, for example, about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.,
glycerol), and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules that can be used
to prepare covalently attached chimeric polypeptides multimers.
Also encompassed by the present invention are chimeric polypeptides
covalently attached to polysialic acid.
[0128] In some embodiments of the present invention, a chimeric
polypeptide is covalently, or chemically, modified to include one
or more water-soluble polymers, including, but not limited to,
polyethylene glycol (PEG), polyoxyethylene glycol, or polypropylene
glycol. See, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192; and 4,179,337. In some embodiments of the
present invention, a chimeric polypeptide comprises one or more
polymers, including, but not limited to, monomethoxy-polyethylene
glycol, dextran, cellulose, another carbohydrate-based polymer,
poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, or
mixtures of such polymers.
[0129] In some embodiments of the present invention, a chimeric
polypeptide is covalently-modified with PEG subunits. In other
embodiments, one or more water-soluble polymers are bonded at one
or more specific positions (for example, at the N-terminus) of the
chimeric polypeptide. In still other embodiments, one or more
water-soluble polymers are randomly attached to one or more side
chains of a chimeric polypeptide. In some embodiments, PEG is used
to improve the therapeutic capacity of a chimeric polypeptide.
Certain such methods are discussed, for example, in U.S. Pat. No.
6,133,426, which is hereby incorporated by reference for any
purpose.
[0130] In embodiments of the present invention wherein the polymer
is PEG, the PEG group can be of any convenient molecular weight,
and can be linear or branched. The average molecular weight of the
PEG group will preferably range from about 2 kD to about 100 kDa,
and more preferably from about 5 kDa to about 50 kDa, e.g., 10, 20,
30, 40, or 50 kDa. The PEG groups will generally be attached to the
chimeric polypeptide via acylation or reductive alkylation through
a reactive group on the PEG moiety (e.g., an aldehyde, amino,
thiol, or ester group) to a reactive group on the chimeric
polypeptide (e.g., an aldehyde, amino, or ester group).
[0131] The PEGylation of a polypeptide, including the chimeric
polypeptides of the present invention, can be specifically carried
out using any of the PEGylation reactions known in the art. Such
reactions are described, for example, in the following references:
Francis et al., (1992), Focus on Growth Factors 3: 4-10; European
Patent Nos. 0 154 316 and 0 401 384; and U.S. Pat. No. 4,179,337.
For example, PEGylation can be carried out via an acylation
reaction or an alkylation reaction with a reactive polyethylene
glycol molecule (or an analogous reactive water-soluble polymer) as
described herein. For the acylation reactions, a selected polymer
should have a single reactive ester group. For reductive
alkylation, a selected polymer should have a single reactive
aldehyde group. A reactive aldehyde is, for example, polyethylene
glycol propionaldehyde, which is water stable, or mono
C.sub.1-C.sub.10 alkoxy or aryloxy derivatives thereof (see, e.g.,
U.S. Pat. No. 5,252,714).
[0132] In some embodiments of the present invention, a useful
strategy for the attachment of the PEG group to a polypeptide
involves combining, through the formation of a conjugate linkage in
solution, a peptide and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The
peptides can be easily prepared with conventional solid phase
synthesis. The peptides are "preactivated" with an appropriate
functional group at a specific site. The precursors are purified
and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous
phase and can be easily monitored by reverse phase analytical HPLC.
The PEGylated peptides can be easily purified by preparative HPLC
and characterized by analytical HPLC, amino acid analysis and laser
desorption mass spectrometry.
[0133] Polysaccharide polymers are another type of water-soluble
polymer that can be used for protein modification. Therefore, the
chimeric polypeptides of the present invention fused to a
polysaccharide polymer form embodiments of the present invention.
Dextrans are polysaccharide polymers comprised of individual
subunits of glucose predominantly linked by alpha 1-6 linkages. The
dextran itself is available in many molecular weight ranges, and is
readily available in molecular weights from about 1 kD to about 70
kD. Dextran is a suitable water-soluble polymer for use as a
vehicle by itself or in combination with another vehicle (e.g., a
Fc). See, e.g., International Publication No. WO 96/11953. The use
of dextran conjugated to therapeutic or diagnostic immunoglobulins
has been reported. See, e.g., European Patent Publication No. 0 315
456, which is hereby incorporated by reference. The present
invention also encompasses the use of dextran of about 1 kD to
about 20 kD.
[0134] In general, chemical modification can be performed under any
suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemically modified
polypeptides will generally comprise the steps of: (a) reacting the
polypeptide with the activated polymer molecule (such as a reactive
ester or aldehyde derivative of the polymer molecule) under
conditions whereby a chimeric polypeptide becomes attached to one
or more polymer molecules, and (b) obtaining the reaction products.
The optimal reaction conditions will be determined based on known
parameters and the desired result. For example, the larger the
ratio of polymer molecules to protein, the greater the percentage
of attached polymer molecule. In one embodiment of the present
invention, chemically modified chimeric polypeptides can have a
single polymer molecule moiety at the amino-terminus (see, e.g.,
U.S. Pat. No. 5,234,784)
[0135] In another embodiment of the present invention, chimeric
polypeptides can be chemically coupled to biotin. The
biotin/chimeric polypeptides are then allowed to bind to avidin,
resulting in tetravalent avidin/biotin/chimeric polypeptides.
Chimeric polypeptides can also be covalently coupled to
dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting
conjugates precipitated with anti-DNP or anti-TNP-IgM to form
decameric conjugates with a valency of 10.
[0136] Generally, conditions that can be alleviated or modulated by
the administration of the present chemically modified chimeric
polypeptides include those described herein for chimeric
polypeptides. However, the chemically modified chimeric
polypeptides disclosed herein can have additional activities,
enhanced or reduced biological activity, or other characteristics,
such as increased or decreased half-life, as compared to unmodified
chimeric polypeptides.
VII. PHARMACEUTICAL COMPOSITIONS
[0137] Therapeutic compositions comprising the disclosed chimeric
polypeptides are within the scope of the present disclosure, and
are specifically contemplated in light of the identification of
several chimeric polypeptides exhibiting desirable properties. Such
chimeric polypeptide pharmaceutical compositions can comprise a
therapeutically effective amount of a chimeric polypeptide in
admixture with a pharmaceutically or physiologically acceptable
formulation agent (e.g., a carrier, formulation material, etc)
selected for suitability with the mode of administration.
[0138] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0139] The pharmaceutical composition can contain formulation
materials for modifying, maintaining, or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine, or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite, or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, or other organic acids), bulking agents (such
as mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid, or hydrogen peroxide), solvents (such as glycerin,
propylene glycol, or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics; PEG; sorbitan esters; polysorbates such
as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as
alkali metal halides--preferably sodium or potassium chloride--or
mannitol sorbitol), delivery vehicles, diluents, excipients and/or
pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical
Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company
1990), and subsequent editions of the same, incorporated herein by
reference for any purpose).
[0140] The optimal pharmaceutical composition will be determined by
a skilled artisan depending upon, for example, the intended route
of administration, delivery format, and desired dosage (see, e.g.,
Remington's Pharmaceutical Sciences, supra). Such compositions can
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the chimeric polypeptide.
[0141] The primary vehicle or carrier in a pharmaceutical
composition can be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier for injection can be water,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can
further include sorbitol or a suitable substitute. In one
embodiment of the present disclosure, chimeric polypeptide
compositions can be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in
the form of a lyophilized cake or an aqueous solution. Further, the
chimeric polypeptide product can be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0142] The chimeric polypeptide pharmaceutical compositions can be
selected for parenteral delivery. Alternatively, the compositions
can be selected for inhalation or for delivery through the
digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is known to those of skill
of the art.
[0143] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at a slightly lower pH, typically within a pH range of from about 5
to about 8.
[0144] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention can be in the
form of a pyrogen-free, parenterally acceptable, aqueous solution
comprising the desired chimeric polypeptide in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a chimeric
polypeptide is formulated as a sterile, isotonic solution, properly
preserved. Yet another preparation can involve the formulation of
the desired molecule with an agent, such as injectable
microspheres, bio-erodible particles, polymeric compounds (such as
polylactic acid or polyglycolic acid), beads, or liposomes, that
provides for the controlled or sustained release of the product
which can then be delivered via a depot injection. Hyaluronic acid
can also be used, and this can have the effect of promoting
sustained duration in the circulation. Other suitable means for the
introduction of the desired molecule include implantable drug
delivery devices.
[0145] In one embodiment, a pharmaceutical composition can be
formulated for inhalation. For example, a chimeric polypeptide can
be formulated as a dry powder for inhalation. Chimeric polypeptide
inhalation solutions can also be formulated with a propellant for
aerosol delivery. In yet another embodiment, solutions can be
nebulized. Pulmonary administration is further described in
International Publication No. WO 94/20069, which describes the
pulmonary delivery of chemically modified proteins.
[0146] It is also contemplated that certain formulations can be
administered orally. In one embodiment of the present invention,
chimeric polypeptides that are administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For
example, a capsule can be designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate
absorption of the chimeric polypeptide. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders can also be employed.
[0147] Another pharmaceutical composition can involve an effective
quantity of chimeric polypeptides in a mixture with non-toxic
excipients that are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions can be prepared in unit-dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0148] Additional chimeric polypeptide pharmaceutical compositions
will be evident to those skilled in the art, including formulations
comprising chimeric polypeptides in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art (see,
e.g., International Publication No. WO 93/15722, which describes
the controlled release of porous polymeric microparticles for the
delivery of pharmaceutical compositions, and Wischke &
Schwendeman, (2008) Int. J. Pharm. 364:298-327, and Freiberg &
Zhu, (2004) Int. J. Pharm. 282:1-18, which discuss
microsphere/microparticle preparation and use).
[0149] Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g. films, or microcapsules. Sustained release matrices
can include polyesters, hydrogels, polylactides (U.S. Pa. No.
3,773,919 and European Patent No. 0 058 481), copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., (1983)
Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer
et al., (1981) J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982,
Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al.,
supra) or poly-D(-)-3-hydroxybutyric acid (European Patent No. 0
133 988). Sustained-release compositions can also include
liposomes, which can be prepared by any of several methods known in
the art. See, e.g., Epstein et al., (1985) Proc. Natl. Acad. Sci.
U.S.A. 82: 3688-92; and European Patent Nos. 0 036 676, 0 088 046,
and 0 143 949.
[0150] The chimeric polypeptide pharmaceutical composition to be
used for in vivo administration typically must be sterile. This can
be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this
method can be conducted either prior to, or following,
lyophilization and reconstitution. The composition for parenteral
administration can be stored in lyophilized form or in a solution.
In addition, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0151] Once the pharmaceutical composition has been formulated, it
can be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations can be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0152] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
can each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0153] The effective amount of a chimeric polypeptide
pharmaceutical composition to be employed therapeutically will
depend, for example, upon the therapeutic context and objectives.
One skilled in the art will appreciate that the appropriate dosage
levels for treatment will thus vary depending, in part, upon the
molecule delivered, the indication for which the chimeric
polypeptide is being used, the route of administration, and the
size (body weight, body surface, or organ size) and condition (the
age and general health) of the patient. Accordingly, the clinician
can titer the dosage and modify the route of administration to
obtain the optimal therapeutic effect. A typical dosage can range
from about 0.1 .mu.g/kg to up to about 100 mg/kg or more, depending
on the factors mentioned above. In other embodiments, the dosage
can range from 0.1 .mu.g/kg up to about 100 mg/kg; or 1 .mu.g/kg up
to about 100 mg/kg; or 5 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20
.mu.g/kg, 25 .mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45
.mu.g/kg, 50 .mu.g/kg, 55 .mu.g/kg, 60 .mu.g/kg, 65 .mu.g/kg, 70
.mu.g/kg, 75 .mu.g/kg, up to about 100 mg/kg. In yet other
embodiments, the dosage can be 50 .mu.g/kg, 100 .mu.g/kg, 150
.mu.g/kg, 200 .mu.g/kg, 250 .mu.g/kg, 300 .mu.g/kg, 350 .mu.g/kg,
400 .mu.g/kg, 450 .mu.g/kg, 500 .mu.g/kg, 550 .mu.g/kg, 600
.mu.g/kg, 650 .mu.g/kg, 700 .mu.g/kg, 750 .mu.g/kg, 800 .mu.g/kg,
850 .mu.g/kg, 900 .mu.g/kg, 950 .mu.g/kg, 100 .mu.g/kg, 200
.mu.g/kg, 300 .mu.g/kg, 400 .mu.g/kg, 500 .mu.g/kg, 600 .mu.g/kg,
700 .mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1000 .mu.g/kg, 2000
.mu.g/kg, 3000 .mu.g/kg, 4000 .mu.g/kg, 5000 .mu.g/kg, 6000
.mu.g/kg, 7000 .mu.g/kg, 8000 .mu.g/kg, 9000 .mu.g/kg, 10 mg/kg or
more.
[0154] The frequency of dosing will depend upon the pharmacokinetic
parameters of the chimeric polypeptide in the formulation being
used. Typically, a clinician will administer the composition until
a dosage is reached that achieves the desired effect. The
composition can therefore be administered as a single dose, as two
or more doses (which may or may not contain the same amount of the
desired molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages can be ascertained through use of
appropriate dose-response data.
[0155] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally; through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems (which may also be injected); or by
implantation devices. Where desired, the compositions can be
administered by bolus injection or continuously by infusion, or by
implantation device.
[0156] Alternatively or additionally, the composition can be
administered locally via implantation of a membrane, sponge, or
other appropriate material onto which the desired molecule has been
absorbed or encapsulated. Where an implantation device is used, the
device can be implanted into any suitable tissue or organ, and
delivery of the desired molecule can be via diffusion,
timed-release bolus, or continuous administration.
VIII. THERAPEUTIC AND OTHER USES OF A CHIMERIC POLYPEPTIDE
[0157] Chimeric polypeptides can be used to treat, diagnose,
ameliorate, or prevent a number of diseases, disorders, or
conditions, including, but not limited to metabolic disorders and
oncology-related disorders. In one embodiment, the metabolic
disorder to be treated is diabetes, e.g., type 2 diabetes. In
another embodiment, the metabolic disorder is obesity. Other
embodiments include metabolic conditions or disorders such as
dyslipidimia; hypertension; hepatosteaotosis, such as non-alcoholic
steatohepatitis (NASH); cardiovascular disease, such as
atherosclerosis; and aging. In another embodiment the
oncology-related disorder is a form of cancer.
[0158] In application, a disorder or condition such as diabetes or
obesity can be treated by administering a chimeric polypeptide as
described herein to a patient in need thereof in the amount of a
therapeutically effective dose. The administration can be performed
as described herein, such as by IV injection, intraperitoneal
injection, intramuscular injection, or orally in the form of a
tablet or liquid formation. In most situations, a desired dosage
can be determined by a clinician, as described herein, and can
represent a therapeutically effective dose of the chimeric
polypeptide. It will be apparent to those of skill in the art that
a therapeutically effective dose of a given chimeric polypeptide
will depend, inter alia, upon the administration schedule, the unit
dose of antigen administered, whether the nucleic acid molecule or
polypeptide is administered in combination with other therapeutic
agents, the immune status and the health of the recipient. The term
"therapeutically effective dose," as used herein, means that amount
of a given chimeric polypeptide that elicits the biological or
medicinal response in a tissue system, animal, or human being
sought by a researcher, medical doctor, or other clinician, which
includes alleviation of the symptoms of a disease or disorder being
treated.
IX. ANTIGEN BINDING PROTEINS
[0159] As used herein, an antigen binding protein is a protein
comprising a portion that binds to an antigen and, optionally, a
scaffold or framework portion that allows the antigen binding
portion to adopt a conformation that promotes binding of the
antigen binding protein to the antigen. Examples of antigen binding
proteins include human antibody, a humanized antibody; a chimeric
antibody; a recombinant antibody; a single chain antibody; a
diabody; a triabody; a tetrabody; a Fab fragment; a F(ab')2
fragment; an IgD antibody; an IgE antibody; an IgM antibody; an
IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4
antibody, and fragments thereof The antigen binding protein can
comprise, for example, an alternative protein scaffold or
artificial scaffold with grafted CDRs or CDR derivatives. Such
scaffolds include, but are not limited to, antibody-derived
scaffolds comprising mutations introduced to, for example,
stabilize the three-dimensional structure of the antigen binding
protein as well as wholly synthetic scaffolds comprising, for
example, a biocompatible polymer. See, e.g., Korndorfer et al.,
2003, Proteins: Structure, Function, and Bioinformatics,
53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654
(2004). In addition, peptide antibody mimetics ("PAMs") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronectin components as a scaffold.
[0160] Antigen binding proteins that specifically bind to the
chimeric polypeptides of the present invention but do not
specifically bind to wild-type polypeptide scaffolds are
contemplated and are within the scope of the present disclosure. An
antigen binding protein (e.g., an antibody) is said to
"specifically bind" its target antigen when the dissociation
constant (K.sub.D) is .ltoreq.10.sup.-8 M. The antibody
specifically binds antigen with "high affinity" when the K.sub.D is
.ltoreq.5.times.10.sup.-9 M, and with "very high affinity" when the
K.sub.D is .ltoreq.5.times.10.sup.-10 M.
[0161] When an antigen binding protein is an antibody, the antibody
can be polyclonal, including monospecific polyclonal; monoclonal
(MAbs); recombinant; chimeric; humanized, such as
complementarity-determining region (CDR)-grafted; human; single
chain; and/or bispecific; as well as fragments; variants; or
chemically modified molecules thereof Antibody fragments include
those portions of the antibody that specifically bind to an epitope
on a chimeric polypeptide. Examples of such fragments include Fab
and F(ab') fragments generated by enzymatic cleavage of full-length
antibodies. Other binding fragments include those generated by
recombinant DNA techniques, such as the expression of recombinant
plasmids containing nucleic acid sequences encoding antibody
variable regions.
[0162] Polyclonal antibodies directed toward a chimeric polypeptide
generally are produced in animals (e.g., rabbits or mice) by means
of multiple subcutaneous or intraperitoneal injections of the
chimeric polypeptide and an adjuvant. It can be useful to conjugate
a chimeric polypeptide to a carrier protein that is immunogenic in
the species to be immunized, such as keyhole limpet hemocyanin,
serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
Also, aggregating agents such as alum are used to enhance the
immune response. After immunization, the animals are bled and the
serum is assayed for anti-chimeric polypeptide antibody titer.
[0163] Monoclonal antibodies directed toward chimeric polypeptides
can be produced using any method that provides for the production
of antibody molecules by continuous cell lines in culture. Examples
of suitable methods for preparing monoclonal antibodies include the
hybridoma methods of Kohler et al., 1975, Nature 256: 495-97 and
the human B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:
3001; Brodeur et al., Monoclonal Antibody Production Techniques and
Applications 51-63 (Marcel Dekker, Inc., 1987). Also provided by
the invention are hybridoma cell lines that produce monoclonal
antibodies reactive with chimeric polypeptides.
[0164] The anti-chimeric polypeptide antibodies of the invention
can be employed in any known assay method, such as competitive
binding assays, direct and indirect sandwich assays, and
immunoprecipitation assays (see, e.g., Sola, Monoclonal Antibodies:
A Manual of Techniques 147-158, CRC Press, Inc., 1987),
incorporated herein by reference in its entirety) for the detection
and quantitation of chimeric polypeptide polypeptides. The
antibodies will bind chimeric polypeptides with an affinity that is
appropriate for the assay method being employed.
[0165] For diagnostic applications, in certain embodiments,
anti-chimeric polypeptide antibodies can be labeled with a
detectable moiety. The detectable moiety can be any one that is
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety can be a radioisotope,
such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.125I,
.sup.99Tc, .sup.111In, or .sup.67Ga; a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin; or an enzyme, such as alkaline
phosphatase, .beta.-galactosidase, or horseradish peroxidase (Bayer
et al., (1990) Meth. Enz. 184: 138-63).
[0166] Competitive binding assays rely on the ability of a labeled
standard (e.g., a chimeric polypeptide, or an immunologically
reactive portion thereof) to compete with the test sample analyte
(e.g., a chimeric polypeptide) for binding with a limited amount of
anti-chimeric polypeptide antibody. The amount of a chimeric
polypeptide in the test sample is inversely proportional to the
amount of standard that becomes bound to the antibodies. To
facilitate determining the amount of standard that becomes bound,
the antibodies typically are insolubilized before or after the
competition, so that the standard and analyte that are bound to the
antibodies can conveniently be separated from the standard and
analyte that remain unbound.
[0167] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody that is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody can itself be labeled with a detectable moiety
(direct sandwich assays) or can be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0168] The anti-chimeric polypeptide antibodies of the present
invention are also useful for in vivo imaging. An antibody labeled
with a detectable moiety can be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host assayed. The antibody can be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0169] The invention also relates to a kit comprising anti-chimeric
polypeptide antibodies and other reagents useful for detecting
chimeric polypeptide levels in biological samples. Such reagents
can include a detectable label, blocking serum, positive and
negative control samples, and detection reagents. Such a kit can
further comprise a set of instructions indicating how the reagents
and kit can be used.
EXAMPLES
[0170] The Examples that follow are illustrative of specific
embodiments of the invention, and various uses thereof. They are
set forth for explanatory purposes only, and should not be
construed as limiting the scope of the disclosed invention in any
way.
Example 1
Expression and Purification of Recombinant Scaffold Polypeptides
and Chimeric Proteins
[0171] Nucleotide sequences encoding wild type FGF19 without the
secretory leader peptide (residues 23-216, SEQ ID NO:4) and
chimeric polypeptides were cloned into the pET30 vector (Novagen).
Briefly, nucleotides for wild type FGF19 and chimeric polypeptides
were generated through polymerase chain reaction (PCR), both PCR
products and pET30 vector were digested with restriction enzyme Nde
I and BamH I and ligated with ligase. DNA constructs were
transformed into BL21(DE3) E .coli (Novagen). Protein expression
was induced with IPTG at 37.degree. C. The purification process was
the same as previously described (Wu et al., (2008) J. Biol Chem.
283:33304-9). FGF21 without the secretory leader peptide, (residues
29-209, SEQ ID NO:10) was purified as previously described (Xu et
al., (2008) Diabetes 58:250-59).
[0172] A description of some of the polypeptides that were
generated is shown in Table 2:
TABLE-US-00002 TABLE 2 SEQ ID Sequence NO: SEQ ID Identifier
Composition of Sequence NT NO: PP FGF19 hFGF19 1 2 Mature FGF19
hFGF19 lacking signal sequence 3 4 Mature FGF19 + hFGF19 lacking
signal sequence with 5 6 N-terminal Met N-terminal Met added FGF21
hFGF21 7 8 Mature FGF21 hFGF21 lacking signal sequence 9 10 Mature
FGF21 + hFGF21 lacking signal sequence with 11 12 N-terminal Met
N-terminal Met added FGF23 hFGF23 13 14 Mature FGF23 hFGF23 lacking
signal sequence 15 16 Mature FGF23 + hFGF23 lacking signal sequence
with 17 18 N-terminal Met N-terminal Met added FGF19dCTD
M::hFGF19(23-177) 19 20 FGF19/21-1 M::hFGF19(23-80)::hFGF21(82-209)
21 22 FGF19/21-2 M::hFGF19(23-49)::hFGF21(52-209) 23 24 FGF19/21-3
M::hFGF19(23-42)::hFGF21(45-209) 25 26 FGF19/21-4
M::hFGF19(23-37)::hFGF21(42-209) 27 28 FGF19/21-5
M::hFGF19(23-32)::hFGF21(37-209) 29 30 FGF21/19.sup.38-42
M::hFGF21(29-41)::hFGF19(38- 31 32 42)::hFGF21(45-209)
FGF19/21.sup.42-44 M::hFGF19(23-37)::hFGF21(42- 33 34
44)::hFGF19(43-216) FGF19-1 M::hFGF19(23-49)::hFGF21(52- 35 36
58)::hFGF19(58-216) FGF19-2 M::hFGF19(23-145)::hFGF21(147- 37 38
161)::hFGF19(163-216) FGF19-3 M::hFGF19(23-49)::hFGF21(52- 39 40
58)::hFGF19(58-145)::hFGF21(147- 161)::hFGF19(163-216) FGF19-4
M::hFGF19(23-37)::hFGF21(42- 41 42 44)::hFGF19(43-49)::hFGF21(52-
58)::hFGF19(58-216) FGF19-5 M::hFGF19(23-37)::hFGF21(42- 43 44
44)::hFGF19(43-145)::hFGF21(148- 162)::hFGF19(163-216) FGF19-6
M::hFGF19(23-37)::hFGF21(42- 45 46 44)::hFGF19(43-49)::hFGF21(52-
58)::hFGF19(58-145)::hFGF21(148- 162)::hFGF19(163-216)
[0173] In Table 2, each construct is presented in the N to C
terminal direction; "M" indicates an N-terminal methionine, hFGF19
(X-Y) indicates a region of human FGF19 stretching between residue
X and residue Y of a wild type FGF19 amino acid sequence, and
hFGF21 (X-Y) indicates a region of human FGF21 stretching between
residue X and residue Y of a wild type FGF21 amino acid sequence.
For example, in the case of FGF19/21-1 in Table 2, this sequence
comprises M::hFGF19(23-80)::hFGF21(82-209) means sequence is
composed of methionine, followed by human FGF19 sequences 23 to 80,
then followed by human FGF21 sequences 82 to 209.
Example 2
Experimental Methods
[0174] The following experimental methods were employed in Examples
3-10.
2.1 Western-blot Analysis of FGF Signaling
[0175] L6 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum and
penicillin/streptomycin. Cells were transfected with expression
vectors using the Lipofectamine 2000 transfection reagent
(Invitrogen) according to the manufacturer's protocol.
[0176] Analysis of FGF signaling in L6 cells were performed as
described before. Cell cultures were collected 10 min after the
treatment of FGF19 or chimeras and snap frozen in liquid nitrogen,
homogenized in the lysis buffer and subjected to western blot
analysis using anti-phospho-p44/42 MAP kinase (ERK1/2) antibody and
anti-ERK antibody (Cell Signaling).
2.2 MSD Assay for FGF Signaling
[0177] L6 cells plated in 24 well plates (10.sup.6 cells/well) were
transfected with various FGF receptors, including FGFR1c and FGFR4
and .alpha.Klotho or .beta.Klotho and serum starved in 0.2% bovine
serum albumin overnight before FGF treatment. Media was aspirated
after 10 min and plates were snap frozen in liquid nitrogen. Cells
in each well were lysed in 60 .mu.l of complete lysis buffer and
total and phosphorylated ERK was measured using MSD whole cell
lysate Phospho-ERK1/2 kit (Meso Scale Discovery, Gaithersburg, Md.)
according to the manufacturer's instructions.
2.3 Glucose Uptake Assay
[0178] 3T3L1 preadipocytes (ATCC CL-173) were cultured and induced
to differentiate. Glucose uptake was assayed as described in
Kharitonenkov, et al., (2005) J Clin Invest 115, 1627-1635.
2.4 In Vivo Hepatocyte BrdU Labeling Assay
[0179] For all BrDU studies described herein, on day 1 of the study
an osmotic minipump (ALZET.RTM., model 1007D) containing
5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, Mo.)
(16 mg/mL) was implanted subcutaneously into each of 7-10 8
week-old female FVB mouse (Charles River Laboratories, Charles
River, Mass.). Each mouse was given an IP injection of either
phosphate-buffered saline (PBS; vehicle), or various proteins as
indicated daily at 2 mg/kg/day beginning on day 2 of the study and
continuing for 6 consecutive days. Samples of liver and duodenum
were collected from each mouse on the day following the last IP
injection and placed in 10% neutral-buffered formalin in
preparation for paraffin-embedding, sectioning, and light
microscopic evaluation. Sections of all collected tissues were
stained by an immunohistochemical method described herein to
visualize BrdU incorporation as a marker of mitotic activity.
Tissue sections were examined at random by routine light microscopy
without knowledge of treatment group. The number of hepatocyte
nuclei stained for BrdU incorporation was assigned a score on a
semiquantitative scale where 0=no increase above expected levels in
vehicle-treated (control) mice and .+-.=equivocal, 1=minimal,
2=mild, 3=moderate, and 4=marked increase above control levels. The
localization (centrilobular or diffusely scattered through hepatic
lobules) of the hepatocytes stained for BrdU incorporation was also
recorded.
[0180] Cellular incorporation of BrdU was detected by digesting
deparaffinized tissue sections with 0.1% Protease (Sigma, St.
Louis, Mo.) and treating the sections with 2N hydrochloric acid.
Sections were blocked with CAS BLOCK (Zymed Laboratories, San
Francisco, Calif.), incubated with rat antibody to BrdU (Accurate,
Westbury, N.Y.; catalog no. OBT0030, lot no. H9180), and bound rat
antibody was detected with biotinylated rabbit antibody to rat IgG
(Vector Laboratories, Burlingame, Calif.; catalog no. BA 4001, lot
no. S0907). Tissue sections were quenched with Peroxidase Blocking
Solution (DAKO Corp., Carpinteria, Calif.) and retained biotin was
detected with Vectastain Elite ABC kit (Vector Laboratories).
Reaction sites were visualized with DAB+Substrate-Chromagen System
(DAKO Corp.). Sections were counterstained with hematoxylin.
Example 3
FGF21 Does not Increase Hepatocyte Proliferation In Vivo
[0181] Because FGF21 is in the same subfamily with FGF19, and both
show significant similarities in receptor/co-receptor requirements
and in regulation of glucose and lipid metabolism, the effects of
each on hepatocyte proliferation was studied. Using in vivo BrdU
labeling, enhanced hepatocyte proliferation around the central vein
was observed in FGF19 transgenic animals as well as in
nontransgenic animals 6 days post daily injection of recombinant
FGF19 protein. See, Nicholes et al., (2002) Am J Pathol 160,
2295-2307. Using a similar BrdU labeling method, we examined the
effects of FGF21 treatment on hepatocyte proliferation and compared
its activity to that of FGF19. As shown in FIG. 1A,
histopathological examination of the liver sections from FGF19
treated animals showed increased BrdU labeled hepatocytes
concentrating in centrilobular regions of hepatic lobules,
consistent with published observations (see, e.g., Nicholes et al.,
(2002) Am J Pathol 160, 2295-2307). In contrast, livers from FGF21
treated animals did not show increased numbers of BrdU-labeled
hepatocytes in pericentral regions, nor was increased BrdU labeling
noted in any other area of the liver, suggesting that FGF21 did not
enhance hepatocyte proliferation under the conditions tested and,
therefore, is distinct from FGF19. FIG. 1B graphically depicts the
incorporation level of the BrdU label at the conclusion of the
experiment, and highlights the observation that FGF19 led to BrdU
incorporation, while the PBS control and FGF21 did not.
Example 4
FGF19, but not FGF21, Activates FGFR4 Mediated ERK Phosphorylation,
and Selective Activation of FGFR4 in Liver Induces Pericentral
Hepatocyte Proliferation
[0182] To better understand the mechanism for FGF19 induced
hepatocyte proliferation and its differences from FGF21, the
receptor and co-receptor requirements between FGF19 and FGF21 were
first compared. The rat myoblast cell line L6, which expresses very
low levels of endogenous FGF receptors, was transfected with
FGFR1c, 2c, 3c or 4 together with .beta.Klotho. Receptor activation
was determined by Western blot analysis of phospho-ERK levels in
treated cells. As shown in FIGS. 2A-2D, while both FGF19 and FGF21
were able to activate FGFR1c (FIG. 2A), 2c (FIG. 2B), and 3c (FIG.
2C), only FGF19 and not FGF21 induced ERK phosphorylation via FGFR4
(FIG. 2D) (see, e.g., Kurosu et al., (2007) J. Biol Chem. 282,
26687-26695, and Lin et al., (2007) J. Bio. Chem. 282,
27277-27284). Given that FGFR4 is the predominant receptor
expressed in hepatocytes, the effect of FGFR4 activation as
measured by ERK phosphorylation on the enhanced hepatocyte
proliferation observed in FGF19 treated animals was studied.
[0183] In this experiment a variant of FGF19, FGF19dCTD, identified
as a selective FGFR4 agonist, was employed. As illustrated in FIG.
3A, FGF19dCTD is a truncated form of FGF19 in which the C-terminal
residues 178-216 have been removed. Because this region is critical
for co-receptor .beta.Klotho interaction, FGF19dCTD cannot activate
FGFRs 1c, 2c, and 3c which depend on .beta.Klotho for activation by
both FGF19 and FGF21 (FIG. 3B; see also Wu et al., (2008) J. Biol
Chem. 283(48):33304-9). FGF19dCTD can, however, still activate
FGFR4 both in vitro (FIG. 3B) and in vivo. Accordingly FGF19dCTD
was employed to examine the effects of selective FGFR4 activation
on hepatocyte proliferation. Analysis of BrdU immunostained liver
sections from FGF19dCTD treated animals also showed enhanced BrdU
labeling indicating increased mitotic activity almost as defined as
was observed in animals treated with wild type FGF19. Thus,
activation of FGFR4 alone can be sufficient to cause increased
hepatocyte proliferation.
Example 5
Identification of a Region of FGF19 That is Critical for FGFR4
Activation
[0184] Upon consideration of the results shown in Example 4, the
region(s) of FGF19 responsible for FGFR4 signaling were identified
and studied. Because FGF19 and FGF21 share significant sequence
homology but differ in the ability to activate FGFR4 signaling,
chimeric proteins comprising regions of FGF19 and FGF21 were
generated. The approach taken was to sequentially replace regions
of an FGF21 wild type sequence with regions of FGF19, in order to
identify the region responsible for FGFR4 activity.
[0185] Results from experiments using FGF19dCTD indicated that the
C-terminal region of FGF19 is not essential for FGFR4 activation;
therefore, a series of FGF19/FGF21 chimeric polypeptides was
generated which sequentially replaced the N-terminal region of
FGF21 with the corresponding region of FGF19; these chimeric
polypeptides are illustrated graphically in FIG. 4A. The properties
of these chimeric polypeptides were then assessed in in vitro
receptor activity assays, adipocyte glucose uptake assays, and in
vivo hepatocyte proliferation assays. As shown in the top panel of
FIG. 4B, all the chimeric polypeptides activated ERK
phosphorylation in L6 cells transfected with FGFR1c and
.beta.Klotho. Consistently, because FGFR1c is the predominant
receptor expressed in adipocytes, all the chimeric polypeptides
induced glucose uptake in differentiated mouse 3T3-L1 adipocytes
with similar potency and efficacy (see FIG. 4C). These results
suggest that all the chimeric polypeptides are functional, and that
fusions between FGF19 and FGF21 yielded properly folded and active
proteins.
[0186] These chimeric polypeptides, however, displayed differences
in FGFR4 selective assays. For example, in L6 cells transfected
with FGFR4 and .beta.Klotho, ERK-phosphorylation was only observed
with the chimeric polypeptides FGF19/21-1, FGF19/21-2 and
FGF19/21-3, which share FGF19 residues 23-42 (FIG. 4B).
ERK-phosphorylation was not observed with FGF19/21-4 or FGF19/21-5,
which comprise shorter N-terminal fragments derived from FGF19
(FIG. 4B, bottom panel). These results indicate that critical FGFR4
activating residues are contained within FGF19 residues 38 to
42.
[0187] The effects of these chimeric polypeptides on hepatocyte
proliferation were then tested in an in vivo BrdU incorporation
assay. Examination of BrdU immunostained liver sections from
treated animals showed that, like FGF19, the chimeric polypeptides
FGF19/21-1, FGF19/21-2, FGF19/21-3, and FGF19/21-4 all exhibited
increased BrdU labeling in the pericentral hepatocytes, however,
such increases were not observed with animals treated with
FGF19/21-5 and FGF19/21-6 (FIG. 4D). Therefore, BrdU labeling
correlated directly with each molecule's ability to activate FGFR4
mediated ERK phosphorylation (as shown in FIG. 4A).
Example 6
Residues 38-42 of FGF19 Confer FGFR4 Activation and Increased
Hepatocyte Proliferation
[0188] A comparison between FGF19/21-4 (containing the first 15
residues from a mature wild type FGF19 polypeptide) and FGF19/21-5
(containing the first 10 residues from a mature wild type FGF19
polypeptide) revealed a difference in each chimeric polypeptide's
ability to activate FGFR4 and to induce hepatocyte proliferation.
Because the two chimeric polypeptides only differ by 5 amino acids,
a study was undertaken to determine whether these 5 residues,
namely the residues at positions 38-42 a full length FGF19
(residues 16-20 in the mature form) were sufficient to confer FGFR4
activation. Another chimeric polypeptide designated
"FGF21/19.sup.38-42" (SEQ ID NO:32), comprising a FGF21 scaffold,
in which residues 38-42 of a full length wild type FGF19 amino acid
sequence (residues 16-20 in the mature form) replaced residues
42-44 of a full length FGF21 (residues 14-16 in the mature form),
was constructed and is depicted graphically in FIG. 5A. Similar to
FGF19 and FGF21, FGF21/19.sup.38-42 induced ERK-phosphorylation in
L6 cells transfected with FGFR1c and .beta.Klotho (FIG. 5B) and was
active in adipocyte glucose uptake assays. Similar to FGF19,
however in contrast to FGF21, the FGF21/19.sup.38-42 chimeric
polypeptide induced ERK-phosphorylation in L6 cells transfected
with FGFR4 and .beta.Klotho (FIG. 5B). Histopathological
examination analysis of liver sections from FGF21/19.sup.38-42
treated animals showed enhanced BrdU labeling in pericentral
hepatocytes similar to FGF19 treatment, distinct from FGF21 (FIG.
5C). These results indicate that introduction of these 5 residues
from FGF19 conferred a gain-of-function phenotype on FGF21 with
respect to FGFR4 activation in vitro and induction of hepatocyte
proliferation in vivo.
Example 7
Replacing Residues 38-42 from FGF19 Does Not Completely Abolish
FGFR4 Activation and Hepatocyte Proliferation
[0189] Using a FGF19 C-terminal truncation variant and novel
FGF19/FGF21 chimeric molecules, it was determined that hepatocyte
FGFR4 activation measured by ERK phosphorylation may lead to
increased hepatocyte proliferation. In side-by-side direct
comparison studies, it was also determined that FGF21 is different
from FGF19 and it lacks the ability to activate FGFR4 and does not
induce hepatocyte proliferation as measured using in vivo BrdU
labeling. In addition, these observations demonstrated the
importance of the FGF19 N-terminal region to FGFR4 activation, and
identified residues 38-42 of full length FGF19 to be sufficient to
confer FGFR4 activation and to increase hepatocyte proliferation,
as a construct in which the replacement of the corresponding region
in FGF21 with these 5 amino acid residues (the construct designated
FGF21/19.sup.38-42 in FIG. 6A) provided gain-of-function activity
to FGF21 in the form of FGFR4 activation and induction of increased
hepatocyte proliferation (FIG. 6A). Additionally, the issue of
whether the mutations in this region of FGF19 would abolish FGF19's
ability to activate FGFR4 and eliminate its ability to induce
hepatocyte proliferation was studied.
[0190] The alignment of FGF19, FGF21 and FGF23 around these 5 amino
acid residues is shown in FIG. 6B. These 5 residues are underlined
in FGF19 sequence, the corresponding region in FGF23 contains only
3 amino acids, WGG, and similarly, the corresponding region of
FGF21 contains only 3 amino acids, G.sup.42Q.sup.43V.sup.44. A
construct comprising the swap of this region between FGF19 and
FGF21 was constructed as shown in FIG. 6A. For FGF21/19.sup.38-42,
as described previously, the residues GQV at positions 42-44 in
full length FGF21 (positions 14-16 in mature FGF21) were replaced
with the corresponding FGF19 residues WGDPI (SEQ ID NO:49) at
positions 38-42 of full length FGF19 (positions 16-20 of mature
FGF19); and for FGF19/21.sup.42-44 (SEQ ID NO:34), which is the
reverse swap, the residues WGDPI at positions 38-42 in full length
FGF19 (positions 16-20 in mature FGF19) were replaced with the
corresponding residues GQV found at positions 42-44 of full length
FGF21 (positions 14-16 of mature FGF21). If this region is the only
region that contributes to FGFR4 activation, the substitution of
FGF21 sequence into FGF19 would abolish that activity.
[0191] To test the activities of these chimeric FGF molecules in
receptor activation assay, the previously described rat myoblast
cell line L6, which expresses negligible levels of endogenous FGF
receptors and .beta.Klotho, was utilized. FGFRs were either
transfected alone or together with .beta.Klotho, and the signaling
was monitored by the ERK phosphorylation levels (Wu et al., (2008)
J. Biol. Chem. 283(48):33304-9). In this assay format,
FGF19/21.sup.42-44 still activated FGFR4 signaling in the presence
of co-receptor .beta.Klotho and its ability to activate
FGFR1c/.beta.Klotho complex was also unaffected. See FIGS. 6A and
6C.
[0192] The effects of FGF19/21.sup.42-44 on hepatocyte
proliferation was examined in vivo by measuring the incorporation
of the label BrdU into hepatocytes post daily intraperitoneally
(i.p.) injection of the recombinant protein for 7 days. Consistent
with the previously observed link between liver FGFR4 activation
and enhanced hepatocyte proliferation, histopathological
examination of liver sections from FGF19/21.sup.42-44 treated
animals showed enhanced BrdU labeling in pericentral hepatocytes
similar to FGF19 (FIG. 6E). There results indicate that additional
regions of FGF19 can independently contribute to FGFR4
activation.
[0193] One surprising finding is that FGF19/21.sup.42-44 is no
longer able to activate FGFR4 in the absence of .beta.Klotho (FIG.
6C lower panel FGFR4 alone transfection). This suggests that
heparin induced FGF19/21.sup.42-44/FGFR4 activation has been
affected by this substitution. This is further confirmed by
solid-phase binding assay where addition of heparin no longer
stimulates FGF19/21.sup.42-44 interaction with FGFR4 (FIG. 6D)
similar to effects observed with mutations in the heparin binding
sites of .beta.1-.beta.2 and .beta.10-.beta.12 regions shown in
FIG. 7. Since FGF21/19.sup.38-42 does not interact with or activate
FGFR4 in the presence of heparin (FIG. 6D), the effect of the
N-terminal 5 residues of FGF19 (38-42) on heparin induced FGFR4
interaction may be an indirect effect.
Example 8
Replacing Heparin Binding Loops in FGF19 Abolished
.beta.Klotho-Independent FGFR4 Activation by FGF19
[0194] FGF19 subfamily members have reduced affinity to
heparin/heparin sulfate, and the presence of co-receptors a or
.beta.Klotho facilitate the binding and activation of FGFRs by this
subfamily members to compensate for the weak heparin binding
affinity. The only exception is the FGF19/FGFR4 interaction. At
relatively high concentrations of heparin, FGF19 can bind and
activate FGFR4 in the absence of .beta.Klotho in both in vitro and
in vivo.
[0195] The published apo-FGF19 and FGF23 structures (PDB codes:2P23
and 2P39; Goetz et al., (2007) Mol. Cell. Biol. 27:3417-28)
provided some insights into the weakened affinity toward heparin
for this subfamily. The 131-132 loop (SEQ ID NOs:52, 54 and 56) and
1310-1312 regions (SEQ ID NOs:58, 60 and 62), which are shown in
FIG. 7 and have been shown to be responsible for high affinity
binding of heparin by other FGF family members, are much larger in
this subfamily and could potentially form steric clashes with
heparin in the ternary complex with FGFR, and therefore result in
lower affinity toward heparin (Goetz et al., (2007) Mol. Cell.
Biol. 27:3417-28).
[0196] A FGF21 model built based on the published apo-FGF19
structure revealed that in addition to the potential steric clash,
the surface charges for FGF21 in these regions are also less
favorable for heparin binding and may explain the even lower
affinity of FGF21 toward heparin compared with FGF19 (Goetz et al.,
(2007) Mol. Cell. Biol. 27:3417-28). Since this is one of the major
difference between FGF19 and FGF21, a modeled FGF19/FGFR structure
based on the published FGF2/FGFR1 complex structure (PDB code:
1FQ9) revealed that these putative heparin binding domains are
positioned opposite to the 5 amino acid residues at positions 38-42
in full length FGF19 and may also contact the receptor
(Schlessinger et al., (2000) Mol. Cell 6:743-50). In light of this
observation, the issue of whether these regions contribute to FGFR4
activation by FGF19 was studied.
[0197] To examine the differences between FGF19 and FGF21 in the
putative heparin binding domains, chimeric constructs in which the
heparin interacting .beta.1-.beta.2 loop and .beta.10-.beta.12
segments in FGF19 were replaced with the corresponding sequences
from FGF21 were designed and expressed. These chimeric constructs
are shown graphically in FIG. 8A. In FIG. 8A, FGF19-1 corresponds
to a chimera in which residues 52-58 of full length FGF21
(positions 24-30 in mature FGF21) replaced residues 50-57 of FGF19
(positions 28-35 in mature FGF19), FGF19-2 corresponds to a chimera
in which residues 147-161 of full length FGF21 (positions 119-133
in mature FGF21) replaced residues 146-162 of FGF19 (positions
124-140 of mature FGF19) and FGF19-3 corresponds to a chimera in
which residues 52-58 of full length FGF21 (positions 24-30 of
mature FGF21) replaced residues 50-57 of full length FGF19
(positions 28-35 in mature FGF19) and residues 147-161 of full
length FGF21 (positions 119-133 in mature FGF21) replaced residues
146-162 of full length FGF19 (positions 124-140 of mature FGF21).
These chimeric polypeptides were used to investigate the
contribution of these domains to heparin binding and FGFR4
activation.
[0198] As demonstrated by the results of a solid-phase binding
assay, replacing the .beta.1-.beta.2 loop and .beta.10-.beta.12
segment of FGF19 individually or in combination abolished heparin
induced FGF19/FGFR4 interaction (FIG. 8B) while preserving the
ability to interact with FGFR4 in the presence of .beta.Klotho
(FIG. 8C), is consistent with the roles of these two regions in
interacting with heparin.
[0199] To further evaluate these findings in a functional assay,
receptors were again transfected into L6 cells. FGFR4 was either
transfected alone or together with .beta.Klotho, and the signaling
was monitored by the ERK phosphorylation levels. Consistent with
solid-phase binding results, in contrast to FGF19, the chimeric
substitutions in these putative heparin binding domains abolished
heparin dependent FGFR4 activation (FIG. 8D, lower panel), while
.beta.Klotho dependent FGFR1c and FGFR4 activation were preserved
(FIG. 8D upper panels). These results indicate that the mutations
in the putative heparin binding domain indeed abolished heparin
dependent receptor activities.
[0200] It has been shown that wild type FGF19 can activate FGFR4
either through heparin or .beta.Klotho. One variant of FGF19,
namely FGF19dCTD, can selectively activate FGFR4 only in a heparin
dependent manner, and this activation still induced enhanced
hepatocyte proliferation. In the case of FGF19-1 mutant described
herein, the heparin dependent FGFR4 activation was abolished while
preserving .beta.Klotho dependent FGFR4 activation. With respect to
FGF19dCTD and FGF19-1, each appeared to retain part of the wild
type FGF19 function, and although both are able to activate FGFR4,
signaling is mediated through different cofactors.
[0201] The issue of whether there is a qualitative difference in
FGFR4 signaling mediated through .beta.Klotho versus signaling
mediated through heparin with respect to stimulation of hepatocyte
proliferation was then studied. Histopathological examination of
liver sections from FGF19-1 treated animals showed enhanced BrdU
labeling in pericentral hepatocytes similar to FGF19 treatment
(FIG. 8E), suggesting that both heparin and .beta.Klotho induced
FGFR4 activation results in enhanced hepatocyte proliferation. As
is the case with wild type FGF19, FGF19-1 is also still active in
other metabolic assays, able to induce glucose uptake into
adipocyte cells and reduced plasma glucose levels in an ob/ob
diabetic animal model, indicating that heparin domain mutations did
not affect other FGF 19 mediated functions.
Example 9
A Chimeric Protein in Which Residues 38-42 of Full Length FGF19 and
both Heparin Binding Regions in FGF19 are Replaced Exhibits
Decreased FGFR4 Activation and Hepatocyte Proliferation
[0202] The single changes in the 5 amino acid region of residues
38-42 of full length FGF19 (positions 14-20 in mature FGF19) and
the heparin binding domains did not completely abolish FGFR4
activation, so a chimeric protein in which the replacement of all
three of these regions was studied. Additional chimeric
polypeptides combining residues 38-42 of full length FGF19 and one
or both of the heparin interaction regions were constructed and
expressed. These chimeric polypeptides were designated FGF19-4,
FGF19-5 and FGF19-6, respectively, and are shown graphically in
FIG. 9A. The activities of these chimeric polypeptides were tested
in vitro and in vivo assays.
[0203] These combination chimeric polypeptides were no longer able
to activate FGFR4 signaling in L6 cells in the absence or presence
of .beta.Klotho but were still able to activate FGFR1c signaling
(FIG. 9B), therefore, selectively abolishing FGFR4 activity.
Consistent with this observation, histopathological examination of
liver sections from FGF19-4, -5, and -6, treated animals did not
show increased numbers of BrdU-labeled hepatocytes in pericentral
regions, nor was increased BrdU labeling noted in any other area of
the liver (FIG. 9C). Therefore, enhanced hepatocyte proliferation
associated with wild type FGF19 was abolished by the combined
mutagenesis of the 5 FGF19 amino acid residues WGDPI and heparin
domains.
[0204] To rule out the possibility that the lack of positive BrdU
labeling is due to differences in the degradation and clearance of
the chimeric proteins in serum, the serum concentration of the
chimeras was measured at various time points after injection into
the mice and similar pharmacokinetic properties of the chimeric
proteins to wild type FGF19 were observed.
Example 10
Chimeric Molecules Lacking the Ability to Activate FGFR4 Can Still
Regulate Glucose Homeostasis
[0205] Since the chimeric FGF19 molecules FGF19-4, FGF19-5, and
FGF19-6, which comprise the combined substitutions of the 5 amino
acids from positions 38-42 of full length FGF19, namely residues
WGDPI, and also one or both of the heparin binding domains, were
still able to activate FGFR1c/.beta.Klotho receptor signaling in L6
cells (FIG. 9B), their ability to regulate glucose homeostasis was
tested.
[0206] The effect of these chimeric proteins on glucose uptake into
adipocytes was first tested. Similar to wild type FGF19 protein,
the chimeric proteins were also able to stimulate glucose uptake
independent of insulin into 3T3L1 adipocytes in vitro (FIG.
10A).
[0207] To further investigate the ability of the chimeric proteins
to regulate glucose homeostasis, ob/ob mice were injected
intraperitoneally with FGF19 or FGF19-4 and blood glucose levels
were measured at 0, 1, 3, and 5 hrs post injection; the values are
reported as area under the curve (AUC) means .+-.S.E.M. over this
time period (FIG. 10B). Plasma glucose levels were significantly
reduced in mice injected with both FGF19 and FGF19-4 with
comparable potency and efficacy (FIG. 10B). These results indicate
that FGF19-4 only selectively lost its ability to induce FGFR4
mediated hepatocyte proliferation, but retained its ability to
modulate glucose regulation.
[0208] Replacing the .beta.1-.beta.2 loop segment of FGF19 only
(FGF19-1) abolished heparin induced FGF19/FGFR4 interaction while
preserving the ability to interact with FGFR4 in the presence of
.beta.Klotho. Similar to wild type FGF19, FGF19-1 is also still
active in other metabolic assays, able to induce glucose uptake
into adipocyte cells (FIG. 10C) and reduced plasma glucose levels
in ob/ob diabetic animals model (FIG. 10D), suggesting that heparin
domain mutations did not affect other FGF19 mediated functions.
Example 11
Pharmacokinetic Analysis of the Chimeric Proteins
[0209] The pharmacokinetic profiles of the chimeric constructs
FGF19/21-1, FGF19/21-2, FGF19/21-3, FGF19/21-4 and FGF19/21-5 were
studied. The following protocol was employed. Following i.p.
injections of 2 mg/kg FGF19/21 chimeric proteins (n=5), C57BL6
mouse serum samples were collected 15 min, 1 hour, 3 hours, and 6
hours after the injections. FGF19/21 chimeric protein
concentrations were determined by an enzyme-linked immunosorbent
assay (ELISA) developed at Amgen. The antibodies used as capture
and detection reagents were generated in-house. A mouse monoclonal
antibody raised against human FGF21 was used as the capture
antibody and was specific for an epitope near the C-terminus on
human FGF21. A biotin-conjugated rabbit polyclonal antibody raised
against human FGF21 was used as the detection antibody and
recognized multiple epitopes on human FGF21.
[0210] The ELISA was performed as follows. The capture antibody was
bound onto a 96-well polystyrene microplate. Standards and quality
control samples were prepared by spiking the FGF19/21 chimera into
mouse plasma. Standards, quality controls, matrix blank, and
unknown samples were loaded into the wells after pretreatment in
assay buffer. After a two hour incubation followed by washing, the
biotin-conjugated detection antibody was added to the wells. After
a one hour incubation followed by washing, a
streptavidin-horseradish peroxidase (HRP) conjugate (R&D
Systems, Inc) was added to the wells. After a 30 minute incubation
followed by washing, a tetramethylbenzidine (TMB) peroxidase
substrate solution was added to the wells. In the presence of HRP,
a colorimetric signal was produced that was proportional to the
amount of FGF19/21 chimera bound by the capture antibody. The color
development was stopped and the intensity of the color (optical
density, OD) was measured at 450-650 nm with a plate reader. The
conversion of OD units to concentration for the unknown samples was
achieved through a software-mediated comparison to a standard curve
assayed on the same plate. The data were regressed using SoftMax
Pro 5 (Molecular Devices Corp.) data reduction package.
[0211] The results of the study are presented in FIG. 11.
Example 12
Deletion or Mutation of FGF19 Residue W38 Abolishes FGFR4 and
FGFR1c Function
[0212] 16 mutants within the residues WGDPI (SEQ ID NO:49) region
at positions 38-42 of the FGF19-1 polypeptide (in which residues
50-57 of FGF19 were replaced with residues 52-58 of FGF21; see FIG.
8) were expressed and purified as described in Example 1. L6 cells
transfected with either FGFR1c/.beta.Klotho or FGFR4/.beta.Klotho
were treated with those purified proteins. Activities of mutants on
FGFR1c or FGFR4 were determined by measuring ERK phosphorylation
levels 15 min after treatments and are summarized in FIG. 12.
[0213] The bar graphs of FIG. 13 reflects the level of
FGFR1c-mediated activity of FGF19 mutants administered at
concentrations of 0, 2.5, 16 and 100 nM. The bar graphs of FIG. 14
reflects the level of FGFR1c-mediated activity of FGF19 mutants
administered at concentrations of 0, 2.5, 7.4, 44, 67 and 200
nM.
[0214] The bar graphs of FIG. 15 reflect the level of
FGFR4-mediated activity of FGF19 mutants administered at
concentrations of 0, 2.5, 16 and 100 nM. The bar graphs of FIG. 15
reflects the level of FGFR4-mediated activity of FGF 19 mutants
administered at concentrations of 0, 2.5, 7.4, 44, 67 and 200
nM.
[0215] Deletion of W38, P41, and 142 abolished activation of both
FGFR1c and FGFR4 receptor by the mutant FGF19 proteins, while
deletion of G39 reduced activity on both receptors and deletion of
D40 selectively removed FGFR1c activity with much less effect on
FGFR4 activity.
[0216] Each of the 5 amino acids was then individually mutated to
alanine to study their involvement in receptor activation. While
some of the mutations in the GDPI (SEQ ID NO:71) sequence to
alanine affected either potency and/or efficacy, only W38A
completely abolished activation of both FGFR1c and FGFR4.
[0217] The results of this study indicate that W38 is a critical
residue for FGF19-induced FGFR activation. The results further
indicate that deletion or mutagenesis of this residue to
selectively decrease or remove FGFR4-mediated activity from FGF19
would require a change from wild type at position 38, particularly
a deletion or mutation. This decrease in FGFR4-mediated activity
may be achievable by mutating or deleting W38 alone or may require
additional deletions or mutations in the WGDPI or surrounding
region. One such example is FGF19-4 (residues 38-42 of FGF19 were
replaced with residues 42-44 of FGF21 and residues 50-57 of FGF19
were replaced with residues 52-58 of FGF21; see FIG. 8) in which
concurrent deletion of W38, P41 and mutation of I42V resulted in
such selective FGFR1c-mediated activity.
[0218] By mitigating the mitogenicity of FGF19, a mutant form of
FGF19 comprising a mutation or deletion at position 38 could make
FGF19 a therapeutically relevant molecule and an attractive
candidate for pharmaceutical development.
Sequence CWU 1
1
711651DNAHomo sapiensCDS(1)..(651) 1atg cgg agc ggg tgt gtg gtg gtc
cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val Val Val
His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg gcc ggg
cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val Ala Gly
Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt cac tac
ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144His Val His Tyr
Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 acc tcc
ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc 192Thr Ser
Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60
gac ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg ctg
240Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu
65 70 75 80 gag atc aag gca gtc gct ctg cgg acc gtg gcc atc aag ggc
gtg cac 288Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly
Val His 85 90 95 agc gtg cgg tac ctc tgc atg ggc gcc gac ggc aag
atg cag ggg ctg 336Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys
Met Gln Gly Leu 100 105 110 ctt cag tac tcg gag gaa gac tgt gct ttc
gag gag gag atc cgc cca 384Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro 115 120 125 gat ggc tac aat gtg tac cga tcc
gag aag cac cgc ctc ccg gtc tcc 432Asp Gly Tyr Asn Val Tyr Arg Ser
Glu Lys His Arg Leu Pro Val Ser 130 135 140 ctg agc agt gcc aaa cag
cgg cag ctg tac aag aac aga ggc ttt ctt 480Leu Ser Ser Ala Lys Gln
Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150 155 160 cca ctc tct
cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct 528Pro Leu Ser
His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170 175 gag
gac ctc agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg 576Glu
Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu 180 185
190 gag acc gac agc atg gac cca ttt ggg ctt gtc acc gga ctg gag gcc
624Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala
195 200 205 gtg agg agt ccc agc ttt gag aag taa 651Val Arg Ser Pro
Ser Phe Glu Lys 210 215 2216PRTHomo sapiens 2Met Arg Ser Gly Cys
Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu Ala
Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 His
Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40
45 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala
50 55 60 Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser
Leu Leu 65 70 75 80 Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile
Lys Gly Val His 85 90 95 Ser Val Arg Tyr Leu Cys Met Gly Ala Asp
Gly Lys Met Gln Gly Leu 100 105 110 Leu Gln Tyr Ser Glu Glu Asp Cys
Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 Asp Gly Tyr Asn Val Tyr
Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 Leu Ser Ser Ala
Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150 155 160 Pro
Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170
175 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu
180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu
Glu Ala 195 200 205 Val Arg Ser Pro Ser Phe Glu Lys 210 215
3585DNAHomo sapiensCDS(1)..(585) 3cgt cca ctt gct ttt tct gat gct
ggt cca cac gtt cac tac ggc tgg 48Arg Pro Leu Ala Phe Ser Asp Ala
Gly Pro His Val His Tyr Gly Trp 1 5 10 15 ggc gac ccc atc cgc ctg
cgg cac ctg tac acc tcc ggc ccc cac ggg 96Gly Asp Pro Ile Arg Leu
Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30 ctc tcc agc tgc
ttc ctg cgc atc cgt gcc gac ggc gtc gtg gac tgc 144Leu Ser Ser Cys
Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40 45 gcg cgg
ggc cag agc gcg cac agt ttg ctg gag atc aag gca gtc gct 192Ala Arg
Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala 50 55 60
ctg cgg acc gtg gcc atc aag ggc gtg cac agc gtg cgg tac ctc tgc
240Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys
65 70 75 80 atg ggc gcc gac ggc aag atg cag ggg ctg ctt cag tac tcg
gag gaa 288Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser
Glu Glu 85 90 95 gac tgt gct ttc gag gag gag atc cgc cca gat ggc
tac aat gtg tac 336Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly
Tyr Asn Val Tyr 100 105 110 cga tcc gag aag cac cgc ctc ccg gtc tcc
ctg agc agt gcc aaa cag 384Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu Ser Ser Ala Lys Gln 115 120 125 cgg cag ctg tac aag aac aga ggc
ttt ctt cca ctc tct cat ttc ctg 432Arg Gln Leu Tyr Lys Asn Arg Gly
Phe Leu Pro Leu Ser His Phe Leu 130 135 140 ccc atg ctg ccc atg gtc
cca gag gag cct gag gac ctc agg ggc cac 480Pro Met Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His 145 150 155 160 ttg gaa tct
gac atg ttc tct tcg ccc ctg gag acc gac agc atg gac 528Leu Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170 175 cca
ttt ggg ctt gtc acc gga ctg gag gcc gtg agg agt ccc agc ttt 576Pro
Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe 180 185
190 gag aag taa 585Glu Lys 4194PRTHomo sapiens 4Arg Pro Leu Ala Phe
Ser Asp Ala Gly Pro His Val His Tyr Gly Trp 1 5 10 15 Gly Asp Pro
Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly 20 25 30 Leu
Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys 35 40
45 Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala
50 55 60 Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr
Leu Cys 65 70 75 80 Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln
Tyr Ser Glu Glu 85 90 95 Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro
Asp Gly Tyr Asn Val Tyr 100 105 110 Arg Ser Glu Lys His Arg Leu Pro
Val Ser Leu Ser Ser Ala Lys Gln 115 120 125 Arg Gln Leu Tyr Lys Asn
Arg Gly Phe Leu Pro Leu Ser His Phe Leu 130 135 140 Pro Met Leu Pro
Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His 145 150 155 160 Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp 165 170
175 Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe
180 185 190 Glu Lys 5588DNAHomo sapiensCDS(1)..(588) 5atg cgt cca
ctt gct ttt tct gat gct ggt cca cac gtt cac tac ggc 48Met Arg Pro
Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly 1 5 10 15 tgg
ggc gac ccc atc cgc ctg cgg cac ctg tac acc tcc ggc ccc cac 96Trp
Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His 20 25
30 ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc gac ggc gtc gtg gac
144Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
35 40 45 tgc gcg cgg ggc cag agc gcg cac agt ttg ctg gag atc aag
gca gtc 192Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys
Ala Val 50 55 60 gct ctg cgg acc gtg gcc atc aag ggc gtg cac agc
gtg cgg tac ctc 240Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser
Val Arg Tyr Leu 65 70 75 80 tgc atg ggc gcc gac ggc aag atg cag ggg
ctg ctt cag tac tcg gag 288Cys Met Gly Ala Asp Gly Lys Met Gln Gly
Leu Leu Gln Tyr Ser Glu 85 90 95 gaa gac tgt gct ttc gag gag gag
atc cgc cca gat ggc tac aat gtg 336Glu Asp Cys Ala Phe Glu Glu Glu
Ile Arg Pro Asp Gly Tyr Asn Val 100 105 110 tac cga tcc gag aag cac
cgc ctc ccg gtc tcc ctg agc agt gcc aaa 384Tyr Arg Ser Glu Lys His
Arg Leu Pro Val Ser Leu Ser Ser Ala Lys 115 120 125 cag cgg cag ctg
tac aag aac aga ggc ttt ctt cca ctc tct cat ttc 432Gln Arg Gln Leu
Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe 130 135 140 ctg ccc
atg ctg ccc atg gtc cca gag gag cct gag gac ctc agg ggc 480Leu Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly 145 150 155
160 cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag acc gac agc atg
528His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
165 170 175 gac cca ttt ggg ctt gtc acc gga ctg gag gcc gtg agg agt
ccc agc 576Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser
Pro Ser 180 185 190 ttt gag aag taa 588Phe Glu Lys 195 6195PRTHomo
sapiens 6Met Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His
Tyr Gly 1 5 10 15 Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr
Ser Gly Pro His 20 25 30 Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val Val Asp 35 40 45 Cys Ala Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys Ala Val 50 55 60 Ala Leu Arg Thr Val Ala
Ile Lys Gly Val His Ser Val Arg Tyr Leu 65 70 75 80 Cys Met Gly Ala
Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu 85 90 95 Glu Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val 100 105 110
Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys 115
120 125 Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His
Phe 130 135 140 Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly 145 150 155 160 His Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu Thr Asp Ser Met 165 170 175 Asp Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser 180 185 190 Phe Glu Lys 195
7630DNAHomo sapiensCDS(1)..(630) 7atg gac tcg gac gag acc ggg ttc
gag cac tca gga ctg tgg gtt tct 48Met Asp Ser Asp Glu Thr Gly Phe
Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 gtg ctg gct ggt ctt ctg
ctg gga gcc tgc cag gca cat cca att cca 96Val Leu Ala Gly Leu Leu
Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 gat tct tct cca
tta tta caa ttc ggg ggc caa gtc cgg cag cgg tac 144Asp Ser Ser Pro
Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr 35 40 45 ctc tac
aca gat gat gcc cag cag aca gaa gcc cac ctg gag atc agg 192Leu Tyr
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg 50 55 60
gag gat ggg acg gtg ggg ggc gct gct gac cag agc ccc gaa agt ctc
240Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
65 70 75 80 ctg cag ctg aaa gcc ttg aag ccg gga gtt att caa atc ttg
gga gtc 288Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu
Gly Val 85 90 95 aag aca tcc agg ttc ctg tgc cag cgg cca gat ggg
gcc ctg tat gga 336Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
Ala Leu Tyr Gly 100 105 110 tcg ctc cac ttt gac cct gag gcc tgc agc
ttc cgg gag ctg ctt ctt 384Ser Leu His Phe Asp Pro Glu Ala Cys Ser
Phe Arg Glu Leu Leu Leu 115 120 125 gag gac gga tac aat gtt tac cag
tcc gaa gcc cac ggc ctc ccg ctg 432Glu Asp Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His Gly Leu Pro Leu 130 135 140 cac ctg cca ggg aac aag
tcc cca cac cgg gac cct gca ccc cga gga 480His Leu Pro Gly Asn Lys
Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155 160 cca gct cgc
ttc ctg cca cta cca ggc ctg ccc ccc gca ccc ccg gag 528Pro Ala Arg
Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu 165 170 175 cca
ccc gga atc ctg gcc ccc cag ccc ccc gat gtg ggc tcc tcg gac 576Pro
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 180 185
190 cct ctg agc atg gtg gga cct tcc cag ggc cga agc ccc agc tac gct
624Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
195 200 205 tcc tga 630Ser 8209PRTHomo sapiens 8Met Asp Ser Asp Glu
Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 Val Leu Ala
Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr 35 40
45 Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
50 55 60 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
Ser Leu 65 70 75 80 Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
Ile Leu Gly Val 85 90 95 Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly Ala Leu Tyr Gly 100 105 110 Ser Leu His Phe Asp Pro Glu Ala
Cys Ser Phe Arg Glu Leu Leu Leu 115 120 125 Glu Asp Gly Tyr Asn Val
Tyr Gln Ser Glu Ala His Gly Leu Pro Leu 130 135 140 His Leu Pro Gly
Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155 160 Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu 165 170
175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser
Tyr Ala 195 200
205 Ser 9546DNAHomo sapiensCDS(1)..(546) 9cac ccc atc cct gac tcc
agt cct ctc ctg caa ttc ggg ggc caa gtc 48His Pro Ile Pro Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 cgg cag cgg tac
ctc tac aca gat gat gcc cag cag aca gaa gcc cac 96Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 ctg gag
atc agg gag gat ggg acg gtg ggg ggc gct gct gac cag agc 144Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45
ccc gaa agt ctc ctg cag ctg aaa gcc ttg aag ccg gga gtt att caa
192Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60 atc ttg gga gtc aag aca tcc agg ttc ctg tgc cag cgg cca
gat ggg 240Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly 65 70 75 80 gcc ctg tat gga tcg ctc cac ttt gac cct gag gcc
tgc agc ttc cgg 288Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala
Cys Ser Phe Arg 85 90 95 gag ctg ctt ctt gag gac gga tac aat gtt
tac cag tcc gaa gcc cac 336Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val
Tyr Gln Ser Glu Ala His 100 105 110 ggc ctc ccg ctg cac ctg cca ggg
aac aag tcc cca cac cgg gac cct 384Gly Leu Pro Leu His Leu Pro Gly
Asn Lys Ser Pro His Arg Asp Pro 115 120 125 gca ccc cga gga cca gct
cgc ttc ctg cca cta cca ggc ctg ccc ccc 432Ala Pro Arg Gly Pro Ala
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 gca ccc ccg gag
cca ccc gga atc ctg gcc ccc cag ccc ccc gat gtg 480Ala Pro Pro Glu
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 ggc
tcc tcg gac cct ctg agc atg gtg gga cct tcc cag ggc cga agc 528Gly
Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser 165 170
175 ccc agc tac gct tcc tga 546Pro Ser Tyr Ala Ser 180 10181PRTHomo
sapiens 10His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly
Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln
Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly
Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu
Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115
120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
Pro 130 135 140 Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly
Pro Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180
11549DNAHomo sapiensCDS(1)..(549) 11atg cac ccc atc cct gac tcc agt
cct ctc ctg caa ttc ggg ggc caa 48Met His Pro Ile Pro Asp Ser Ser
Pro Leu Leu Gln Phe Gly Gly Gln 1 5 10 15 gtc cgg cag cgg tac ctc
tac aca gat gat gcc cag cag aca gaa gcc 96Val Arg Gln Arg Tyr Leu
Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala 20 25 30 cac ctg gag atc
agg gag gat ggg acg gtg ggg ggc gct gct gac cag 144His Leu Glu Ile
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln 35 40 45 agc ccc
gaa agt ctc ctg cag ctg aaa gcc ttg aag ccg gga gtt att 192Ser Pro
Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile 50 55 60
caa atc ttg gga gtc aag aca tcc agg ttc ctg tgc cag cgg cca gat
240Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
65 70 75 80 ggg gcc ctg tat gga tcg ctc cac ttt gac cct gag gcc tgc
agc ttc 288Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser Phe 85 90 95 cgg gag ctg ctt ctt gag gac gga tac aat gtt tac
cag tcc gaa gcc 336Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr
Gln Ser Glu Ala 100 105 110 cac ggc ctc ccg ctg cac ctg cca ggg aac
aag tcc cca cac cgg gac 384His Gly Leu Pro Leu His Leu Pro Gly Asn
Lys Ser Pro His Arg Asp 115 120 125 cct gca ccc cga gga cca gct cgc
ttc ctg cca cta cca ggc ctg ccc 432Pro Ala Pro Arg Gly Pro Ala Arg
Phe Leu Pro Leu Pro Gly Leu Pro 130 135 140 ccc gca ccc ccg gag cca
ccc gga atc ctg gcc ccc cag ccc ccc gat 480Pro Ala Pro Pro Glu Pro
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 145 150 155 160 gtg ggc tcc
tcg gac cct ctg agc atg gtg gga cct tcc cag ggc cga 528Val Gly Ser
Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg 165 170 175 agc
ccc agc tac gct tcc tga 549Ser Pro Ser Tyr Ala Ser 180 12182PRTHomo
sapiens 12Met His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln 1 5 10 15 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln
Gln Thr Glu Ala 20 25 30 His Leu Glu Ile Arg Glu Asp Gly Thr Val
Gly Gly Ala Ala Asp Gln 35 40 45 Ser Pro Glu Ser Leu Leu Gln Leu
Lys Ala Leu Lys Pro Gly Val Ile 50 55 60 Gln Ile Leu Gly Val Lys
Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp 65 70 75 80 Gly Ala Leu Tyr
Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 85 90 95 Arg Glu
Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala 100 105 110
His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp 115
120 125 Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu
Pro 130 135 140 Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp 145 150 155 160 Val Gly Ser Ser Asp Pro Leu Ser Met Val
Gly Pro Ser Gln Gly Arg 165 170 175 Ser Pro Ser Tyr Ala Ser 180
13756DNAHomo sapiensCDS(1)..(756) 13atg ttg ggg gcc cgc ctc agg ctc
tgg gtc tgt gcc ttg tgc agc gtc 48Met Leu Gly Ala Arg Leu Arg Leu
Trp Val Cys Ala Leu Cys Ser Val 1 5 10 15 tgc agc atg agc gtc ctc
aga gcc tat ccc aat gcc tcc cca ctg ctc 96Cys Ser Met Ser Val Leu
Arg Ala Tyr Pro Asn Ala Ser Pro Leu Leu 20 25 30 ggc tcc agc tgg
ggt ggc ctg atc cac ctg tac aca gcc aca gcc agg 144Gly Ser Ser Trp
Gly Gly Leu Ile His Leu Tyr Thr Ala Thr Ala Arg 35 40 45 aac agc
tac cac ctg cag atc cac aag aat ggc cat gtg gat ggc gca 192Asn Ser
Tyr His Leu Gln Ile His Lys Asn Gly His Val Asp Gly Ala 50 55 60
ccc cat cag acc atc tac agt gcc ctg atg atc aga tca gag gat gct
240Pro His Gln Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu Asp Ala
65 70 75 80 ggc ttt gtg gtg att aca ggt gtg atg agc aga aga tac ctc
tgc atg 288Gly Phe Val Val Ile Thr Gly Val Met Ser Arg Arg Tyr Leu
Cys Met 85 90 95 gat ttc aga ggc aac att ttt gga tca cac tat ttc
gac ccg gag aac 336Asp Phe Arg Gly Asn Ile Phe Gly Ser His Tyr Phe
Asp Pro Glu Asn 100 105 110 tgc agg ttc caa cac cag acg ctg gaa aac
ggg tac gac gtc tac cac 384Cys Arg Phe Gln His Gln Thr Leu Glu Asn
Gly Tyr Asp Val Tyr His 115 120 125 tct cct cag tat cac ttc ctg gtc
agt ctg ggc cgg gcg aag aga gcc 432Ser Pro Gln Tyr His Phe Leu Val
Ser Leu Gly Arg Ala Lys Arg Ala 130 135 140 ttc ctg cca ggc atg aac
cca ccc ccg tac tcc cag ttc ctg tcc cgg 480Phe Leu Pro Gly Met Asn
Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg 145 150 155 160 agg aac gag
atc ccc cta att cac ttc aac acc ccc ata cca cgg cgg 528Arg Asn Glu
Ile Pro Leu Ile His Phe Asn Thr Pro Ile Pro Arg Arg 165 170 175 cac
acc cgg agc gcc gag gac gac tcg gag cgg gac ccc ctg aac gtg 576His
Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val 180 185
190 ctg aag ccc cgg gcc cgg atg acc ccg gcc ccg gcc tcc tgt tca cag
624Leu Lys Pro Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys Ser Gln
195 200 205 gag ctc ccg agc gcc gag gac aac agc ccg atg gcc agt gac
cca tta 672Glu Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala Ser Asp
Pro Leu 210 215 220 ggg gtg gtc agg ggc ggt cga gtg aac acg cac gct
ggg gga acg ggc 720Gly Val Val Arg Gly Gly Arg Val Asn Thr His Ala
Gly Gly Thr Gly 225 230 235 240 ccg gaa ggc tgc cgc ccc ttc gcc aag
ttc atc tag 756Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe Ile 245 250
14251PRTHomo sapiens 14Met Leu Gly Ala Arg Leu Arg Leu Trp Val Cys
Ala Leu Cys Ser Val 1 5 10 15 Cys Ser Met Ser Val Leu Arg Ala Tyr
Pro Asn Ala Ser Pro Leu Leu 20 25 30 Gly Ser Ser Trp Gly Gly Leu
Ile His Leu Tyr Thr Ala Thr Ala Arg 35 40 45 Asn Ser Tyr His Leu
Gln Ile His Lys Asn Gly His Val Asp Gly Ala 50 55 60 Pro His Gln
Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu Asp Ala 65 70 75 80 Gly
Phe Val Val Ile Thr Gly Val Met Ser Arg Arg Tyr Leu Cys Met 85 90
95 Asp Phe Arg Gly Asn Ile Phe Gly Ser His Tyr Phe Asp Pro Glu Asn
100 105 110 Cys Arg Phe Gln His Gln Thr Leu Glu Asn Gly Tyr Asp Val
Tyr His 115 120 125 Ser Pro Gln Tyr His Phe Leu Val Ser Leu Gly Arg
Ala Lys Arg Ala 130 135 140 Phe Leu Pro Gly Met Asn Pro Pro Pro Tyr
Ser Gln Phe Leu Ser Arg 145 150 155 160 Arg Asn Glu Ile Pro Leu Ile
His Phe Asn Thr Pro Ile Pro Arg Arg 165 170 175 His Thr Arg Ser Ala
Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val 180 185 190 Leu Lys Pro
Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys Ser Gln 195 200 205 Glu
Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala Ser Asp Pro Leu 210 215
220 Gly Val Val Arg Gly Gly Arg Val Asn Thr His Ala Gly Gly Thr Gly
225 230 235 240 Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe Ile 245 250
15684DNAHomo sapiensCDS(1)..(684) 15tat ccc aat gcc tcc cca ctg ctc
ggc tcc agc tgg ggt ggc ctg atc 48Tyr Pro Asn Ala Ser Pro Leu Leu
Gly Ser Ser Trp Gly Gly Leu Ile 1 5 10 15 cac ctg tac aca gcc aca
gcc agg aac agc tac cac ctg cag atc cac 96His Leu Tyr Thr Ala Thr
Ala Arg Asn Ser Tyr His Leu Gln Ile His 20 25 30 aag aat ggc cat
gtg gat ggc gca ccc cat cag acc atc tac agt gcc 144Lys Asn Gly His
Val Asp Gly Ala Pro His Gln Thr Ile Tyr Ser Ala 35 40 45 ctg atg
atc aga tca gag gat gct ggc ttt gtg gtg att aca ggt gtg 192Leu Met
Ile Arg Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly Val 50 55 60
atg agc aga aga tac ctc tgc atg gat ttc aga ggc aac att ttt gga
240Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe Gly
65 70 75 80 tca cac tat ttc gac ccg gag aac tgc agg ttc caa cac cag
acg ctg 288Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln
Thr Leu 85 90 95 gaa aac ggg tac gac gtc tac cac tct cct cag tat
cac ttc ctg gtc 336Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr
His Phe Leu Val 100 105 110 agt ctg ggc cgg gcg aag aga gcc ttc ctg
cca ggc atg aac cca ccc 384Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu
Pro Gly Met Asn Pro Pro 115 120 125 ccg tac tcc cag ttc ctg tcc cgg
agg aac gag atc ccc cta att cac 432Pro Tyr Ser Gln Phe Leu Ser Arg
Arg Asn Glu Ile Pro Leu Ile His 130 135 140 ttc aac acc ccc ata cca
cgg cgg cac acc cgg agc gcc gag gac gac 480Phe Asn Thr Pro Ile Pro
Arg Arg His Thr Arg Ser Ala Glu Asp Asp 145 150 155 160 tcg gag cgg
gac ccc ctg aac gtg ctg aag ccc cgg gcc cgg atg acc 528Ser Glu Arg
Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg Met Thr 165 170 175 ccg
gcc ccg gcc tcc tgt tca cag gag ctc ccg agc gcc gag gac aac 576Pro
Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp Asn 180 185
190 agc ccg atg gcc agt gac cca tta ggg gtg gtc agg ggc ggt cga gtg
624Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg Val
195 200 205 aac acg cac gct ggg gga acg ggc ccg gaa ggc tgc cgc ccc
ttc gcc 672Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro
Phe Ala 210 215 220 aag ttc atc tag 684Lys Phe Ile 225 16227PRTHomo
sapiens 16Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly Gly
Leu Ile 1 5 10 15 His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His
Leu Gln Ile His 20 25 30 Lys Asn Gly His Val Asp Gly Ala Pro His
Gln Thr Ile Tyr Ser Ala 35 40 45 Leu Met Ile Arg Ser Glu Asp Ala
Gly Phe Val Val Ile Thr Gly Val 50 55 60 Met Ser Arg Arg Tyr Leu
Cys Met Asp Phe Arg Gly Asn Ile Phe Gly 65 70 75 80 Ser His Tyr Phe
Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr Leu 85 90 95 Glu Asn
Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu Val 100 105 110
Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro Pro 115
120 125 Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu Ile
His 130
135 140 Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg Ser Ala Glu Asp
Asp 145 150 155 160 Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro Arg
Ala Arg Met Thr 165 170 175 Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu
Pro Ser Ala Glu Asp Asn 180 185 190 Ser Pro Met Ala Ser Asp Pro Leu
Gly Val Val Arg Gly Gly Arg Val 195 200 205 Asn Thr His Ala Gly Gly
Thr Gly Pro Glu Gly Cys Arg Pro Phe Ala 210 215 220 Lys Phe Ile 225
17687DNAHomo sapiensCDS(1)..(687) 17atg tat ccc aat gcc tcc cca ctg
ctc ggc tcc agc tgg ggt ggc ctg 48Met Tyr Pro Asn Ala Ser Pro Leu
Leu Gly Ser Ser Trp Gly Gly Leu 1 5 10 15 atc cac ctg tac aca gcc
aca gcc agg aac agc tac cac ctg cag atc 96Ile His Leu Tyr Thr Ala
Thr Ala Arg Asn Ser Tyr His Leu Gln Ile 20 25 30 cac aag aat ggc
cat gtg gat ggc gca ccc cat cag acc atc tac agt 144His Lys Asn Gly
His Val Asp Gly Ala Pro His Gln Thr Ile Tyr Ser 35 40 45 gcc ctg
atg atc aga tca gag gat gct ggc ttt gtg gtg att aca ggt 192Ala Leu
Met Ile Arg Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly 50 55 60
gtg atg agc aga aga tac ctc tgc atg gat ttc aga ggc aac att ttt
240Val Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe
65 70 75 80 gga tca cac tat ttc gac ccg gag aac tgc agg ttc caa cac
cag acg 288Gly Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His
Gln Thr 85 90 95 ctg gaa aac ggg tac gac gtc tac cac tct cct cag
tat cac ttc ctg 336Leu Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln
Tyr His Phe Leu 100 105 110 gtc agt ctg ggc cgg gcg aag aga gcc ttc
ctg cca ggc atg aac cca 384Val Ser Leu Gly Arg Ala Lys Arg Ala Phe
Leu Pro Gly Met Asn Pro 115 120 125 ccc ccg tac tcc cag ttc ctg tcc
cgg agg aac gag atc ccc cta att 432Pro Pro Tyr Ser Gln Phe Leu Ser
Arg Arg Asn Glu Ile Pro Leu Ile 130 135 140 cac ttc aac acc ccc ata
cca cgg cgg cac acc cgg agc gcc gag gac 480His Phe Asn Thr Pro Ile
Pro Arg Arg His Thr Arg Ser Ala Glu Asp 145 150 155 160 gac tcg gag
cgg gac ccc ctg aac gtg ctg aag ccc cgg gcc cgg atg 528Asp Ser Glu
Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg Met 165 170 175 acc
ccg gcc ccg gcc tcc tgt tca cag gag ctc ccg agc gcc gag gac 576Thr
Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp 180 185
190 aac agc ccg atg gcc agt gac cca tta ggg gtg gtc agg ggc ggt cga
624Asn Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg
195 200 205 gtg aac acg cac gct ggg gga acg ggc ccg gaa ggc tgc cgc
ccc ttc 672Val Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg
Pro Phe 210 215 220 gcc aag ttc atc tag 687Ala Lys Phe Ile 225
18228PRTHomo sapiens 18Met Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser
Ser Trp Gly Gly Leu 1 5 10 15 Ile His Leu Tyr Thr Ala Thr Ala Arg
Asn Ser Tyr His Leu Gln Ile 20 25 30 His Lys Asn Gly His Val Asp
Gly Ala Pro His Gln Thr Ile Tyr Ser 35 40 45 Ala Leu Met Ile Arg
Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly 50 55 60 Val Met Ser
Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe 65 70 75 80 Gly
Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr 85 90
95 Leu Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu
100 105 110 Val Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met
Asn Pro 115 120 125 Pro Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu
Ile Pro Leu Ile 130 135 140 His Phe Asn Thr Pro Ile Pro Arg Arg His
Thr Arg Ser Ala Glu Asp 145 150 155 160 Asp Ser Glu Arg Asp Pro Leu
Asn Val Leu Lys Pro Arg Ala Arg Met 165 170 175 Thr Pro Ala Pro Ala
Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp 180 185 190 Asn Ser Pro
Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg 195 200 205 Val
Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro Phe 210 215
220 Ala Lys Phe Ile 225 19534DNAArtificialtruncated protein 19atg
cgg agc ggg tgt gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met
Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10
15 tgg ctg gcc gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca
96Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
20 25 30 cac gtt cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac
ctg tac 144His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His
Leu Tyr 35 40 45 acc tcc ggc ccc cac ggg ctc tcc agc tgc ttc ctg
cgc atc cgt gcc 192Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala 50 55 60 gac ggc gtc gtg gac tgc gcg cgg ggc cag
agc gcg cac agt ttg ctg 240Asp Gly Val Val Asp Cys Ala Arg Gly Gln
Ser Ala His Ser Leu Leu 65 70 75 80 gag atc aag gca gtc gct ctg cgg
acc gtg gcc atc aag ggc gtg cac 288Glu Ile Lys Ala Val Ala Leu Arg
Thr Val Ala Ile Lys Gly Val His 85 90 95 agc gtg cgg tac ctc tgc
atg ggc gcc gac ggc aag atg cag ggg ctg 336Ser Val Arg Tyr Leu Cys
Met Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105 110 ctt cag tac tcg
gag gaa gac tgt gct ttc gag gag gag atc cgc cca 384Leu Gln Tyr Ser
Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 gat ggc
tac aat gtg tac cga tcc gag aag cac cgc ctc ccg gtc tcc 432Asp Gly
Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140
ctg agc agt gcc aaa cag cgg cag ctg tac aag aac aga ggc ttt ctt
480Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu
145 150 155 160 cca ctc tct cat ttc ctg ccc atg ctg ccc atg gtc cca
gag gag cct 528Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro
Glu Glu Pro 165 170 175 gag taa 534Glu 20177PRTArtificialSynthetic
Construct 20Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala
Gly Leu 1 5 10 15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro 20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr 35 40 45 Thr Ser Gly Pro His Gly Leu Ser
Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60 Asp Gly Val Val Asp Cys
Ala Arg Gly Gln Ser Ala His Ser Leu Leu 65 70 75 80 Glu Ile Lys Ala
Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His 85 90 95 Ser Val
Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105 110
Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115
120 125 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val
Ser 130 135 140 Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg
Gly Phe Leu 145 150 155 160 Pro Leu Ser His Phe Leu Pro Met Leu Pro
Met Val Pro Glu Glu Pro 165 170 175 Glu 21624DNAArtificialchimeric
fusion protein 21atg cgg agc ggg tgt gtg gtg gtc cac gta tgg atc
ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val Val Val His Val Trp Ile
Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg gcc ggg cgt cca ctt gct
ttt tct gat gct ggt cca 96Trp Leu Ala Val Ala Gly Arg Pro Leu Ala
Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt cac tac ggc tgg ggc gac
ccc atc cgc ctg cgg cac ctg tac 144His Val His Tyr Gly Trp Gly Asp
Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 acc gat gat gcc cag cag
aca gaa tgc ttc ctg cgc atc cgt gcc gac 192Thr Asp Asp Ala Gln Gln
Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp 50 55 60 ggc gtc gtg gac
tgc gcg cgg ggc cag agc gcg cac agt ttg ctg gag 240Gly Val Val Asp
Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu 65 70 75 80 atc aag
gca gtc gct ctg cgg acc gtg gcc atc aag ggc gtg cac agc 288Ile Lys
Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser 85 90 95
gtg cgg tac ctc tgc atg ggc gcc gac ggc aag atg cag ggg ctg ctt
336Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu
100 105 110 cag tac tcg gag gaa gac tgt gct ttc gag gag ctg ctt ctt
gag gac 384Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Leu Leu Leu
Glu Asp 115 120 125 gga tac aat gtt tac cag tcc gaa gcc cac ggc ctc
ccg ctg cac ctg 432Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu
Pro Leu His Leu 130 135 140 cca ggg aac aag tcc cca cac cgg gac cct
gca ccc cga gga cca gct 480Pro Gly Asn Lys Ser Pro His Arg Asp Pro
Ala Pro Arg Gly Pro Ala 145 150 155 160 cgc ttc ctg cca cta cca ggc
ctg ccc ccc gca ccc ccg gag cca ccc 528Arg Phe Leu Pro Leu Pro Gly
Leu Pro Pro Ala Pro Pro Glu Pro Pro 165 170 175 gga atc ctg gcc ccc
cag ccc ccc gat gtg ggc tcc tcg gac cct ctg 576Gly Ile Leu Ala Pro
Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu 180 185 190 agc atg gtg
gga cct tcc cag ggc cga agc ccc agc tac gct tcc tga 624Ser Met Val
Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 205
22207PRTArtificialSynthetic Construct 22Met Arg Ser Gly Cys Val Val
Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu Ala Val Ala
Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 His Val His
Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 Thr
Asp Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp 50 55
60 Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu
65 70 75 80 Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val
His Ser 85 90 95 Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met
Gln Gly Leu Leu 100 105 110 Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu
Glu Leu Leu Leu Glu Asp 115 120 125 Gly Tyr Asn Val Tyr Gln Ser Glu
Ala His Gly Leu Pro Leu His Leu 130 135 140 Pro Gly Asn Lys Ser Pro
His Arg Asp Pro Ala Pro Arg Gly Pro Ala 145 150 155 160 Arg Phe Leu
Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro 165 170 175 Gly
Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu 180 185
190 Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195
200 205 23627DNAArtificialchimeric fusion protein 23atg cgg agc ggg
tgt gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg
gcc gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
cac gtt cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac
144His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr
35 40 45 acc tcc ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc
cgt gcc 192Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile
Arg Ala 50 55 60 gac ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg
cac agt ttg ctg 240Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala
His Ser Leu Leu 65 70 75 80 cag ctg aaa gcc ttg aag ccg gga gtt att
caa atc ttg gga gtc aag 288Gln Leu Lys Ala Leu Lys Pro Gly Val Ile
Gln Ile Leu Gly Val Lys 85 90 95 aca tcc agg ttc ctg tgc cag cgg
cca gat ggg gcc ctg tat gga tcg 336Thr Ser Arg Phe Leu Cys Gln Arg
Pro Asp Gly Ala Leu Tyr Gly Ser 100 105 110 ctc cac ttt gac cct gag
gcc tgc agc ttc cgg gag ctg ctt ctt gag 384Leu His Phe Asp Pro Glu
Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu 115 120 125 gac gga tac aat
gtt tac cag tcc gaa gcc cac ggc ctc ccg ctg cac 432Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His 130 135 140 ctg cca
ggg aac aag tcc cca cac cgg gac cct gca ccc cga gga cca 480Leu Pro
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro 145 150 155
160 gct cgc ttc ctg cca cta cca ggc ctg ccc ccc gca ccc ccg gag cca
528Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro
165 170 175 ccc gga atc ctg gcc ccc cag ccc ccc gat gtg ggc tcc tcg
gac cct 576Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser
Asp Pro 180 185 190 ctg agc atg gtg gga cct tcc cag ggc cga agc ccc
agc tac gct tcc 624Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro
Ser Tyr Ala Ser 195 200 205 tga 62724208PRTArtificialSynthetic
Construct 24Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala
Gly Leu 1 5 10 15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro 20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr 35 40 45 Thr Ser Gly Pro His Gly Leu Ser
Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60 Asp Gly Val Val Asp Cys
Ala Arg Gly Gln Ser Ala His Ser Leu Leu 65 70 75 80 Gln Leu Lys Ala
Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys 85 90 95 Thr Ser
Arg Phe
Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser 100 105 110 Leu His
Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu 115 120 125
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His 130
135 140 Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
Pro 145 150 155 160 Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala
Pro Pro Glu Pro 165 170 175 Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp
Val Gly Ser Ser Asp Pro 180 185 190 Leu Ser Met Val Gly Pro Ser Gln
Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 205
25624DNAArtificialchimeric fusion protein 25atg cgg agc ggg tgt gtg
gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val
Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg
gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val
Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt
cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144His Val
His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45
acc gat gat gcc cag cag aca gaa gcc cac ctg gag atc agg gag gat
192Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
50 55 60 ggg acg gtg ggg ggc gct gct gac cag agc ccc gaa agt ctc
ctg cag 240Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
Leu Gln 65 70 75 80 ctg aaa gcc ttg aag ccg gga gtt att caa atc ttg
gga gtc aag aca 288Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu
Gly Val Lys Thr 85 90 95 tcc agg ttc ctg tgc cag cgg cca gat ggg
gcc ctg tat gga tcg ctc 336Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
Ala Leu Tyr Gly Ser Leu 100 105 110 cac ttt gac cct gag gcc tgc agc
ttc cgg gag ctg ctt ctt gag gac 384His Phe Asp Pro Glu Ala Cys Ser
Phe Arg Glu Leu Leu Leu Glu Asp 115 120 125 gga tac aat gtt tac cag
tcc gaa gcc cac ggc ctc ccg ctg cac ctg 432Gly Tyr Asn Val Tyr Gln
Ser Glu Ala His Gly Leu Pro Leu His Leu 130 135 140 cca ggg aac aag
tcc cca cac cgg gac cct gca ccc cga gga cca gct 480Pro Gly Asn Lys
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala 145 150 155 160 cgc
ttc ctg cca cta cca ggc ctg ccc ccc gca ccc ccg gag cca ccc 528Arg
Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro 165 170
175 gga atc ctg gcc ccc cag ccc ccc gat gtg ggc tcc tcg gac cct ctg
576Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
180 185 190 agc atg gtg gga cct tcc cag ggc cga agc ccc agc tac gct
tcc tga 624Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
Ser 195 200 205 26207PRTArtificialSynthetic Construct 26Met Arg Ser
Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp
Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25
30 His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr
35 40 45 Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
Glu Asp 50 55 60 Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
Ser Leu Leu Gln 65 70 75 80 Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
Ile Leu Gly Val Lys Thr 85 90 95 Ser Arg Phe Leu Cys Gln Arg Pro
Asp Gly Ala Leu Tyr Gly Ser Leu 100 105 110 His Phe Asp Pro Glu Ala
Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp 115 120 125 Gly Tyr Asn Val
Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu 130 135 140 Pro Gly
Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala 145 150 155
160 Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro
165 170 175 Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
Pro Leu 180 185 190 Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser
Tyr Ala Ser 195 200 205 27624DNAArtificialchimeric fusion protein
27atg cgg agc ggg tgt gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc
48Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1
5 10 15 tgg ctg gcc gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt
cca 96Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly
Pro 20 25 30 cac gtt cac tac ggc tgg ggc gac ccc atc cgg cag cgg
tac ctc tac 144His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Gln Arg
Tyr Leu Tyr 35 40 45 aca gat gat gcc cag cag aca gaa gcc cac ctg
gag atc agg gag gat 192Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu
Glu Ile Arg Glu Asp 50 55 60 ggg acg gtg ggg ggc gct gct gac cag
agc ccc gaa agt ctc ctg cag 240Gly Thr Val Gly Gly Ala Ala Asp Gln
Ser Pro Glu Ser Leu Leu Gln 65 70 75 80 ctg aaa gcc ttg aag ccg gga
gtt att caa atc ttg gga gtc aag aca 288Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln Ile Leu Gly Val Lys Thr 85 90 95 tcc agg ttc ctg tgc
cag cgg cca gat ggg gcc ctg tat gga tcg ctc 336Ser Arg Phe Leu Cys
Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu 100 105 110 cac ttt gac
cct gag gcc tgc agc ttc cgg gag ctg ctt ctt gag gac 384His Phe Asp
Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp 115 120 125 gga
tac aat gtt tac cag tcc gaa gcc cac ggc ctc ccg ctg cac ctg 432Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu 130 135
140 cca ggg aac aag tcc cca cac cgg gac cct gca ccc cga gga cca gct
480Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
145 150 155 160 cgc ttc ctg cca cta cca ggc ctg ccc ccc gca ccc ccg
gag cca ccc 528Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro
Glu Pro Pro 165 170 175 gga atc ctg gcc ccc cag ccc ccc gat gtg ggc
tcc tcg gac cct ctg 576Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly
Ser Ser Asp Pro Leu 180 185 190 agc atg gtg gga cct tcc cag ggc cga
agc ccc agc tac gct tcc tga 624Ser Met Val Gly Pro Ser Gln Gly Arg
Ser Pro Ser Tyr Ala Ser 195 200 205 28207PRTArtificialSynthetic
Construct 28Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala
Gly Leu 1 5 10 15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro 20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Gln Arg Tyr Leu Tyr 35 40 45 Thr Asp Asp Ala Gln Gln Thr Glu
Ala His Leu Glu Ile Arg Glu Asp 50 55 60 Gly Thr Val Gly Gly Ala
Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln 65 70 75 80 Leu Lys Ala Leu
Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr 85 90 95 Ser Arg
Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu 100 105 110
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp 115
120 125 Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His
Leu 130 135 140 Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg
Gly Pro Ala 145 150 155 160 Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
Ala Pro Pro Glu Pro Pro 165 170 175 Gly Ile Leu Ala Pro Gln Pro Pro
Asp Val Gly Ser Ser Asp Pro Leu 180 185 190 Ser Met Val Gly Pro Ser
Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 205
29618DNAArtificialchimeric fusion protein 29atg cgg agc ggg tgt gtg
gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val
Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg
gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val
Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 ctc ctg
caa ttc ggg ggc caa gtc cgg cag cgg tac ctc tac aca gat 144Leu Leu
Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp 35 40 45
gat gcc cag cag aca gaa gcc cac ctg gag atc agg gag gat ggg acg
192Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr
50 55 60 gtg ggg ggc gct gct gac cag agc ccc gaa agt ctc ctg cag
ctg aaa 240Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
Leu Lys 65 70 75 80 gcc ttg aag ccg gga gtt att caa atc ttg gga gtc
aag aca tcc agg 288Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
Lys Thr Ser Arg 85 90 95 ttc ctg tgc cag cgg cca gat ggg gcc ctg
tat gga tcg ctc cac ttt 336Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu
Tyr Gly Ser Leu His Phe 100 105 110 gac cct gag gcc tgc agc ttc cgg
gag ctg ctt ctt gag gac gga tac 384Asp Pro Glu Ala Cys Ser Phe Arg
Glu Leu Leu Leu Glu Asp Gly Tyr 115 120 125 aat gtt tac cag tcc gaa
gcc cac ggc ctc ccg ctg cac ctg cca ggg 432Asn Val Tyr Gln Ser Glu
Ala His Gly Leu Pro Leu His Leu Pro Gly 130 135 140 aac aag tcc cca
cac cgg gac cct gca ccc cga gga cca gct cgc ttc 480Asn Lys Ser Pro
His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe 145 150 155 160 ctg
cca cta cca ggc ctg ccc ccc gca ccc ccg gag cca ccc gga atc 528Leu
Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile 165 170
175 ctg gcc ccc cag ccc ccc gat gtg ggc tcc tcg gac cct ctg agc atg
576Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met
180 185 190 gtg gga cct tcc cag ggc cga agc ccc agc tac gct tcc tga
618Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 205
30205PRTArtificialSynthetic Construct 30Met Arg Ser Gly Cys Val Val
Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu Ala Val Ala
Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp 35 40 45 Asp
Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr 50 55
60 Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys
65 70 75 80 Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
Ser Arg 85 90 95 Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly
Ser Leu His Phe 100 105 110 Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu
Leu Leu Glu Asp Gly Tyr 115 120 125 Asn Val Tyr Gln Ser Glu Ala His
Gly Leu Pro Leu His Leu Pro Gly 130 135 140 Asn Lys Ser Pro His Arg
Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe 145 150 155 160 Leu Pro Leu
Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile 165 170 175 Leu
Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met 180 185
190 Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 195 200 205
31636DNAArtificialchimeric fusion protein 31atg gac tcg gac gag acc
ggg ttc gag cac tca gga ctg tgg gtt tct 48Met Asp Ser Asp Glu Thr
Gly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 gtg ctg gct ggt
ctt ctg ctg gga gcc tgc cag gca cat cca att cca 96Val Leu Ala Gly
Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 gat tct
tct cca tta tta caa ttc ggg tgg ggc gac ccc atc cgg cag 144Asp Ser
Ser Pro Leu Leu Gln Phe Gly Trp Gly Asp Pro Ile Arg Gln 35 40 45
cgg tac ctc tac aca gat gat gcc cag cag aca gaa gcc cac ctg gag
192Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu
50 55 60 atc agg gag gat ggg acg gtg ggg ggc gct gct gac cag agc
ccc gaa 240Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
Pro Glu 65 70 75 80 agt ctc ctg cag ctg aaa gcc ttg aag ccg gga gtt
att caa atc ttg 288Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val
Ile Gln Ile Leu 85 90 95 gga gtc aag aca tcc agg ttc ctg tgc cag
cgg cca gat ggg gcc ctg 336Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly Ala Leu 100 105 110 tat gga tcg ctc cac ttt gac cct
gag gcc tgc agc ttc cgg gag ctg 384Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg Glu Leu 115 120 125 ctt ctt gag gac gga tac
aat gtt tac cag tcc gaa gcc cac ggc ctc 432Leu Leu Glu Asp Gly Tyr
Asn Val Tyr Gln Ser Glu Ala His Gly Leu 130 135 140 ccg ctg cac ctg
cca ggg aac aag tcc cca cac cgg gac cct gca ccc 480Pro Leu His Leu
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro 145 150 155 160 cga
gga cca gct cgc ttc ctg cca cta cca ggc ctg ccc ccc gca ccc 528Arg
Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro 165 170
175 ccg gag cca ccc gga atc ctg gcc ccc cag ccc ccc gat gtg ggc tcc
576Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser
180 185 190 tcg gac cct ctg agc atg gtg gga cct tcc cag ggc cga agc
ccc agc 624Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
Pro Ser 195 200 205 tac gct tcc tga 636Tyr Ala Ser 210
32211PRTArtificialSynthetic Construct 32Met Asp Ser Asp Glu Thr Gly
Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 Val Leu Ala Gly Leu
Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 Asp Ser Ser
Pro Leu Leu Gln Phe Gly Trp Gly Asp Pro Ile Arg Gln 35 40 45 Arg
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 50 55
60 Ile Arg Glu Asp Gly
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 65 70 75 80 Ser Leu Leu
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 85 90 95 Gly
Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu 100 105
110 Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu
115 120 125 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
Gly Leu 130 135 140 Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg
Asp Pro Ala Pro 145 150 155 160 Arg Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro Ala Pro 165 170 175 Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val Gly Ser 180 185 190 Ser Asp Pro Leu Ser
Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser 195 200 205 Tyr Ala Ser
210 33645DNAArtificialchimeric fusion protein 33atg cgg agc ggg tgt
gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys
Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc
gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala
Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac
gtt cac tac ggc ggc caa gtc cgc ctg cgg cac ctg tac acc tcc 144His
Val His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Ser 35 40
45 ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc gac ggc
192Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
50 55 60 gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg ctg
gag atc 240Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu
Glu Ile 65 70 75 80 aag gca gtc gct ctg cgg acc gtg gcc atc aag ggc
gtg cac agc gtg 288Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly
Val His Ser Val 85 90 95 cgg tac ctc tgc atg ggc gcc gac ggc aag
atg cag ggg ctg ctt cag 336Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln 100 105 110 tac tcg gag gaa gac tgt gct ttc
gag gag gag atc cgc cca gat ggc 384Tyr Ser Glu Glu Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly 115 120 125 tac aat gtg tac cga tcc
gag aag cac cgc ctc ccg gtc tcc ctg agc 432Tyr Asn Val Tyr Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Ser 130 135 140 agt gcc aaa cag
cgg cag ctg tac aag aac aga ggc ttt ctt cca ctc 480Ser Ala Lys Gln
Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu 145 150 155 160 tct
cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct gag gac 528Ser
His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp 165 170
175 ctc agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag acc
576Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr
180 185 190 gac agc atg gac cca ttt ggg ctt gtc acc gga ctg gag gcc
gtg agg 624Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala
Val Arg 195 200 205 agt ccc agc ttt gag aag taa 645Ser Pro Ser Phe
Glu Lys 210 34214PRTArtificialSynthetic Construct 34Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
His Val His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Ser 35
40 45 Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp
Gly 50 55 60 Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu
Leu Glu Ile 65 70 75 80 Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser Val 85 90 95 Arg Tyr Leu Cys Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln 100 105 110 Tyr Ser Glu Glu Asp Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly 115 120 125 Tyr Asn Val Tyr Arg
Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser 130 135 140 Ser Ala Lys
Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu 145 150 155 160
Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp 165
170 175 Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr 180 185 190 Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg 195 200 205 Ser Pro Ser Phe Glu Lys 210
35648DNAArtificialchimeric fusion protein 35atg cgg agc ggg tgt gtg
gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val
Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg
gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val
Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt
cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144His Val
His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45
acc gat gat gcc cag cag aca gaa tgc ttc ctg cgc atc cgt gcc gac
192Thr Asp Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp
50 55 60 ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg
ctg gag 240Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu
Leu Glu 65 70 75 80 atc aag gca gtc gct ctg cgg acc gtg gcc atc aag
ggc gtg cac agc 288Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser 85 90 95 gtg cgg tac ctc tgc atg ggc gcc gac ggc
aag atg cag ggg ctg ctt 336Val Arg Tyr Leu Cys Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu 100 105 110 cag tac tcg gag gaa gac tgt gct
ttc gag gag gag atc cgc cca gat 384Gln Tyr Ser Glu Glu Asp Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp 115 120 125 ggc tac aat gtg tac cga
tcc gag aag cac cgc ctc ccg gtc tcc ctg 432Gly Tyr Asn Val Tyr Arg
Ser Glu Lys His Arg Leu Pro Val Ser Leu 130 135 140 agc agt gcc aaa
cag cgg cag ctg tac aag aac aga ggc ttt ctt cca 480Ser Ser Ala Lys
Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro 145 150 155 160 ctc
tct cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct gag 528Leu
Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu 165 170
175 gac ctc agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag
576Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
180 185 190 acc gac agc atg gac cca ttt ggg ctt gtc acc gga ctg gag
gcc gtg 624Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val 195 200 205 agg agt ccc agc ttt gag aag taa 648Arg Ser Pro
Ser Phe Glu Lys 210 215 36215PRTArtificialSynthetic Construct 36Met
Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10
15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His
Leu Tyr 35 40 45 Thr Asp Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg
Ile Arg Ala Asp 50 55 60 Gly Val Val Asp Cys Ala Arg Gly Gln Ser
Ala His Ser Leu Leu Glu 65 70 75 80 Ile Lys Ala Val Ala Leu Arg Thr
Val Ala Ile Lys Gly Val His Ser 85 90 95 Val Arg Tyr Leu Cys Met
Gly Ala Asp Gly Lys Met Gln Gly Leu Leu 100 105 110 Gln Tyr Ser Glu
Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp 115 120 125 Gly Tyr
Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu 130 135 140
Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro 145
150 155 160 Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu
Pro Glu 165 170 175 Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser
Ser Pro Leu Glu 180 185 190 Thr Asp Ser Met Asp Pro Phe Gly Leu Val
Thr Gly Leu Glu Ala Val 195 200 205 Arg Ser Pro Ser Phe Glu Lys 210
215 37645DNAArtificialchimeric fusion protein 37atg cgg agc ggg tgt
gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys
Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc
gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala
Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac
gtt cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144His
Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40
45 acc tcc ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc
192Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala
50 55 60 gac ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt
ttg ctg 240Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser
Leu Leu 65 70 75 80 gag atc aag gca gtc gct ctg cgg acc gtg gcc atc
aag ggc gtg cac 288Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile
Lys Gly Val His 85 90 95 agc gtg cgg tac ctc tgc atg ggc gcc gac
ggc aag atg cag ggg ctg 336Ser Val Arg Tyr Leu Cys Met Gly Ala Asp
Gly Lys Met Gln Gly Leu 100 105 110 ctt cag tac tcg gag gaa gac tgt
gct ttc gag gag gag atc cgc cca 384Leu Gln Tyr Ser Glu Glu Asp Cys
Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 gat ggc tac aat gtg tac
cga tcc gag aag cac cgc ctc ccg gtc tcc 432Asp Gly Tyr Asn Val Tyr
Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 ctg cca ggg aac
aag tcc cca cac cgg gac cct gca ccc cga gga cca 480Leu Pro Gly Asn
Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro 145 150 155 160 tct
cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct gag gac 528Ser
His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp 165 170
175 ctc agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag acc
576Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr
180 185 190 gac agc atg gac cca ttt ggg ctt gtc acc gga ctg gag gcc
gtg agg 624Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala
Val Arg 195 200 205 agt ccc agc ttt gag aag taa 645Ser Pro Ser Phe
Glu Lys 210 38214PRTArtificialSynthetic Construct 38Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35
40 45 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg
Ala 50 55 60 Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His
Ser Leu Leu 65 70 75 80 Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala
Ile Lys Gly Val His 85 90 95 Ser Val Arg Tyr Leu Cys Met Gly Ala
Asp Gly Lys Met Gln Gly Leu 100 105 110 Leu Gln Tyr Ser Glu Glu Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 Leu Pro Gly
Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro 145 150 155 160
Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp 165
170 175 Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr 180 185 190 Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg 195 200 205 Ser Pro Ser Phe Glu Lys 210
39642DNAArtificialchimeric fusion protein 39atg cgg agc ggg tgt gtg
gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val
Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg
gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val
Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt
cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144His Val
His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45
acc gat gat gcc cag cag aca gaa tgc ttc ctg cgc atc cgt gcc gac
192Thr Asp Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp
50 55 60 ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg
ctg gag 240Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu
Leu Glu 65 70 75 80 atc aag gca gtc gct ctg cgg acc gtg gcc atc aag
ggc gtg cac agc 288Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser 85 90 95 gtg cgg tac ctc tgc atg ggc gcc gac ggc
aag atg cag ggg ctg ctt 336Val Arg Tyr Leu Cys Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu 100 105 110 cag tac tcg gag gaa gac tgt gct
ttc gag gag gag atc cgc cca gat 384Gln Tyr Ser Glu Glu Asp Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp 115 120 125 ggc tac aat gtg tac cga
tcc gag aag cac cgc ctc ccg gtc tcc ctg 432Gly Tyr Asn Val Tyr Arg
Ser Glu Lys His Arg Leu Pro Val Ser Leu 130 135 140 cca ggg aac aag
tcc cca cac cgg gac cct gca ccc cga gga cca tct 480Pro Gly Asn Lys
Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ser 145 150 155 160 cat
ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct gag gac ctc 528His
Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu 165 170
175 agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag acc gac
576Arg Gly His Leu Glu Ser Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp 180 185 190 agc atg gac cca ttt
ggg ctt gtc acc gga ctg gag gcc gtg agg agt 624Ser Met Asp Pro Phe
Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser 195 200 205 ccc agc ttt
gag aag taa 642Pro Ser Phe Glu Lys 210 40213PRTArtificialSynthetic
Construct 40Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala
Gly Leu 1 5 10 15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro 20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu Arg His Leu Tyr 35 40 45 Thr Asp Asp Ala Gln Gln Thr Glu
Cys Phe Leu Arg Ile Arg Ala Asp 50 55 60 Gly Val Val Asp Cys Ala
Arg Gly Gln Ser Ala His Ser Leu Leu Glu 65 70 75 80 Ile Lys Ala Val
Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser 85 90 95 Val Arg
Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu 100 105 110
Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp 115
120 125 Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser
Leu 130 135 140 Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg
Gly Pro Ser 145 150 155 160 His Phe Leu Pro Met Leu Pro Met Val Pro
Glu Glu Pro Glu Asp Leu 165 170 175 Arg Gly His Leu Glu Ser Asp Met
Phe Ser Ser Pro Leu Glu Thr Asp 180 185 190 Ser Met Asp Pro Phe Gly
Leu Val Thr Gly Leu Glu Ala Val Arg Ser 195 200 205 Pro Ser Phe Glu
Lys 210 41642DNAArtificialchimeric fusion protein 41atg cgg agc ggg
tgt gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg
gcc gtg gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
cac gtt cac tac ggc ggc caa gtc cgc ctg cgg cac ctg tac acc gat
144His Val His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Asp
35 40 45 gat gcc cag cag aca gaa tgc ttc ctg cgc atc cgt gcc gac
ggc gtc 192Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp
Gly Val 50 55 60 gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg
ctg gag atc aag 240Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu
Leu Glu Ile Lys 65 70 75 80 gca gtc gct ctg cgg acc gtg gcc atc aag
ggc gtg cac agc gtg cgg 288Ala Val Ala Leu Arg Thr Val Ala Ile Lys
Gly Val His Ser Val Arg 85 90 95 tac ctc tgc atg ggc gcc gac ggc
aag atg cag ggg ctg ctt cag tac 336Tyr Leu Cys Met Gly Ala Asp Gly
Lys Met Gln Gly Leu Leu Gln Tyr 100 105 110 tcg gag gaa gac tgt gct
ttc gag gag gag atc cgc cca gat ggc tac 384Ser Glu Glu Asp Cys Ala
Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr 115 120 125 aat gtg tac cga
tcc gag aag cac cgc ctc ccg gtc tcc ctg agc agt 432Asn Val Tyr Arg
Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser 130 135 140 gcc aaa
cag cgg cag ctg tac aag aac aga ggc ttt ctt cca ctc tct 480Ala Lys
Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser 145 150 155
160 cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct gag gac ctc
528His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu
165 170 175 agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag
acc gac 576Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp 180 185 190 agc atg gac cca ttt ggg ctt gtc acc gga ctg gag
gcc gtg agg agt 624Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser 195 200 205 ccc agc ttt gag aag taa 642Pro Ser Phe
Glu Lys 210 42213PRTArtificialSynthetic Construct 42Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
His Val His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Asp 35
40 45 Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg Ala Asp Gly
Val 50 55 60 Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu
Glu Ile Lys 65 70 75 80 Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly
Val His Ser Val Arg 85 90 95 Tyr Leu Cys Met Gly Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln Tyr 100 105 110 Ser Glu Glu Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly Tyr 115 120 125 Asn Val Tyr Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser 130 135 140 Ala Lys Gln
Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser 145 150 155 160
His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu 165
170 175 Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr
Asp 180 185 190 Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala
Val Arg Ser 195 200 205 Pro Ser Phe Glu Lys 210
43639DNAArtificialchimeric fusion protein 43atg cgg agc ggg tgt gtg
gtg gtc cac gta tgg atc ctg gcc ggc ctc 48Met Arg Ser Gly Cys Val
Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc gtg
gcc ggg cgt cca ctt gct ttt tct gat gct ggt cca 96Trp Leu Ala Val
Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac gtt
cac tac ggc ggc caa gtc cgc ctg cgg cac ctg tac acc tcc 144His Val
His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Ser 35 40 45
ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc gac ggc
192Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly
50 55 60 gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt ttg ctg
gag atc 240Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu
Glu Ile 65 70 75 80 aag gca gtc gct ctg cgg acc gtg gcc atc aag ggc
gtg cac agc gtg 288Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly
Val His Ser Val 85 90 95 cgg tac ctc tgc atg ggc gcc gac ggc aag
atg cag ggg ctg ctt cag 336Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys
Met Gln Gly Leu Leu Gln 100 105 110 tac tcg gag gaa gac tgt gct ttc
gag gag gag atc cgc cca gat ggc 384Tyr Ser Glu Glu Asp Cys Ala Phe
Glu Glu Glu Ile Arg Pro Asp Gly 115 120 125 tac aat gtg tac cga tcc
gag aag cac cgc ctc ccg gtc tcc ctg cca 432Tyr Asn Val Tyr Arg Ser
Glu Lys His Arg Leu Pro Val Ser Leu Pro 130 135 140 ggg aac aag tcc
cca cac cgg gac cct gca ccc cga gga cca tct cat 480Gly Asn Lys Ser
Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ser His 145 150 155 160 ttc
ctg ccc atg ctg ccc atg gtc cca gag gag cct gag gac ctc agg 528Phe
Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg 165 170
175 ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg gag acc gac agc
576Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser
180 185 190 atg gac cca ttt ggg ctt gtc acc gga ctg gag gcc gtg agg
agt ccc 624Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg
Ser Pro 195 200 205 agc ttt gag aag taa 639Ser Phe Glu Lys 210
44212PRTArtificialSynthetic Construct 44Met Arg Ser Gly Cys Val Val
Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu Ala Val Ala
Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 His Val His
Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr Thr Ser 35 40 45 Gly
Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly 50 55
60 Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile
65 70 75 80 Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His
Ser Val 85 90 95 Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln
Gly Leu Leu Gln 100 105 110 Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu
Glu Ile Arg Pro Asp Gly 115 120 125 Tyr Asn Val Tyr Arg Ser Glu Lys
His Arg Leu Pro Val Ser Leu Pro 130 135 140 Gly Asn Lys Ser Pro His
Arg Asp Pro Ala Pro Arg Gly Pro Ser His 145 150 155 160 Phe Leu Pro
Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg 165 170 175 Gly
His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser 180 185
190 Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro
195 200 205 Ser Phe Glu Lys 210 45636DNAArtificialchimeric fusion
protein 45atg cgg agc ggg tgt gtg gtg gtc cac gta tgg atc ctg gcc
ggc ctc 48Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala
Gly Leu 1 5 10 15 tgg ctg gcc gtg gcc ggg cgt cca ctt gct ttt tct
gat gct ggt cca 96Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser
Asp Ala Gly Pro 20 25 30 cac gtt cac tac ggc ggc caa gtc cgc ctg
cgg cac ctg tac acc gat 144His Val His Tyr Gly Gly Gln Val Arg Leu
Arg His Leu Tyr Thr Asp 35 40 45 gat gcc cag cag aca gaa tgc ttc
ctg cgc atc cgt gcc gac ggc gtc 192Asp Ala Gln Gln Thr Glu Cys Phe
Leu Arg Ile Arg Ala Asp Gly Val 50 55 60 gtg gac tgc gcg cgg ggc
cag agc gcg cac agt ttg ctg gag atc aag 240Val Asp Cys Ala Arg Gly
Gln Ser Ala His Ser Leu Leu Glu Ile Lys 65 70 75 80 gca gtc gct ctg
cgg acc gtg gcc atc aag ggc gtg cac agc gtg cgg 288Ala Val Ala Leu
Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg 85 90 95 tac ctc
tgc atg ggc gcc gac ggc aag atg cag ggg ctg ctt cag tac 336Tyr Leu
Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr 100 105 110
tcg gag gaa gac tgt gct ttc gag gag gag atc cgc cca gat ggc tac
384Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr
115 120 125 aat gtg tac cga tcc gag aag cac cgc ctc ccg gtc tcc ctg
cca ggg 432Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu
Pro Gly 130 135 140 aac aag tcc cca cac cgg gac cct gca ccc cga gga
cca tct cat ttc 480Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
Pro Ser His Phe 145 150 155 160 ctg ccc atg ctg ccc atg gtc cca gag
gag cct gag gac ctc agg ggc 528Leu Pro Met Leu Pro Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly 165 170 175 cac ttg gaa tct gac atg ttc
tct tcg ccc ctg gag acc gac agc atg 576His Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met 180 185 190 gac cca ttt ggg ctt
gtc acc gga ctg gag gcc gtg agg agt ccc agc 624Asp Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser 195 200 205 ttt gag aag
taa 636Phe Glu Lys 210 46211PRTArtificialSynthetic Construct 46Met
Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10
15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
20 25 30 His Val His Tyr Gly Gly Gln Val Arg Leu Arg His Leu Tyr
Thr Asp 35 40 45 Asp Ala Gln Gln Thr Glu Cys Phe Leu Arg Ile Arg
Ala Asp Gly Val 50 55 60 Val Asp Cys Ala Arg Gly Gln Ser Ala His
Ser Leu Leu Glu Ile Lys 65 70 75 80 Ala Val Ala Leu Arg Thr Val Ala
Ile Lys Gly Val His Ser Val Arg 85 90 95 Tyr Leu Cys Met Gly Ala
Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr 100 105 110 Ser Glu Glu Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr 115 120 125 Asn Val
Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Pro Gly 130 135 140
Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ser His Phe 145
150 155 160 Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu
Arg Gly 165 170 175 His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu
Thr Asp Ser Met 180 185 190 Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala Val Arg Ser Pro Ser 195 200 205 Phe Glu Lys 210 4721PRTHomo
sapiens 47Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Asp Pro Ile
Arg Leu 1 5 10 15 Arg His Leu Tyr Thr 20 4819PRTHomo sapiens 48Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr 1 5 10
15 Leu Tyr Thr 495PRTHomo sapiens 49Trp Gly Asp Pro Ile 1 5
5018PRTHomo sapiens 50Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly
Gly Leu Ile His Leu 1 5 10 15 Tyr Thr 5114PRTHomo sapiens 51Leu Tyr
Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu 1 5 10 528PRTHomo
sapiens 52Ser Gly Pro His Gly Leu Ser Ser 1 5 5313PRTHomo sapiens
53Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 1 5 10
547PRTHomo sapiens 54Asp Asp Ala Gln Gln Thr Glu 1 5 5512PRTHomo
sapiens 55Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His Leu 1 5 10
566PRTHomo sapiens 56Ala Thr Ala Arg Asn Ser 1 5 5726PRTHomo
sapiens 57Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp
Pro Ala 1 5 10 15 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu 20 25
5817PRTHomo sapiens 58Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn
Arg Gly
Phe Leu Pro 1 5 10 15 Leu 5928PRTHomo sapiens 59Leu Pro Val Ser Leu
Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn 1 5 10 15 Arg Gly Phe
Leu Pro Leu Ser His Phe Leu Pro Met 20 25 6015PRTHomo sapiens 60Pro
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro 1 5 10 15
6127PRTHomo sapiens 61Phe Leu Val Ser Leu Gly Arg Ala Lys Arg Ala
Phe Leu Pro Gly Met 1 5 10 15 Asn Pro Pro Pro Tyr Ser Gln Phe Leu
Ser Arg 20 25 6216PRTHomo sapiens 62Gly Arg Ala Lys Arg Ala Phe Leu
Pro Gly Met Asn Pro Pro Pro Tyr 1 5 10 15 634PRTArtificial
Sequencelinker peptide 63Gly Gly Gly Gly 1 645PRTArtificial
Sequencepeptide linker 64Gly Gly Gly Gly Gly 1 5 6515PRTArtificial
Sequencepeptide linker 65Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Gly Ser 1 5 10 15 6615PRTArtificial Sequencepeptide
linker 66Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15 678PRTArtificial Sequencepeptide linker 67Gly Gly Gly
Lys Gly Gly Gly Gly 1 5 688PRTArtificial Sequencepeptide linker
68Gly Gly Gly Asn Gly Ser Gly Gly 1 5 698PRTArtificial
Sequencepeptide linker 69Gly Gly Gly Cys Gly Gly Gly Gly 1 5
705PRTArtificial Sequencepeptide linker 70Gly Pro Asn Gly Gly 1 5
714PRTHomo sapiens 71Gly Asp Pro Ile 1
* * * * *