U.S. patent application number 15/910332 was filed with the patent office on 2018-07-12 for mutated fibroblast growth factor (fgf) 1 and methods of use.
This patent application is currently assigned to Salk Institute for Biological Studies. The applicant listed for this patent is Salk Institute for Biological Studies. Invention is credited to Annette Atkins, Michael Downes, Ronald M. Evans, Jae Myoung Suh, Ruth T. Yu.
Application Number | 20180193418 15/910332 |
Document ID | / |
Family ID | 52826689 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180193418 |
Kind Code |
A1 |
Suh; Jae Myoung ; et
al. |
July 12, 2018 |
MUTATED FIBROBLAST GROWTH FACTOR (FGF) 1 AND METHODS OF USE
Abstract
The present disclosure provides FGF1 mutant proteins, such as
those having an N-terminal deletion, point mutation(s), or
combinations thereof, which can reduce blood glucose in a mammal.
Such mutant FGF1 proteins can be part of a chimeric protein that
includes a .beta.-Klotho-binding protein, an FGFR1c-binding
protein, a .beta.-Klotho-binding protein and a FGFR1c-binding
protein, a C-terminal region from FGF19 or FGF21. In some examples,
mutant FGF1 proteins have reduced mitogenic activity. Also provided
are nucleic acid molecules that encode such proteins, and vectors
and cells that include such nucleic acids. Methods of using the
disclosed molecules to reduce blood glucose levels are also
provided.
Inventors: |
Suh; Jae Myoung; (San Diego,
CA) ; Downes; Michael; (San Diego, CA) ;
Evans; Ronald M.; (La Jolla, CA) ; Atkins;
Annette; (San Diego, CA) ; Yu; Ruth T.; (La
Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Salk Institute for Biological Studies |
La Jolla |
CA |
US |
|
|
Assignee: |
Salk Institute for Biological
Studies
La Jolla
CA
|
Family ID: |
52826689 |
Appl. No.: |
15/910332 |
Filed: |
March 2, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14520178 |
Oct 21, 2014 |
9925241 |
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15910332 |
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61893766 |
Oct 21, 2013 |
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61949945 |
Mar 7, 2014 |
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61975530 |
Apr 4, 2014 |
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62019185 |
Jun 30, 2014 |
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62046038 |
Sep 4, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61P 3/04 20180101; A61P 9/12 20180101; A61K 31/422 20130101; A61P
3/10 20180101; A61K 31/4439 20130101; A61P 15/00 20180101; A61P
3/08 20180101; A61K 45/06 20130101; A61P 3/06 20180101; A61K 31/421
20130101; A61P 1/16 20180101; A61P 29/00 20180101; C07K 14/501
20130101; A61K 31/7088 20130101; A61K 31/4439 20130101; A61K
2300/00 20130101; A61K 31/421 20130101; A61K 2300/00 20130101; A61K
31/422 20130101; A61K 2300/00 20130101; A61K 38/1825 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/50 20060101 C07K014/50; A61K 31/7088 20060101
A61K031/7088; A61K 45/06 20060101 A61K045/06; A61K 31/421 20060101
A61K031/421; A61K 31/4439 20060101 A61K031/4439; A61K 31/422
20060101 A61K031/422 |
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. DK057978, DK090962, HL088093, HL105278 and ES010337 awarded by
The National Institutes of Health, National Human Genome Research
Institute. The government has certain rights in the invention.
Claims
1. An isolated mutated mature fibroblast growth factor (FGF) 1
protein comprising: a deletion of at least six consecutive
N-terminal amino acids; a point mutation at R35; and a point
mutation at K112, K113, K118, R119 or combinations thereof, wherein
the amino acid numbering is based on SEQ ID NO: 5
2. The isolated mutated mature FGF1 protein of claim 1, wherein the
a deletion of at least six contiguous N-terminal amino acids
comprises deletion of at least 9 consecutive N-terminal amino
acids.
3. The isolated mutated mature FGF1 protein of claim 1, wherein the
a deletion of at least six contiguous N-terminal amino acids
comprises deletion of 9 consecutive N-terminal amino acids.
4. The isolated mutated mature FGF1 protein of claim 1, wherein the
point mutation at R35 is R35E.
5. The isolated mutated mature FGF1 protein of claim 1, wherein the
point mutation at K112 is K112D, K112E, or K112Q.
6. The isolated mutated mature FGF1 protein of claim 1, wherein the
point mutation at K113 is K113D, K113E, or K113Q.
7. The isolated mutated mature FGF1 protein of claim 1, wherein the
point mutation at R119 is R119G, R119V, or R119E.
8. The isolated mutated mature FGF1 protein of claim 1, wherein the
isolated protein comprises a point mutation at both K112 and
K113.
9. The isolated mutated mature FGF1 protein of claim 1, wherein the
isolated protein comprises a point mutation at: K113; K118; R119;
K113 and R119; or K118 and R119,
10. The isolated mutated mature FGF1 protein of claim 8, wherein
the point mutation at K112 is K112Q and the point mutation at K113
is K113Q.
11. The isolated mutated mature FGF1 protein of claim 1, wherein
the protein comprises at least 90% sequence identity to SEQ ID NO:
194.
12. The isolated mutated mature FGF1 protein of claim 1, wherein
the protein comprises at least 95% sequence identity to SEQ ID NO:
194.
13. The isolated mutated mature FGF1 protein of claim 1, wherein
the protein comprises at least 98% sequence identity to SEQ ID NO:
194.
14. A method of reducing blood glucose in a mammal, comprising:
administering to the subject a therapeutically effective amount of
the isolated mutated mature FGF1 protein of claim 1, thereby
reducing blood glucose in the mammal.
15. The method of claim 14, wherein the administering is
subcutaneous, intraperitoneal, intramuscular, or intravenous.
16. The method of claim 14, wherein the amount of FGF1 protein
administered is at least 0.5 mg/kg.
17. The method of claim 14, wherein the method further comprises
administering an additional therapeutic compound.
18. The method of claim 17, wherein the additional therapeutic
compound is an alpha-glucosidase inhibitor, amylin agonist,
dipeptidyl-peptidase 4 (DPP-4) inhibitor, meglitinide,
sulfonylurea, or a peroxisome proliferator-activated receptor
(PPAR)-gamma agonist.
19. The method of claim 18, wherein the PPAR-gamma agonist is a
thiazolidinedione (TZD), aleglitazar, farglitazar, muraglitazar, or
tesaglitazar.
20. The method of claim 19, wherein the TZD is pioglitazone,
rosiglitazone, rivoglitazone, or troglitazone.
21. The method of claim 14, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/520,178 filed Oct. 21, 2014, which claims priority to U.S.
Provisional Application No. 61/893,766 filed Oct. 21, 2013, U.S.
Provisional Application No. 61/949,945 filed Mar. 7, 2014, U.S.
Provisional Application No. 61/975,530 filed Apr. 4, 2014, U.S.
Provisional Application No. 62/019,185 filed Jun. 30, 2014, and
U.S. Provisional Application No. 62/046,038 filed Sep. 4, 2014, all
herein incorporated by reference in their entireties.
FIELD
[0003] This application provides mutated FGF1 proteins,
FGFR1c-binding protein multimers, nucleic acids encoding such
proteins, and methods of their use, for example to treat a
metabolic disease.
BACKGROUND
[0004] Type 2 diabetes and obesity are leading causes of mortality
and are associated with the Western lifestyle, which is
characterized by excessive nutritional intake and lack of exercise.
A central player in the pathophysiology of these diseases is the
nuclear hormone receptor (NHR) PPAR.gamma., a lipid sensor and
master regulator of adipogenesis. PPAR.gamma. is also the molecular
target for the thiazolidinedione (TZD)-class of insulin
sensitizers, which command a large share of the current oral
anti-diabetic drug market. However, there are numerous side effects
associated with the use of TZDs such as weight gain, liver
toxicity, upper respiratory tract infection, headache, back pain,
hyperglycemia, fatigue, sinusitis, diarrhea, hypoglycemia, mild to
moderate edema, and anemia. Thus, the identification of new insulin
sensitizers is needed.
SUMMARY
[0005] It is shown herein that mutants of fibroblast growth factor
(FGF) 1 having reduced or eliminated mitogenic activity can be used
to reduce blood glucose in a mammal. Based on these observations,
methods for reducing blood glucose in a mammal, for example to
treat a metabolic disease, are disclosed. Such FGF1 mutants can
have an N-terminal truncation, point mutations, or combinations
thereof, for example to reduce the mitogenic activity of the native
FGF1 protein. Such FGF1 mutants can be used alone or in combination
with other agents, such as other glucose reducing agents, such as
thiazolidinedione.
[0006] In some examples, the FGF1 mutant is part of a chimeric
protein, such as one that includes at least 10, at least 20, at
least 30, at least 40, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, or at
least 50 contiguous amino acids from a C-terminal end of FGF19 or
FGF21.
[0007] In some examples, the FGF1 mutant is part of a chimeric
protein, such as one that includes at least 10, at least 20, at
least 30, at least 35, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 120, at
least 150, at least 180, or at least 200 amino acids (such as
20-500, 20 to 250, 30 to 200, 35 to 180, 37 to 90, or 37 to 180
amino acids) of a protein that selectively binds to beta-Klotho
(.beta.-Klotho), such as SEQ ID NO: 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145 or 146.
[0008] In some examples, the FGF1 mutant is part of a chimeric
protein, such as one that includes at least 10, at least 20, at
least 30, at least 35, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 120, at
least 150, at least 180, or at least 200 amino acids (such as
20-500, 20 to 250, 30 to 200, 35 to 180, 37 to 90, or 37 to 180
amino acids) of a protein that selectively binds to FGFR1c, such as
SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or multimers
thereof (e.g., dimers, timers), such as SEQ ID NO: 190.
[0009] In some examples, the FGF1 mutant is part of a chimeric
protein, such as one that includes at least 10, at least 20, at
least 30, at least 35, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 120, at
least 150, at least 180, or at least 200 amino acids (such as
20-500, 20 to 250, 30 to 200, 35 to 180, 37 to 90, or 37 to 180
amino acids) of a protein that selectively binds to .beta.-Klotho,
and that includes at least 10, at least 20, at least 30, at least
35, at least 40, at least 50, at least 60, at least 70, at least
80, at least 90, at least 100, at least 120, at least 150, at least
180, or at least 200 amino acids (such as 20-500, 20 to 250, 30 to
200, 35 to 180, 37 to 90, or 37 to 180 amino acids) of a protein
that selectively binds to FGFR1c, such as SEQ ID NO: 168, 169, 170
or 171.
[0010] In some examples, chimeric proteins include a linker between
the FGF1 mutant and the FGF19, FGF21, FGFR1c-binding, or
.beta.-Klotho-binding sequence. In some examples, use of the
disclosed methods result in one or more of: reduction in
triglycerides, decrease in insulin resistance, reduction of
hyperinsulinemia, increase in glucose tolerance, or reduction of
hyperglycemia in a mammal.
[0011] Provided herein are mutated FGF1 proteins, which can include
deletion of an N-terminal portion of FGF1, point mutations (such as
amino acid substitutions, deletions, additions, or combinations
thereof), or combinations of N-terminal deletions and point
mutations, and methods of their use to lower glucose, for example
to treat a metabolic disease. In some examples, such mutations
reduce the mitogenicity of mature FGF1 (e.g., SEQ ID NO: 5), such
as a reduction of at least 20%, at least 50%, at least 75% or at
least 90%. In some examples, the mutant FGF1 protein is a truncated
version of the mature protein (e.g., SEQ ID NO: 5), which can
include for example deletion of at least 5, at least 6, at least
10, at least 11, at least 12, at least 13, or at least 20
consecutive N-terminal amino acids. In some examples, one or more
of the deleted N-terminal amino acids are replaced with
corresponding amino acids from FGF21 (or any FGF having low
affinity for FGFR4, including FGF3, FGF5, FGF7, FGF9 and FGF10),
such as at least 1, at least 2, at least 3, at least 4, at least 5,
at least 10, or at least 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 corresponding amino acids
from FGF21 (e.g., see SEQ ID NOS: 21, 219, 221, 222 and 223). In
some examples, the mutant FGF1 protein is a mutated version of the
mature protein (e.g., SEQ ID NO: 5), such as one containing at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9 or at least 10 amino acid
substitutions (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 substitutions), such
as one or more of those shown in Table 1. In some examples, the
mutant FGF1 protein includes both an N-terminal truncation and
point mutations. In some examples, the mutant FGF1 protein includes
at least 120 consecutive amino acids from amino acids 5-141 of FGF1
(e.g., of SEQ ID NO: 2 or 4), (which in some examples can include
1-20 point mutations, such as substitutions, deletions, or
additions).
[0012] In some examples, the FGF1 mutants provided herein are used
to generate a chimeric protein, such as an FGF1/FGF21, FGF1/FGF19,
FGF1/.beta.-Klotho-binding protein, FGF1/FGFR1c-binding protein or
FGF1/.beta.-Klotho-binding protein/FGFR1c-binding protein. For
example, the C-terminal end or the N-terminal end of the disclosed
FGF1 mutants can be joined directly or indirectly to the N-terminal
end of a C-terminal fragment of FGF21 or FGF19, such as SEQ ID NO:
86 or 100, respectively. Similarly, the C-terminal end of the
disclosed FGF1 mutants can be joined directly or indirectly to the
N-terminal end of a .beta.-Klotho binding domain (such as SEQ ID
NO: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146 or .beta.-Klotho binding portion of SEQ ID NO: 168, 169, 170 or
171), or the N-terminal end of the disclosed FGF1 mutants can be
joined directly or indirectly to the C-terminal end of a
.beta.-Klotho-binding domain. In addition, the C-terminal end of
the disclosed FGF1 mutants can be joined directly or indirectly to
the N-terminal end of a FGFR1c-binding domain (such as SEQ ID NO:
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167 or 190), or the N-terminal
end of the disclosed FGF1 mutants can be joined directly or
indirectly to the C-terminal end of a FGFR1c-binding domain. In
some examples, the C-terminal end of the disclosed FGF1 mutants can
be joined directly or indirectly to an FGFR1c-binding domain (such
as any of SEQ ID NOS: 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 190 or
FGFR1c-binding portion of 168, 169, 170 or 171) and a
.beta.-Klotho-binding domain, the N-terminal end of the disclosed
FGF1 mutants can be joined directly or indirectly to the C-terminal
end of a FGFR1c-binding domain and a .beta.-Klotho-binding domain,
or both (such as SEQ ID NO: 168, 169, 170 or 171). Such chimeric
proteins can be used to reduce blood glucose in a mammal, for
example to treat a metabolic disease.
[0013] Specific exemplary FGF1 mutant proteins are shown in SEQ ID
NOS: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 113, 114, 115, 116, 117, 118, 119, 120, 191,
192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,
205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,
218, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237 and 238, which can be used to generate any of the chimeras
provided herein. Specific exemplary FGF1/FGF21 chimeras are shown
in SEQ ID NOS: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 219,
221, 222, and 223. Specific exemplary FGF1/FGF19 chimeras are shown
in SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 220, and 224. Specific exemplary
FGF1/.beta.-Klotho-binding chimeras are shown in FIGS. 23-25 (and
in SEQ ID NOS: 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, and 187). Specific exemplary FGF1/FGFR1c-binding chimeras are
shown in FIGS. 23J and 241 (and in SEQ ID NOS: 188 and 189).
Specific exemplary .beta.-Klotho-binding/FGFR1c-binding chimeras
that can be linked directly or indirectly to an N- or C-terminal
end of a FGF1 mutant protein are shown in SEQ ID NOS: 168, 169, 170
and 171.
[0014] Also provided are FGFR1c-binding protein dimers and
multimers (such as trimers) and their use to treat metabolic
disease. An example is shown in FIG. 25E (also see SEQ ID NO:
190).
[0015] Also provided are isolated nucleic acid molecules encoding
the disclosed mutant FGF1 proteins (which includes chimeras), and
the FGFR1c binding proteins. Vectors and cells that include such
nucleic acid molecules are also provided.
[0016] Methods of using the disclosed mutant FGF1 proteins and
FGFR1c binding protein multimers (or nucleic acid molecules
encoding such) are provided, such as a mutated mature FGF1 protein
having a deletion of at least six contiguous N-terminal amino
acids, at least one point mutation, or combinations thereof, for
example to reduce or eliminate mitogenic activity. In some examples
the methods include administering a therapeutically effective
amount of a disclosed mutant FGF1 protein or FGFR1c binding protein
(or nucleic acid molecules encoding such) to reduce blood glucose
in a mammal, such as a decrease of at least 5%. In some examples
the methods include administering a therapeutically effective
amount of a disclosed mutant FGF1 protein or FGFR1c binding protein
multimer (or nucleic acid molecules encoding such) to treat a
metabolic disease in a mammal. Exemplary metabolic diseases that
can be treated with the disclosed methods include but are not
limited to: diabetes (such as type 2 diabetes, non-type 2 diabetes,
type 1 diabetes, latent autoimmune diabetes (LAD), or maturity
onset diabetes of the young (MODY)), polycystic ovary syndrome
(PCOS), metabolic syndrome (MetS), obesity, non-alcoholic
steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD),
dyslipidemia (e.g., hyperlipidemia), and cardiovascular diseases
(e.g., hypertension). In some examples, one or more of these
diseases are treated simultaneously with the disclosed FGF21
mutants. Also provided are methods of reducing fed and fasting
blood glucose, improving insulin sensitivity and glucose tolerance,
reducing systemic chronic inflammation, ameliorating hepatic
steatosis in a mammal, reducing food intake, or combinations
thereof, by administering a therapeutically effective amount of a
disclosed mutant FGF1 protein or FGFR1c binding protein multimer
(or nucleic acid molecules encoding such).
[0017] The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows an exemplary wild-type mature FGF1 sequence
(SEQ ID NO: 5), N-terminal deletions that can be made to mature
FGF1 (SEQ ID NOS: 7, 8 and 9), point mutations that can be made to
mature FGF1 (SEQ ID NOS: 10 and 11), and mutations to the heparan
binding domain of FGF1 (SEQ ID NOS: 12 and 13).
[0019] FIGS. 2A and 2B are graphs showing blood glucose levels of
(A) ob/ob mice treated with control vehicle (PBS, open symbols),
rFGF1 (0.5 mg/kg subcutaneous, filled symbols), or
rFGF1.sup..DELTA.NT (0.5 mg/kg subcutaneous, dashed line, n=8-12).
This shows that the non-mitogenic FGF1 variant rFGF1.sup..DELTA.NT
(SEQ ID NO: 7) has equivalent efficacy as wild-type FGF1 in
lowering blood glucose levels in ob/ob diabetic mice. (B) Diet
induced obese (DIO) mice treated with control vehicle (PBS, open
bars), rFGF1 (0.5 mg/kg subcutaneous, filled bars), or
rFGF1.sup..DELTA.NT (0.5 mg/kg subcutaneous, striped bars) at
indicated times (n=10). This shows that the non-mitogenic FGF1
variant rFGF1.sup..DELTA.NT has equivalent efficacy as wild-type
FGF1 in lowering blood glucose levels in high fat diet fed obese
mice. Subcutaneous injections of vehicle (PBS) or rFGF1 (0.5 mg/kg)
were performed on ad lib fed mice. Values are means.+-.SEM.
Statistics by two-tailed t test. *P<0.05, **P<0.01.
[0020] FIG. 2C is a graph showing food intake in DIO mice after
control vehicle (PBS, open bar), rFGF1 (0.5 mg/kg subcutaneous,
filled bars), or rFGF1.sup..DELTA.NT (0.5 mg/kg subcutaneous,
striped bar) treatment (n=5).
[0021] FIGS. 3A-3D show how an exemplary wild-type mature FGF1
sequence (SEQ ID NO: 5) can be mutated to include mutations that
increase thermostability of FGF1 (M1, M2 and M3 deletions, SEQ ID
NOS: 22, 28, and 40, respectively), which can be combined with FGF1
N-terminal deletions and/or point mutations (SEQ ID NOS: 23, 24,
25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50 and 51).
[0022] FIGS. 4A-4B show additional FGF1 mutant sequences that can
be generated from an exemplary wild-type mature FGF1 sequence (SEQ
ID NO: 5) to include N-terminal deletions and/or point mutations
(SEQ ID NOS: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, and 66).
[0023] FIGS. 5A-5B show additional FGF1 mutant sequences that can
be generated from an exemplary wild-type mature FGF1 sequence (SEQ
ID NO: 5) to include N-terminal deletions and/or point mutations
(SEQ ID NOS: 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, and 84).
[0024] FIGS. 6A-6B show FGF21 (SEQ ID NO: 20) and a C-terminal
portion of FGF21 (SEQ ID NO: 86) that binds to beta-klotho, and how
they can be attached to FGF1 mutants described herein to form
FGF1/FGF21 chimeric proteins (SEQ ID NOS: 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, and 98). The FGF1/FGF21 chimeras shown can
further include one or more of K12V and N95V FGF1 non mitogenic
mutations (or other mutations disclosed herein, such as those
listed in Table 1) that have longer glucose lowering duration.
[0025] FIGS. 7A-7B show FGF19 (SEQ ID NO: 99) and a C-terminal
portion of FGF19 (SEQ ID NO: 100) that binds to beta-klotho, and
how they can be attached to FGF1 mutants described herein to form
FGF1/FGF19 chimeric proteins (SEQ ID NOS: 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, and 112). The FGF1/FGF19 chimeras
shown can further include one or more of K12V and N95V FGF1 non
mitogenic mutations that have longer glucose lowering duration.
[0026] FIG. 8 is a digital image showing the effect of
intracellular signaling with M1 thru M5 peptides (SEQ ID NOS: 22,
28, 40, 54 and 212, respectively). HEK293 cells were serum starved
and then treated with the indicated peptides at 10 ng/ml
concentration for 15 min. Total cell lysates were subject to
western blots with indicated antibodies.
[0027] FIG. 9 is a digital image showing peptides NT1 (SEQ ID NO:
7), NT2 (SEQ ID NO: 8), or NT3 (SEQ ID NO: 9) KN (SEQ ID NO: 10),
and KLE (SEQ ID NO: 11) and their ability to affect intracellular
signaling. HEK293 cells were serum starved and then treated with
the indicated peptides at 10 ng/ml concentration for 15 min. Total
cell lysates were subject to western blots with indicated
antibodies.
[0028] FIG. 10 is a digital image showing peptides FGF1 (SEQ ID NO:
5) and NT1 (SEQ ID NO: 7) and their ability to affect intracellular
signaling. HEK293 cells were serum starved and then treated with
the indicated peptides at 10 ng/ml concentration for 15 min. Total
cell lysates were subject to western blots with indicated
antibodies.
[0029] FIGS. 11A and 11B are graphs showing the glucose lowering
effects for M1, M2, and M3 in ob/ob mice. Mice were 5 mo old
C57BL/6J ob/ob on normal chow. The peptides were injected SQ (0.5
mg/kg).
[0030] FIG. 12 shows in vivo glucose lowering effects correlate
with FGFR mediated signaling. Mice were 5 mo old C57BL/6J ob/ob on
normal chow. The peptides NT1 (SEQ ID NO: 7) and NT2 (SEQ ID NO:
8), were injected SQ (0.5 mg/kg).
[0031] FIG. 13 is a digital image showing that the in vivo glucose
lowering effect correlate with FGFR mediated signaling. Serum
starved HEK 293 cells were treated with indicated peptides (10
ng/ml) for 15 min and subject to western blot.
FGF1.sup..DELTA.NTPrep1 and FGF1.sup..DELTA.NTPrep2 are the same
sequence, just independent preparations of the protein.
[0032] FIG. 14 is a graph showing blood glucose levels 0 to 120 hrs
following administration of a single injection of FGF1-KLE (SEQ ID
NO: 11) or FGF1-KN (SEQ ID NO: 10). The FGF1-KN mutant retained the
ability to lower glucose for 120 hrs while FGF1-KLE fails to lower
glucose.
[0033] FIG. 15A compares the dose response of downstream FGFR
signaling induced by rFGF1 (SEQ ID NO: 5) and NT1
(rFGF1.sup..DELTA.NT, SEQ ID NO: 7). FIG. 15B is the same as FIG.
2C. FIG. 15C compares the dose response of rFGF1 and NT1 in
lowering glucose in ob/ob mice. A. Western blot showing
intracellular signaling in serum starved HEK293 cells after a 15
min treatment with the indicated concentrations of PBS (vehicle),
rFGF1.sup..DELTA.NT, or rFGF1. B. Food intake in DIO mice during 24
hr period after injection of control vehicle (PBS, open bar), rFGF1
(0.5 mg/kg subcutaneous, filled bars), or rFGF.sup..DELTA.NT (0.5
mg/kg subcutaneous, striped bar, n=5). C. Dose response of glucose
lowering effects of subcutaneously delivered rFGF1.sup..DELTA.NT
(striped bars) in comparison to rFGF1 (filled bars) in 12 week old
ob/ob mice (n=6-12). ***P<0.005.
[0034] FIG. 16 is a bar graph showing blood glucose levels 0 hr, 16
hrs, or 24 hrs following administration of PBS, NT1
(FGF1.sup..DELTA.NT, SEQ ID NO: 7), NT2 (FGF1.sup..DELTA.NT2, SEQ
ID NO: 8), or NT3 (FGF1.sup..DELTA.NT3, SEQ ID NO: 9). Note that if
the N-terminus is truncated at 14 amino acids, glucose lowering
ability is substantially decreased (NT2). Mice were 5 mo old
C57BL/6J ob/ob on normal chow. The peptides were injected SQ (0.5
mg/kg).
[0035] FIGS. 17A and 17B are bar graphs showing that NT1 (SEQ ID
NO: 7) fails to lower blood glucose levels in HFD-fed aP2-Cre;
FGFR1 f/f mice (mutant FGFR1 KO mice). Blood glucose levels in 8
month old HFD-fed wildtype FGFR1 f/f (control open bars) or
adipose-specific FGFR1 knockout (mutant, R1 KO, aP2-Cre; FGFR1 f/f,
filled bars) mice after NT1 treatment (murine rFGF1.sup..DELTA.NT,
0.5 mg/kg subcutaneous injection, n=5 per group). Values are
means.+-.SEM. (A) shows the raw blood glucose levels, (B) shows the
data normalized to initial blood glucose as 100%.
[0036] FIGS. 18A and 18B are bar graphs showing that mouse rFGF1
(amino acids 1-15 of SEQ ID NO 4) fails to lower blood glucose
levels in HFD-fed aP2-Cre; FGFR1 f/f mice (FGFR1 KO, filled bars).
Blood glucose levels in 8 month old HFD-fed wild type (FGFR1 f/f,
black bars) or adipose-specific FGFR1 knockout (R1 KO, aP2-Cre;
FGFR1f/f, dotted bars) mice after rFGF1 treatment (murine rFGF1,
0.5 mg/kg subcutaneous injection, n-=5 per group). Values are
means.+-.SEM. (A) shows the raw blood glucose levels, (B) shows the
data normalized to initial blood glucose as 100%. Murine FGF1
(amino acids 1-15 of SEQ ID NO 4) is .about.96% homologous to the
human sequence (SEQ ID NO: 5)
[0037] FIG. 19 is a bar graph showing that FGF1 mutations K118E
(SEQ ID NO: 12) and K118N (SEQ ID NO: 13) fail to lower blood
glucose levels in DIO mice. Blood glucose levels in 7 months
HFD-fed C57BL/6J mice after PBS (open bar), K118E (filled bar), and
K118N (hatched bar) treatment (0.5 mg/kg subcutaneous injection,
n=4-8 per group). Values are means.+-.SEM.
[0038] FIG. 20 shows a native FGF1 sequence (SEQ ID NO: 5) and
eight heparan binding mutant FGF1 KKK analogs (SEQ ID NOS: 113,
114, 115, 116, 117, 118, 119, and 120).
[0039] FIGS. 21 and 22 show that FGF1 heparan binding mutant KKK
lowers glucose.
[0040] FIGS. 23-26 show exemplary arrangements of FGF1
mutant/.beta.-Klotho-binding chimeras and FGFR1c-binding protein
dimers. Exemplary sequences are shown in SEQ ID NOS: 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188, 189 and 190. Although monomers or dimers of FGFR1c- or
.beta.-Klotho-binding proteins are shown, in some examples greater
multimers are used, such as trimers, etc. In addition, the FGF1
mutant/.beta.-Klotho-binding chimeras can be made into FGF1
mutant/FGFR1c-binding chimeras by replacing the
.beta.-Klotho-binding portion with an FGFR1c-binding portion (e.g.,
as shown in FIGS. 23J and 241 for .DELTA.NT FGF1). Furthermore,
FGFR1c-binding portion(s) can be included in the FGF1
mutant/.beta.-Klotho-binding chimeras (e.g., as shown in FIGS. 23K
and 24J for .DELTA.NT FGF1). The sequence of CC2240 is shown in SEQ
ID NO: 121 and C2987 in SEQ ID NO: 148.
[0041] FIG. 27 shows exemplary FGF1 mutant sequences that include
an R35E substitution (SEQ ID NOS: 191-198).
[0042] FIG. 28 shows exemplary FGF1 mutant sequences that include
an R35V substitution (SEQ ID NOS: 199-206).
[0043] FIG. 29 shows exemplary FGF1 mutant sequences (SEQ ID NOS:
207-211). This free cysteine (C117) forms intermolecular disulfide
bonds that lead to protein aggregation. The mutation to valine is
designed to improve stability, hence it is introduced in
combination with other point mutations. KKKR are putative heparin
binding residues. KY, KE, KEY, KNY are various combinations of
point mutations to residues that interact with the FGF receptors
(K=K12, E=E87, Y=Y94, N=N95).
[0044] FIGS. 30A-30E show exemplary FGF1 mutant sequences that are
mutated to (A) increase stability (SEQ ID NOS: 54, 212-218 and
113), (B) chimeras (SEQ ID NOS: 219-224), (C) increase stability
and decrease mitogenicity (SEQ ID NOS: 225-229, (D) increase
stability and decrease mitogenicity (SEQ ID NOS: 230-233), and (E)
increase stability and decrease mitogenicity (SEQ ID
NOS:234-238).
[0045] FIG. 31 shows an alignment of FGF1 (SEQ ID NO: 5) and FGF2
(SEQ ID NO: 85), with amino acids that form beta strands in bold,
and other relevant residues highlighted and their interaction
noted.
[0046] FIGS. 32A-32D are graphs showing the affect of FGF1
mutations on blood glucose lowering and feeding effects in vivo.
Peptides KN (Salk_004, SEQ ID NO: 10), KKK (Salk_010, SEQ ID NO:
226), FGF1 (SEQ ID NO: 5), KLE (Salk_011, SEQ ID NO: 11),
FGF1.sup..DELTA.NT (NT1) (SEQ ID NO: 7) and FGF1.sup..DELTA.NTKN
(Salk_009, SEQ ID NO: 225) were tested.
[0047] FIGS. 33A-33B are bar graphs showing the affect of FGF1
mutations on (A) blood glucose lowering and (B) feeding effects in
vivo. Peptides FGF1 (SEQ ID NO: 5), Salk_013 (SEQ ID NO: 31), and
Salk_012 (SEQ ID NO: 79) were tested.
[0048] FIGS. 34A-34B are bar graphs showing the affect of FGF1
mutations on (A) blood glucose lowering and (B) feeding effects in
vivo. Peptides Salk_014 (SEQ ID NO: 230), Salk_024 (SEQ ID NO: 84),
Salk_025 (SEQ ID NO: 208), Salk_026 (SEQ ID NO: 209), and Salk_023
(SEQ ID NO: 38) were tested.
[0049] FIGS. 35A-35B are bar graphs showing the affect of FGF1
mutations on (A) blood glucose lowering and (B) feeding effects in
vivo. Peptides Salk_014 (SEQ ID NO: 230), Salk_024 (SEQ ID NO: 84),
Salk_025 (SEQ ID NO: 208), and Salk_026 (SEQ ID NO: 209), and
Salk_023 (SEQ ID NO: 38) were tested.
[0050] FIGS. 36A-36B are bar graphs showing the affect of FGF1
mutations on (A) blood glucose lowering and (B) feeding effects in
vivo. Peptides Salk_014 (SEQ ID NO: 230) and Salk_032 (SEQ ID NO:
215) were tested.
[0051] FIGS. 37A-37B are bar graphs showing the affect of a
FGF1-FGF19 chimera on (A) blood glucose lowering and (B) feeding
effects in vivo. Peptides Salk_014 (SEQ ID NO: 230) and Salk_019
(SEQ ID NO: 224) were tested.
[0052] FIG. 38 is a bar graph showing the affect of a FGF1-FGF21
chimera on (A) blood glucose lowering and (B) feeding effects in
vivo. Peptides FGF1 (SEQ ID NO: 5), FGF1.sup..DELTA.NT (SEQ ID NO:
7), FGF21 (SEQ ID 20) and FGF1-FGF21 chimera (SEQ ID NO: 114+SEQ ID
NO: 86) were tested.
SEQUENCE LISTING
[0053] The nucleic and amino acid sequences are shown using
standard letter abbreviations for nucleotide bases, and three
letter code for amino acids, as defined in 37 C.F.R. 1.822. Only
one strand of each nucleic acid sequence is shown, but the
complementary strand is understood as included by any reference to
the displayed strand. The sequence listing submitted herewith,
generated on Mar. 2, 2018, 296 Kb is herein incorporated by
reference.
[0054] SEQ ID NOS: 1 and 2 provide an exemplary human FGF1 nucleic
acid and protein sequences, respectively. Source: GenBank.RTM.
Accession Nos: BC032697.1 and AAH32697.1. Heparan binding residues
are amino acids 127-129 and 133-134.
[0055] SEQ ID NOS: 3 and 4 provide an exemplary mouse FGF1 nucleic
acid and protein sequences, respectively. Source: GenBank.RTM.
Accession Nos: BC037601.1 and AAH37601.1.
[0056] SEQ ID NO: 5 provides an exemplary mature form of FGF1 (140
aa, sometimes referred to in the art as FGF1 15-154)
[0057] SEQ ID NO: 6 provides an exemplary mature form of FGF1 with
an N-terminal deletion.
[0058] SEQ ID NO: 7 provides an exemplary mature form of FGF1 with
an N-terminal deletion
(FGF1.sup..DELTA.NT(10-140.alpha..alpha.)).
[0059] SEQ ID NO: 8 provides an exemplary mature form of FGF1 with
an N-terminal deletion
(FGF1.sup..DELTA.NT2(14-140.alpha..alpha.)).
[0060] SEQ ID NO: 9 provides an exemplary mature form of FGF1 with
an N-terminal deletion
(FGF1.sup..DELTA.NT3(12-140.alpha..alpha.)).
[0061] SEQ ID NO: 10 provides an exemplary mature form of FGF1 with
point mutations (K12V, N95V, wherein numbering refers to SEQ ID NO:
5) to reduce mitogenic activity.
[0062] SEQ ID NO: 11 provides an exemplary mature form of FGF1 with
point mutations (K12V, L46V, E87V, N95V, P134V, wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity.
[0063] SEQ ID NOS: 12 and 13 provide exemplary mature forms of FGF1
with mutations in the heparan binding domain (K118N or K118E,
respectively, wherein numbering refers to SEQ ID NO: 5). In some
examples these sequences further include MFNLPPG at their
N-terminus. Such proteins have reduced mitogenicity as compared to
wild-type FGF1.
[0064] SEQ ID NOS: 14-17 provide exemplary mutated FGF1 nuclear
export sequences.
[0065] SEQ ID NO: 18 provides a coding sequence for SEQ ID NO:
6.
[0066] SEQ ID NOS: 19 and 20 provide an exemplary human FGF21
nucleic acid and protein sequence. Obtained from GenBank.RTM.
Accession Nos. AY359086 and AAQ89444.1. The mature form of FGF21 is
about amino acids 21-208.
[0067] SEQ ID NO: 21 provides an exemplary N-terminally truncated
form of FGF1, wherein the four N-terminal amino acids are from
FGF21 (amino acids 40-43 of SEQ ID NO: 20).
[0068] SEQ ID NO: 22 provides an exemplary mature form of FGF1 with
point mutations (K12V, C117V and P134V wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability. From Xia et al., PLoS One. 7(11):e48210, 2012.
[0069] SEQ ID NO: 23 (FGF1(1-140.alpha..alpha.)M1a) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95V,
C117V, and P134V wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0070] SEQ ID NO: 24 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M1)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, C117V, and P134V wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0071] SEQ ID NO: 25 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M1a)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, C117V, and P134V wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0072] SEQ ID NO: 26 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M1a)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, N95V, C117V, and P134V wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity, and increase
thermostability.
[0073] SEQ ID NO: 27 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M1a)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, N95V, C117V, and P134V wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity, and increase
thermostability
[0074] SEQ ID NO: 28 (FGF1(1-140.alpha..alpha.)M2) provides an
exemplary mature form of FGF1 with point mutations (L44F, C83T,
C117V, and F132W wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability. From Xia et
al., PLoS One. 7(11):e48210, 2012.
[0075] SEQ ID NO: 29 (FGF1(1-140.alpha..alpha.)M2a) provides an
exemplary mature form of FGF1 with point mutations (L44F, C83T,
N95V, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0076] SEQ ID NO: 30 (FGF1(1-140.alpha..alpha.)M2b) provides an
exemplary mature form of FGF1 with point mutations (K12V, L44F,
C83T, C117V, and F132W wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0077] SEQ ID NO: 31 (FGF1(1-140.alpha..alpha.)M2c) provides an
exemplary mature form of FGF1 with point mutations (K12V, L44F,
C83T, N95V, C117V, and F132W wherein numbering refers to SEQ ID NO:
5) to reduce mitogenic activity and increase thermostability.
[0078] SEQ ID NO: 32 (FGF1.sup..DELTA.NT1(10-140.alpha..alpha.)M2)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (L44F, C83T, C117V, and F132W wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0079] SEQ ID NO: 33 (FGF1.sup..DELTA.NT3(12-140.alpha..alpha.)M2)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (L44F, C83T, C117V, and F132W wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0080] SEQ ID NO: 34 (FGF1.sup..DELTA.NT1(10-140.alpha..alpha.)M2a)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (L44F, C83T, N95V, C117V, and F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0081] SEQ ID NO: 35 (FGF1.sup..DELTA.NT3(12-140.alpha..alpha.)M2a)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (L44F, C83T, N95V, C117V, and F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0082] SEQ ID NO: 36 (FGF1.sup..DELTA.NT1(10-140.alpha..alpha.)M2b)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, L44F, C83T, C117V, and F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0083] SEQ ID NO: 37 (FGF1.sup..DELTA.NT3(12-140.alpha..alpha.)M2b)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, L44F, C83T, C117V, and F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0084] SEQ ID NO: 38 (FGF1.sup..DELTA.NT1(10-140.alpha..alpha.)M2c)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, L44F, C83T, N95V, and C117V, F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0085] SEQ ID NO: 39 (FGF1.sup..DELTA.NT3(12-140.alpha..alpha.)M2c)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, L44F, C83T, N95V, and C117V, F132W wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0086] SEQ ID NO: 40 (FGF1(1-140.alpha..alpha.)M3) provides an
exemplary mature form of FGF1 with mutations (L44F, M67I, L73V,
V109L, L111I, C117V, A103G, R119G .DELTA.104-106, and
.DELTA.120-122, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability. From Xia et al.,
PLoS One. 7(11):e48210, 2012.
[0087] SEQ ID NO: 41 (FGF1(1-140.alpha..alpha.)M3a) provides an
exemplary mature form of FGF1 with mutations (K12V, L44F, M67I,
L73V, V109L, L111I, C117V, A103G, R119G, .DELTA.104-106, and
.DELTA.120-122 wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0088] SEQ ID NO: 42 (FGF1(1-140.alpha..alpha.)M3b) provides an
exemplary mature form of FGF1 with mutations (K12V, L44F, M67I,
L73V, N95V, V109L, L111I, C117V, A103G, R119G, .DELTA.104-106, and
.DELTA.120-122 wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0089] SEQ ID NO: 43 (FGF1(1-140.alpha..alpha.)M3c) provides an
exemplary mature form of FGF1 with mutations (K12V, L44F, M67I,
L73V, N95V, V109L, L111I, C117V, A103G, R119G, .DELTA.104-106, and
.DELTA.120-122 wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0090] SEQ ID NO: 44 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M3)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (L44F, M67I, L73V, V109L, L111I, C117V, A103G, R119G,
.DELTA.104-106, and .DELTA.120-122 wherein numbering refers to SEQ
ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0091] SEQ ID NO: 45 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M3)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (L44F, M67I, L73V, V109L, L111I, C117V, A103G, R119G,
.DELTA.104-106, and .DELTA.120-122 wherein numbering refers to SEQ
ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0092] SEQ ID NO: 46 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M3a)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (K12V, L44F, M67I, L73V, V109L, L111I, C117V, A103G,
R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0093] SEQ ID NO: 47 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M3a)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (K12V, L44F, M67I, L73V, V109L, L111I, C117V, A103G,
R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0094] SEQ ID NO: 48 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M3b)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (L44F, M67I, L73V, N95V, V109L, L111I, C117V, A103G,
R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0095] SEQ ID NO: 49 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M3b)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (L44F, M67I, L73V, N95V, V109L, L111I, C117V, A103G,
R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0096] SEQ ID NO: 50 (FGF1.sup..DELTA.NT1 (1-140.alpha..alpha.)M3c)
provides an exemplary N-terminally truncated form of FGF1 with
mutations (K12V, L44F, M67I, L73V, N95V, V109L, L111I, C117V,
A103G, R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0097] SEQ ID NO: 51 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)M3c)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, L44F, M67I, L73V, N95V, V109L, L111I, C117V,
A103G, R119G, .DELTA.104-106, and .DELTA.120-122 wherein numbering
refers to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0098] SEQ ID NO: 52 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95V, and
K118N wherein numbering refers to SEQ ID NO: 5).
[0099] SEQ ID NO: 53 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95, and
K118E wherein numbering refers to SEQ ID NO: 5).
[0100] SEQ ID NO: 54 FGF1 (1-140.alpha..alpha.) K12V, N95V, C117V
provides an exemplary mature form of FGF1 with point mutations
(K12V, N95V, and C117V wherein numbering refers to SEQ ID NO:
5).
[0101] SEQ ID NO: 55 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95V,
C117V, and K118N wherein numbering refers to SEQ ID NO: 5).
[0102] SEQ ID NO: 56 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95V,
C117V, and K118E wherein numbering refers to SEQ ID NO: 5).
[0103] SEQ ID NO: 57 (FGF1.sup..DELTA.NT (10-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with point
mutations (K12V and N95V, wherein numbering refers to SEQ ID NO:
5).
[0104] SEQ ID NO: 58 (FGF1.sup..DELTA.NT2 (12-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with point
mutations (K12V, and N95V, wherein numbering refers to SEQ ID NO:
5).
[0105] SEQ ID NO: 59 (FGF1.sup..DELTA.NT (10-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (K12V, wherein numbering refers to SEQ ID NO: 5).
[0106] SEQ ID NO: 60 (FGF1.sup..DELTA.NT2 (12-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (K12V, wherein numbering refers to SEQ ID NO: 5).
[0107] SEQ ID NO: 61 (FGF1.sup..DELTA.NT (10-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (N95V, wherein numbering refers to SEQ ID NO: 5).
[0108] SEQ ID NO: 62 (FGF1.sup..DELTA.NT2 (12-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (N95V, wherein numbering refers to SEQ ID NO: 5).
[0109] SEQ ID NO: 63 (FGF1.sup..DELTA.NT (10-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with point
mutations (K12V, N95V, and K118N, wherein numbering refers to SEQ
ID NO: 5).
[0110] SEQ ID NO: 64 (FGF1.sup..DELTA.NT2 (12-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with point
mutations (K12V, N95V, and K118E, wherein numbering refers to SEQ
ID NO: 5).
[0111] SEQ ID NO: 65 (FGF1.sup..DELTA.NT (10-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (K118N, wherein numbering refers to SEQ ID NO: 5).
[0112] SEQ ID NO: 66 (FGF1.sup..DELTA.NT2 (12-140.alpha..alpha.)
provides an exemplary N-terminally truncated FGF1 with a point
mutation (K118E, wherein numbering refers to SEQ ID NO: 5).
[0113] SEQ ID NO: 67 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K9T and N10T
wherein numbering refers to SEQ ID NO: 5).
[0114] SEQ ID NO: 68 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K9T, N10T, and
N95V, wherein numbering refers to SEQ ID NO: 5).
[0115] SEQ ID NO: 69 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K9T, N10T, and
K118N, wherein numbering refers to SEQ ID NO: 5).
[0116] SEQ ID NO: 70 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with a mutant NLS sequence.
[0117] SEQ ID NO: 71 (FGF1.sup..DELTA.NT (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (Q40P and S47I, wherein numbering refers to SEQ ID
NO: 5).
[0118] SEQ ID NO: 72 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (Q40P and S47I, wherein numbering refers to SEQ ID
NO: 5).
[0119] SEQ ID NO: 73 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, Q40P,
S47I, and N95V wherein numbering refers to SEQ ID NO: 5).
[0120] SEQ ID NO: 74 FGF1.sup..DELTA.NT (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, Q40P, S47I, and N95V, wherein numbering
refers to SEQ ID NO: 5).
[0121] SEQ ID NO: 75 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, Q40P, S47I, and N95V, wherein numbering
refers to SEQ ID NO: 5).
[0122] SEQ ID NO: 76 (FGF1.sup..DELTA.NT (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (Q40P, S47I, and H93G, wherein numbering refers to
SEQ ID NO: 5).
[0123] SEQ ID NO: 77 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (Q40P, S47I, and H93G, wherein numbering refers to
SEQ ID NO: 5).
[0124] SEQ ID NO: 78 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, Q40P,
S47I, H93G, and N95V, wherein numbering refers to SEQ ID NO:
5).
[0125] SEQ ID NO: 79 (FGF1.sup..DELTA.NT (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, Q40P, S47I, H93G, and N95V, wherein
numbering refers to SEQ ID NO: 5).
[0126] SEQ ID NO: 80 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (K12V, Q40P, S47I, H93G, and N95V, wherein
numbering refers to SEQ ID NO: 5).
[0127] SEQ ID NO: 81 (FGF1.sup..DELTA.NT (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (C117P and K118V, wherein numbering refers to SEQ
ID NO: 5).
[0128] SEQ ID NO: 82 (FGF1.sup..DELTA.NT3 (1-140.alpha..alpha.)
provides an exemplary N-terminally truncated form of FGF1 with
point mutations (C117P and K118V, wherein numbering refers to SEQ
ID NO: 5).
[0129] SEQ ID NO: 83 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with point mutations (K12V, N95V,
C117P, and K118V, wherein numbering refers to SEQ ID NO: 5).
[0130] SEQ ID NO: 84 (FGF1 (1-140.alpha..alpha.) provides an
exemplary mature form of FGF1 with a point mutation (R35E, wherein
numbering refers to SEQ ID NO: 5). Such an antagonist can be used
to treat hypoglycemia or type I diabetes.
[0131] SEQ ID NO: 85 provides an exemplary portion of an FGF2
protein sequence.
[0132] SEQ ID NO: 86 provides an exemplary C-terminal FGF21 protein
sequence (P.sup.168-S.sup.209 hFGF21.sup.C-tail). This fragment can
be attached at its N-terminus to the C-terminus of any FGF1 mutant
provided herein to generate an FGF1/FGF21 chimera.
[0133] SEQ ID NO: 87 provides an exemplary FGF1/FGF21 chimera,
which contains the K12V and N95V FGF1 point mutations. The FGF21
portion is amino acids 136 to 177.
[0134] SEQ ID NO: 88 provides an exemplary FGF1/FGF21 chimera
(FGF1.sup..DELTA.NT-FGF21.sup.C-tail). The FGF21 portion is amino
acids 127 to 168.
[0135] SEQ ID NO: 89 provides an exemplary FGF1/FGF21 chimera
(FGF1.sup..DELTA.NT3-FGF21.sup.C-tail). The FGF21 portion is amino
acids 125 to 166.
[0136] SEQ ID NO: 90 provides an exemplary FGF1/FGF21 chimera
(M1-FGF21.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF21 portion is amino
acids 127 to 168.
[0137] SEQ ID NO: 91 provides an exemplary FGF1/FGF21 chimera
(M1-FGF21.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF21 portion is amino
acids 125 to 166.
[0138] SEQ ID NO: 92 provides an exemplary FGF1/FGF21 chimera
(M1-FGF21.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF21 portion is amino
acids 136 to 177.
[0139] SEQ ID NO: 93 provides an exemplary FGF1/FGF21 chimera
(M2-FGF21.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF21 portion
is amino acids 127 to 168.
[0140] SEQ ID NO: 94 provides an exemplary FGF1/FGF21 chimera
(M2-FGF21.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF21 portion
is amino acids 125 to 166.
[0141] SEQ ID NO: 95 provides an exemplary FGF1/FGF21 chimera
(M2-FGF21.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF21 portion
is amino acids 136 to 177.
[0142] SEQ ID NO: 96 provides an exemplary FGF1/FGF21 chimera
(M3-FGF21.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF21 portion is amino acids 121 to
162.
[0143] SEQ ID NO: 97 provides an exemplary FGF1/FGF21 chimera
(M3-FGF21.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF21 portion is amino acids 119 to
160.
[0144] SEQ ID NO: 98 provides an exemplary FGF1/FGF21 chimera
(M3-FGF21.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF21 portion is amino acids 130 to
171.
[0145] SEQ ID NO: 99 provides an exemplary FGF19 protein sequence.
The mature form of FGF19 is amino acids 23 to 216.
[0146] SEQ ID NO: 100 provides an exemplary C-terminal FGF19
protein sequence (L.sup.169-K.sup.216 h FGF19C-tail). This fragment
can be attached at its N-terminus to the C-terminus of any FGF1
mutant provided herein to generate an FGF1/FGF19 chimera.
[0147] SEQ ID NO: 101 provides an exemplary FGF1/FGF19 chimera. The
FGF1 portion includes point mutations K.sup.12V, and N.sup.95V. The
FGF19 portion is amino acids 136 to 183.
[0148] SEQ ID NO: 102 provides an exemplary FGF1/FGF19 chimera
(FGF1.sup..DELTA.NT-FGF19.sup.C-tail). The FGF19 portion is amino
acids 127 to 174.
[0149] SEQ ID NO: 103 provides an exemplary FGF1/FGF19 chimera
(FGF1.sup..DELTA.NT3-FGF19.sup.C-tail). The FGF19 portion is amino
acids 125 to 172.
[0150] SEQ ID NO: 104 provides an exemplary FGF1/FGF19 chimera
(M1-FGF19.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF19 portion is amino
acids 136 to 183.
[0151] SEQ ID NO: 105 provides an exemplary FGF1/FGF19 chimera
(M1-FGF19.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF19 portion is amino
acids 127 to 174.
[0152] SEQ ID NO: 106 provides an exemplary FGF1/FGF19 chimera
(M1-FGF19.sup.C-tail). The FGF1 portion includes point mutations
K.sup.12V, C.sup.117V, and P.sup.134V. The FGF19 portion is amino
acids 125 to 172.
[0153] SEQ ID NO: 107 provides an exemplary FGF1/FGF19 chimera
(M2-FGF19.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF19 portion
is amino acids 136 to 183.
[0154] SEQ ID NO: 108 provides an exemplary FGF1/FGF19 chimera
(M2-FGF19.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF19 portion
is amino acids 127 to 174.
[0155] SEQ ID NO: 109 provides an exemplary FGF1/FGF19 chimera
(M2-FGF19.sup.C-tail). The FGF1 portion includes point mutations
L.sup.44F, C.sup.83T, C.sup.117V, and F.sup.132W. The FGF19 portion
is amino acids 125 to 172.
[0156] SEQ ID NO: 110 provides an exemplary FGF1/FGF19 chimera
(M3-FGF19.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF19 portion is amino acids 130 to
177.
[0157] SEQ ID NO: 111 provides an exemplary FGF1/FGF19 chimera
(M3-FGF19.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF19 portion is amino acids 121 to
168.
[0158] SEQ ID NO: 112 provides an exemplary FGF1/FGF19 chimera
(M3-FGF19.sup.C-tail). The FGF1 portion includes mutations
L.sup.44F, M.sup.67I, L.sup.73V, V.sup.109L, L.sup.111I,
C.sup.117V, A.sup.103G, R.sup.119G, .DELTA..sup.104-106 and
.DELTA..sup.120-122. The FGF19 portion is amino acids 119 to
166.
[0159] SEQ ID NO: 113 provides an exemplary FGF1 heparan binding
KKK mutant analog K112D, K113Q, K118V (wherein numbering refers to
SEQ ID NO: 5).
[0160] SEQ ID NO: 114 provides an exemplary FGF1 heparan binding
KKK mutant analog with mutations K112D, K113Q, C117V, K118V
(wherein numbering refers to SEQ ID NO: 5).
[0161] SEQ ID NO: 115 provides an exemplary FGF1 heparan binding
KKK mutant analog with an N-terminal truncation and mutations
K112D, K113Q, K118V (wherein numbering refers to SEQ ID NO: 5).
[0162] SEQ ID NO: 116 provides an exemplary FGF1 heparan binding
KKK mutant analog with an N-terminal truncation and mutations
K112D, K113Q, K118V (wherein numbering refers to SEQ ID NO: 5).
[0163] SEQ ID NO: 117 provides an exemplary FGF1 heparan binding
KKK mutant analog with an N-terminal truncation and mutations
K112D, K113Q, C117V, K118V (wherein numbering refers to SEQ ID NO:
5).
[0164] SEQ ID NO: 118 provides an exemplary FGF1 heparan binding
KKK mutant analog with an N-terminal truncation and mutations
K112D, K113Q, C117V, K118V (wherein numbering refers to SEQ ID NO:
5).
[0165] SEQ ID NO: 119 provides an exemplary FGF1 heparan binding
KKK mutant analog with mutations K12V, N95V, K112D, K113Q, K118V
(wherein numbering refers to SEQ ID NO: 5).
[0166] SEQ ID NO: 120 provides an exemplary FGF1 heparan binding
KKK mutant analog with mutations K12V, N95V, K112D, K113Q, C117V,
K118V (wherein numbering refers to SEQ ID NO: 5).
[0167] SEQ ID NO: 121 provides an exemplary .beta.-Klotho binding
protein dimer sequence (C2240) that can be attached at its N- or
C-terminus directly or indirectly to any of the FGF1 mutants
provided herein to generate a chimeric protein.
[0168] SEQ ID NO: 122 provides an exemplary .beta.-Klotho binding
protein sequence that can be attached at its N- or C-terminus
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein.
[0169] SEQ ID NOs: 123-130 provide exemplary .beta.-Klotho binding
protein sequences that can be attached at their N- or C-termini
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein. In addition, each can be linked to
SEQ ID NO: 122 via a linker and then the resulting chimera attached
at its N- or C-terminus directly or indirectly to any of the FGF1
mutants provided herein to generate a chimeric protein.
[0170] SEQ ID NOs: 131-140 provide exemplary .beta.-Klotho binding
protein sequences that can be attached at their N- or C-termini
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein.
[0171] SEQ ID NO: 141 provides an exemplary .beta.-Klotho binding
protein sequence that can be attached at its N- or C-terminus
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein. In addition, it can be linked to
any of SEQ ID NOS: 142-143 via a linker and then the resulting
chimera attached at its N- or C-terminus directly or indirectly to
any of the FGF1 mutants provided herein to generate a chimeric
protein.
[0172] SEQ ID NO: 142 provides an exemplary .beta.-Klotho binding
protein sequence that can be attached at its N- or C-terminus
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein. In addition, it can be linked to
SEQ ID NO: 141 via a linker and then the resulting chimera attached
at its N- or C-terminus directly or indirectly to any of the FGF1
mutants provided herein to generate a chimeric protein.
[0173] SEQ ID NO: 143 provides an exemplary .beta.-Klotho binding
protein sequence that can be attached at its N- or C-terminus
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein. In addition, it can be linked to
SEQ ID NO: 141 via a linker and then the resulting chimera attached
at its N- or C-terminus directly or indirectly to any of the FGF1
mutants provided herein to generate a chimeric protein.
[0174] SEQ ID NOs: 144-146 provide exemplary .beta.-Klotho binding
protein sequences that can be attached at their N- or C-termini
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein.
[0175] SEQ ID NO: 147 provides an exemplary FGFR1c binding protein
sequence that can be attached at its N- or C-terminus directly or
indirectly to any of the FGF1 mutants provided herein to generate a
chimeric protein. In addition, it can be linked to itself one or
more times to generate an FGFR1c multimer, such as a dimer or a
trimer.
[0176] SEQ ID NO: 148 (C2987) provides an exemplary FGFR1c binding
protein sequence that can be attached at its N- or C-terminus
directly or indirectly to any of the FGF1 mutants provided herein
to generate a chimeric protein. In addition, it can be linked to
itself one or more times to generate an FGFR1c multimer, such as a
dimer or a trimer.
[0177] SEQ ID NOS: 149-167 provide exemplary FGFR1c binding protein
sequences that can be attached at their N- or C-termini directly or
indirectly to any of the FGF1 mutants provided herein to generate a
chimeric protein. In addition, each can be linked to itself one or
more times to generate an FGFR1c multimer, such as a dimer or a
trimer, or combinations of these binding proteins can be linked
together.
[0178] SEQ ID NOs: 168-171 provide exemplary .beta.-Klotho-FGFR1c
binding protein sequences that can be attached at their N- or
C-termini directly or indirectly to any of the FGF1 mutants
provided herein to generate a chimeric protein.
[0179] SEQ ID NO: 172 provides an exemplary WT-FGF1/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 25A.
[0180] SEQ ID NO: 173 provides an exemplary .DELTA.NT
FGF1/.beta.-Klotho binding protein chimera sequence (C2240). This
is represented in FIG. 25B.
[0181] SEQ ID NO: 174 provides an exemplary FGF1 KN/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 25C.
[0182] SEQ ID NO: 175 provides an exemplary FGF1KKK/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 25D.
[0183] SEQ ID NO: 176 provides an exemplary WT-FGF1/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 25F.
[0184] SEQ ID NO: 177 provides an exemplary .DELTA.NT
FGF1/.beta.-Klotho binding protein chimera sequence (C2240) with
two .beta.-Klotho binding protein portions. This is represented in
FIG. 25G.
[0185] SEQ ID NO: 178 provides an exemplary FGF1 KN/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 25H.
[0186] SEQ ID NO: 179 provides an exemplary FGF1 KKK/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 25I.
[0187] SEQ ID NO: 180 provides an exemplary WT-FGF1/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 26A.
[0188] SEQ ID NO: 181 provides an exemplary .DELTA.NT
FGF1/.beta.-Klotho binding protein chimera sequence (C2240). This
is represented in FIG. 26B.
[0189] SEQ ID NO: 182 provides an exemplary FGF1 KN/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 26C.
[0190] SEQ ID NO: 183 provides an exemplary FGF1KKK/.beta.-Klotho
binding protein chimera sequence (C2240). This is represented in
FIG. 26D.
[0191] SEQ ID NO: 184 provides an exemplary WT-FGF1/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 26F.
[0192] SEQ ID NO: 185 provides an exemplary dNT FGF1/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 26F.
[0193] SEQ ID NO: 186 provides an exemplary FGF1 KN/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 25H.
[0194] SEQ ID NO: 187 provides an exemplary FGF1KKK/.beta.-Klotho
binding protein chimera sequence (C2240) with two .beta.-Klotho
binding protein portions. This is represented in FIG. 25I.
[0195] SEQ ID NO: 188 provides an exemplary .DELTA.NT
FGF1/FGFR1c-binding protein chimera sequence (C2987). This is
represented in FIG. 23J.
[0196] SEQ ID NO: 189 provides an exemplary .DELTA.NT
FGF1/FGFR1c-binding protein chimera sequence (C2987). This is
represented in FIG. 241.
[0197] SEQ ID NO: 190 provides an exemplary FGFR1c dimer chimera
sequence (C2987). This is represented in FIG. 25E.
[0198] SEQ ID NO: 191 (FGF1(1-140.alpha..alpha.) R35E, C117V)
provides an exemplary mature form of FGF1 with mutations (R35E and
C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0199] SEQ ID NO: 192 (FGF1(1-140.alpha..alpha.) R35E, C117V, KKK)
provides an exemplary mature form of FGF1 with mutations (R35E,
K112D, K113Q, C117V, and K118V wherein numbering refers to SEQ ID
NO: 5) to reduce mitogenic activity and increase
thermostability.
[0200] SEQ ID NO: 193 (FGF1(1-140.alpha..alpha.) R35E, C117V K12V,
N95V) provides an exemplary mature form of FGF1 with mutations
(K12V, R35E, N95V, and C117V wherein numbering refers to SEQ ID NO:
5) to reduce mitogenic activity and increase thermostability.
[0201] SEQ ID NO: 194 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
R35E, C117V) provides an exemplary N-terminally truncated form of
FGF1 with mutations (R35E and C117V, wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0202] SEQ ID NO: 195 (FGF1.sup..DELTA.NTKN KKK
(10-140.alpha..alpha.)) provides an exemplary N-terminally
truncated form of FGF1 with mutations (K112D, K113Q, K118V, K12V,
N95V, C117V, and R35E, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0203] SEQ ID NO: 196 (FGF1 KKK (KN) (1-140.alpha..alpha.))
provides an exemplary mature form of FGF1 with mutations (K112D,
K113Q, K118V, K12V, N95V, C117V, and R35E, wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0204] SEQ ID NO: 197 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
M2KN) provides an exemplary N-terminally truncated form of FGF1
with mutations (K12V, L44F, R35E, C83T, N95V, C117V, and F132W,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic
activity and increase thermostability.
[0205] SEQ ID NO: 198 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
M2KNKKK) provides an exemplary N-terminally truncated form of FGF1
with mutations (K12V, L44F, R35E, C83T, N95V, C117V, K112D, K113Q,
K118V, and F132W, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0206] SEQ ID NO: 199 (FGF1(1-140.alpha..alpha.) R35V, C117V)
provides an exemplary mature form of FGF1 with mutations (R35V and
C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0207] SEQ ID NO: 200 (FGF1(1-140.alpha..alpha.) R35V, C117V, KKK)
provides an exemplary mature form of FGF1 with mutations (R35V,
K112D, K113Q, C117V, and K118V wherein numbering refers to SEQ ID
NO: 5) to reduce mitogenic activity and increase
thermostability.
[0208] SEQ ID NO: 201 (FGF1(1-140.alpha..alpha.) K12V, R35V, N95V,
C117V) provides an exemplary mature form of FGF1 with mutations
(K12V, R35V, N95V, and C117V wherein numbering refers to SEQ ID NO:
5) to reduce mitogenic activity and increase thermostability.
[0209] SEQ ID NO: 202 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
R35V, C117V) provides an exemplary N-terminally truncated form of
FGF1 with mutations (R35V and C117V, wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0210] SEQ ID NO: 203 (FGF1.sup..DELTA.NTKN KKK
(10-140.alpha..alpha.)) provides an exemplary N-terminally
truncated form of FGF1 with mutations (K112D, K113Q, K118V K12V,
N95V, C117V, and R35V, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0211] SEQ ID NO: 204 (FGF1 KKK (KN) (1-140.alpha..alpha.))
provides an exemplary mature form of FGF1 with mutations (K112D,
K113Q, K118V, K12V, N95V, C117V, and R35V, wherein numbering refers
to SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0212] SEQ ID NO: 205 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
M2KN) provides an exemplary N-terminally truncated form of FGF1
with mutations (K12V, L44F, R35V, C83T, N95V, C117V, and F132W,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic
activity and increase thermostability.
[0213] SEQ ID NO: 206 (FGF1.sup..DELTA.NT1 (10-140.alpha..alpha.)
M2KNKKK) provides an exemplary N-terminally truncated form of FGF1
with mutations (K12V, L44F, R35V, C83T, N95V, C117V, K112D, K113Q,
K118V, and F132W, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0214] SEQ ID NO: 207 (FGF1-140.alpha..alpha.) C117V, KKKR provides
an exemplary mature form of FGF1 with mutations (K112D, K113Q,
C117V, K118V, R119V, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenic activity and increase thermostability.
[0215] SEQ ID NO: 208 (FGF1-140.alpha..alpha.) C117V, KY provides
an exemplary mature form of FGF1 with mutations (K12V, Y94V, C117V,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic
activity and increase thermostability.
[0216] SEQ ID NO: 209 (FGF1-140.alpha..alpha.) C117V, KE provides
an exemplary mature form of FGF1 with mutations (K12V, E87V, C117V,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic
activity and increase thermostability.
[0217] SEQ ID NO: 210 (FGF1-140.alpha..alpha.) C117V, KEY provides
an exemplary mature form of FGF1 with mutations (K12V, E87V, Y94V,
C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0218] SEQ ID NO: 211 (FGF1-140.alpha..alpha.) C117V, KNY provides
an exemplary mature form of FGF1 with mutations (K12V, Y94V, N95V,
C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0219] SEQ ID NO: 212 (FGF1-140.alpha..alpha.) K12V, L46V, E87V,
N95V, C117V, P134V provides an exemplary mature form of FGF1 with
point mutations (K12V, L46V, E87V, N95V, C117V, P134V, wherein
numbering refers to SEQ ID NO: 5) to reduce mitogenic activity and
increase thermostability.
[0220] SEQ ID NO: 213 (FGF1-140.alpha..alpha.) C117V, K118V
provides an exemplary mature form of FGF1 with mutations (C117V and
K118V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0221] SEQ ID NO: 214 (FGF.sup..DELTA.NT1C 10-140.alpha..alpha.)
K12V, N95V, C83T, C117V provides an exemplary N-terminally
truncated form of FGF1 with mutations (K12V, N95V, C83T, and C117V,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenic
activity and increase thermostability.
[0222] SEQ ID NO: 215 (FGF.sup..DELTA.NT1C 10-140.alpha..alpha.)
K12V, N95V, C16T, C83S, C117A, provides an exemplary N-terminally
truncated form of FGF1 with mutations (K12V, N95V, C16T, C83S, and
C117A, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0223] SEQ ID NO: 216 (FGF.sup..DELTA.NT1 10-140.alpha..alpha.)
H21Y, L44F, H102Y, F108Y, C117V, provides an exemplary N-terminally
truncated form of FGF1 with mutations (H21Y, L44F, H102Y, F108Y,
and C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0224] SEQ ID NO: 217 (FGF.sup..DELTA.NT1 10-140.alpha..alpha.)
K12V, H21Y, L44F, N95V, H102Y, F108Y, C117V, provides an exemplary
N-terminally truncated form of FGF1 with mutations (K12V, H21Y,
L44F, N95V, H102Y, F108Y, and C117V, wherein numbering refers to
SEQ ID NO: 5) to reduce mitogenic activity and increase
thermostability.
[0225] SEQ ID NO: 218 (FGF1 1-140.alpha..alpha.) K12V, H21Y, L44F,
N95V, H102Y, F108Y, C117V, provides an exemplary mature form of
FGF1 with mutations (K12V, H21Y, L44F, N95V, H102Y, F108Y, and
C117V, wherein numbering refers to SEQ ID NO: 5) to reduce
mitogenic activity and increase thermostability.
[0226] SEQ ID NO: 219 (wtFGF1.DELTA.HBS-FGF21C-tail) provides an
exemplary mature form of FGF1 with mutations that reduce the
functionality of the heparin binding site to affect serum half-life
and receptor affinity (K112D, K113Q, K118V, wherein numbering
refers to SEQ ID NO: 5) fused to a portion of FGF21 at the
C-terminus (amino acids 136 to 177) to generate a reagent that
combines the metabolic benefits of a .beta. klotho-dependent
agonist (FGF21) and .beta. klotho-independent agonist (FGF1).
[0227] SEQ ID NO: 220 (wtFGF1.DELTA.HBS-FGF19C-tail) provides an
exemplary mature form of FGF1 with mutations that reduce the
functionality of the heparin binding site to affect serum half-life
and receptor affinity (K112D, K113Q, K118V, wherein numbering
refers to SEQ ID NO: 5) fused to a portion of FGF19 at the
C-terminus (amino acids 138 to 183) to generate a reagent that
combines the metabolic benefits of a .beta. klotho-dependent
agonist (FGF19) and .beta. klotho-independent agonist (FGF1).
[0228] SEQ ID NO: 221 provides an exemplary N-terminally truncated
form of FGF1, wherein the 16 N-terminal amino acids are from FGF21
(amino acids 28-43 of SEQ ID NO: 20), and the sequence includes a
C117V mutation.
[0229] SEQ ID NO: 222 provides an exemplary N-terminally truncated
form of FGF1, wherein the four N-terminal amino acids are from
FGF21 (amino acids 40-43 of SEQ ID NO: 20), and the sequence
includes a C117V mutation.
[0230] SEQ ID NO: 223 (wtFGF1-FGF21C-tail) provides an exemplary
mature form of FGF1 fused to a portion of FGF21 at the C-terminus
(amino acids 136 to 177) to generate a reagent that combines the
metabolic benefits of a .beta. klotho-dependent agonist (FGF21) and
.beta. klotho-independent agonist (FGF1).
[0231] SEQ ID NO: 224 (wtFGF1-FGF19C-tail) provides an exemplary
mature form of FGF1 fused to a portion of FGF19 at the C-terminus
(amino acids 138 to 183) to generate a reagent that combines the
metabolic benefits of a .beta. klotho-dependent agonist (FGF19) and
.beta. klotho-independent agonist (FGF1).
[0232] SEQ ID NO: 225 (FGF.sup..DELTA.NT1C 10-140.alpha..alpha.)
K12V, N95V, C117V, provides an exemplary N-terminally truncated
form of FGF1 with mutations (K12V, N95V, and C117V, wherein
numbering refers to SEQ ID NO: 5) to reduce the mitogenicity and
increase the stability of FGF1.
[0233] SEQ ID NO: 226 (FGF1 KKK 1-140.alpha..alpha.) K112D, K113Q,
K118V, provides an exemplary mature form of FGF1 with mutations
(K112D, K113Q, and K118V, wherein numbering refers to SEQ ID NO: 5)
to reduce the mitogenicity and increase the stability of FGF1.
[0234] SEQ ID NO: 227 (FGF1 1-140.alpha..alpha.) K12V, Q40P, S47I,
H93G, N95V, provides an exemplary mature form of FGF1 with
mutations (K12V, Q40P, S47I, H93G, and N95V, wherein numbering
refers to SEQ ID NO: 5) to reduce the mitogenicity and increase the
thermal stability of FGF1.
[0235] SEQ ID NO: 228 (FGF.sup..DELTA.NT 10-140.alpha..alpha.)
K12V, Q40P, S47I, H93G, N95V provides an exemplary N-terminally
truncated form of FGF1 with mutations (K12V, Q40P, S47I, H93G, and
N95V, wherein numbering refers to SEQ ID NO: 5) to reduce the
mitogenicity and increase the thermal stability of FGF1.
[0236] SEQ ID NO: 229 (FGF1 1-140.alpha..alpha.) M2KN K12V, L44F,
C83T, N95V, C117V, F132W provides an exemplary mature form of FGF1
with mutations (K12V, L44F, C83T, N95V, C117V, and F132W, wherein
numbering refers to SEQ ID NO: 5) to reduce the mitogenicity
without increasing the thermal stability of FGF1.
[0237] SEQ ID NO: 230 (FGF1 1-140.alpha..alpha.) C117V provides an
exemplary mature form of FGF1 with mutation (C117V, wherein
numbering refers to SEQ ID NO: 5) to improve the stability of FGF1
by eliminating a free cysteine the can form disulfide brigded
aggregated protein.
[0238] SEQ ID NO: 231 (FGF1 1-140.alpha..alpha.))KKK(KN) K112D,
K113Q, K118V, K12V, N95V, C117V provides an exemplary mature form
of FGF1 with mutations (K112D, K113Q, K118V, K12V, N95V, and C117V,
wherein numbering refers to SEQ ID NO: 5) to reduce mitogenicity
and heparan binding, and decrease the potential for protein
aggregation of FGF1.
[0239] SEQ ID NO: 232 (FGF1 10-140.alpha..alpha.) M2KN K12V, L44F,
C83T, N95V, C117V, F132W, provides an exemplary N-terminally
truncated form of FGF1 with mutations (K12V, L44F, C83T, N95V,
C117V, and F132W, wherein numbering refers to SEQ ID NO: 5) to
reduce mitogenicity and decrease the potential for protein
aggregation of FGF1, without affecting the thermal stability.
[0240] SEQ ID NO: 233 (FGF1 1-140.alpha..alpha.) R35E, C117V,
provides an exemplary mature form of FGF1 with mutations (R35E and
C117V, wherein numbering refers to SEQ ID NO: 5) to manipulate the
receptor binding affinity/specificity and decrease the potential
for protein aggregation of FGF1.
[0241] SEQ ID NO: 234 (FGF1 1-140.alpha..alpha.) KY K12V, Y94V,
C117V, provides an exemplary mature form of FGF1 with mutations
(K12V, Y94V, and C117V, wherein numbering refers to SEQ ID NO: 5)
to manipulate the receptor binding affinity/specificity and
decrease the potential for protein aggregation of FGF1.
[0242] SEQ ID NO: 235 (FGF1 1-140.alpha..alpha.) KE K12V, E87V,
C117V, provides an exemplary mature form of FGF1 with mutations
(K12V, E87V, and C117V, wherein numbering refers to SEQ ID NO: 5)
to manipulate the receptor binding affinity/specificity and
decrease the potential for protein aggregation of FGF1
[0243] SEQ ID NO: 236 (FGF1 1-140.alpha..alpha.) KKKR K112D, K113Q,
C117V, K118V, R119V provides an exemplary mature form of FGF1 with
mutations (K112D, K113Q, C117V, K118V, and R119V, wherein numbering
refers to SEQ ID NO: 5) to reduce the heparan binding
affinity/specificity and decrease the potential for protein
aggregation of FGF1.
[0244] SEQ ID NO: 237 (FGF1 1-140.alpha..alpha.) KN R35E, K12V,
N95V, C117V provides an exemplary mature form of FGF1 with
mutations (R35E, K12V, N95V, and C117V, wherein numbering refers to
SEQ ID NO: 5) to manipulate the receptor binding
affinity/specificity and decrease the potential for protein
aggregation of FGF1.
[0245] SEQ ID NO: 238 (FGF1 10-140.alpha..alpha.) KN R35E, C117V
provides an exemplary N-terminally truncated form of FGF1 with
mutations (R35E and C117V wherein numbering refers to SEQ ID NO: 5)
to manipulate the receptor binding affinity/specificity and
decrease the potential for protein aggregation of FGF1.
DETAILED DESCRIPTION
[0246] The following explanations of terms and methods are provided
to better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. The singular forms "a," "an," and "the" refer to one or
more than one, unless the context clearly dictates otherwise. For
example, the term "comprising a cell" includes single or plural
cells and is considered equivalent to the phrase "comprising at
least one cell." The term "or" refers to a single element of stated
alternative elements or a combination of two or more elements,
unless the context clearly indicates otherwise. As used herein,
"comprises" means "includes." Thus, "comprising A or B," means
"including A, B, or A and B," without excluding additional
elements. Dates of GenBank.RTM. Accession Nos. referred to herein
are the sequences available at least as early as Oct. 21, 2013. All
references and GenBank.RTM. Accession numbers cited herein are
incorporated by reference in their entireties.
[0247] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting.
[0248] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0249] Administration: To provide or give a subject an agent, such
as a mutated FGF1 protein disclosed herein, by any effective route.
Exemplary routes of administration include, but are not limited to,
oral, injection (such as subcutaneous, intramuscular, intradermal,
intraperitoneal, intravenous, and intratumoral), sublingual,
rectal, transdermal, intranasal, vaginal and inhalation routes.
[0250] Beta-Klotho binding domain or protein: A peptide sequence
that binds selectively to .beta.-Klotho (such as human
.beta.-Klotho, OMIM 61135, GenBank.RTM. Accession No. NP_783864.1),
but not to other proteins. .beta.-Klotho is a cofactor for FGF21
activity. Such a binding domain can include one or more monomers
(wherein the monomers can be the same or different .beta.-Klotho
binding proteins), thereby generating a multimer (such as a dimer).
In specific examples, such a domain/protein is not an antibody.
Exemplary .beta.-Klotho binding proteins can be found in SEQ ID
NOS: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145
146, and 168-171 as well as U.S. Pat. No. 8,372,952, U.S.
Publication No. 2013/0197191, and Smith et al., PLoS One 8:e61432,
2013, all herein incorporated by reference.
[0251] A .beta.-Klotho binding protein "specifically binds" to
.beta.-Klotho when the dissociation constant (K.sub.D) is at least
about 1.times.10.sup.-7 M, at least about 1.5.times.10.sup.-7, at
least about 2.times.10.sup.-7, at least about 2.5.times.10.sup.-7,
at least about 3.times.10.sup.-7, at least about at least about
5.times.10.sup.-7 M, at least about 1.times.10.sup.-8 M, at least
about 5.times.10.sup.-8, at least about 1.times.10.sup.-9, at least
about 5.times.10.sup.-9, at least about 1.times.10.sup.-10, or at
least about 5.times.10.sup.-10 M. In one embodiment, K.sub.D is
measured by a radiolabeled antigen binding assay (RIA) performed
with the .beta.-Klotho binding protein and .beta.-Klotho. In
another example, K.sub.D is measured using an ELISA assay.
[0252] C-terminal portion: A region of a protein sequence that
includes a contiguous stretch of amino acids that begins at or near
the C-terminal residue of the protein. A C-terminal portion of the
protein can be defined by a contiguous stretch of amino acids
(e.g., a number of amino acid residues).
[0253] Chimeric protein: A protein that includes at least a portion
of the sequence of a full-length first protein (e.g., FGF1) and at
least a portion of the sequence of a full-length second protein
(e.g., FGF19, FGF21, .beta.-Klotho-binding protein, or
FGF1Rc-binding protein), where the first and second proteins are
different. A chimeric polypeptide also encompasses polypeptides
that include two or more non-contiguous portions derived from the
same polypeptide. The two different peptides can be joined directly
or indirectly, for example using a linker.
[0254] Diabetes mellitus: A group of metabolic diseases in which a
subject has high blood sugar, either because the pancreas does not
produce enough insulin, or because cells do not respond to the
insulin that is produced. Type 1 diabetes results from the body's
failure to produce insulin. This form has also been called
"insulin-dependent diabetes mellitus" (IDDM) or "juvenile
diabetes". Type 2 diabetes results from insulin resistance, a
condition in which cells fail to use insulin properly, sometimes
combined with an absolute insulin deficiency. This form is also
called "non insulin-dependent diabetes mellitus" (NIDDM) or
"adult-onset diabetes." The defective responsiveness of body
tissues to insulin is believed to involve the insulin receptor.
Diabetes mellitus is characterized by recurrent or persistent
hyperglycemia, and in some examples diagnosed by demonstrating any
one of: [0255] a. Fasting plasma glucose level.gtoreq.7.0 mmol/l
(126 mg/dl); [0256] b. Plasma glucose.gtoreq.11.1 mmol/1(200 mg/dL)
two hours after a 75 g oral glucose load as in a glucose tolerance
test; [0257] c. Symptoms of hyperglycemia and casual plasma
glucose.gtoreq.11.1 mmol/l (200 mg/dl); [0258] d. Glycated
hemoglobin (Hb A1C).gtoreq.6.5%
[0259] Effective amount or Therapeutically effective amount: The
amount of agent, such as a mutated FGF1 protein (or nucleic acid
encoding such) disclosed herein, that is an amount sufficient to
prevent, treat (including prophylaxis), reduce and/or ameliorate
the symptoms and/or underlying causes of any of a disorder or
disease. In one embodiment, an "effective amount" is sufficient to
reduce or eliminate a symptom of a disease, such as a diabetes
(such as type II diabetes), for example by lowering blood
glucose.
[0260] Fibroblast Growth Factor 1 (FGF1): OMIM 13220. Includes FGF1
nucleic acid molecules and proteins. A protein that binds to the
FGF receptor, and is also known as the acidic FGF. FGF1 sequences
are publically available, for example from GenBank.RTM. sequence
database (e.g., Accession Nos. NP_00791 and NP_034327 provide
exemplary FGF1 protein sequences, while Accession Nos. NM_000800
and NM_010197 provide exemplary FGF1 nucleic acid sequences). One
of ordinary skill in the art can identify additional FGF1 nucleic
acid and protein sequences, including FGF1 variants.
[0261] Specific examples of native FGF1 sequences are provided in
SEQ ID NOS: 1-5. A native FGF1 sequence is one that does not
include a mutation that alters the normal activity of the protein
(e.g., activity of SEQ ID NO: 2, 4 or SEQ ID NO: 5). A mutated FGF1
is a variant of FGF1 with different or altered biological activity,
such as reduced mitogenicity (e.g., a variant of any of SEQ ID NOS:
1-5, such as one having at least 90%, at least 95%, at least 96%,
at least 97%, at least 98% or at least 99% sequence identity to any
of SEQ ID NOS: 1-5, but is not a native/wild-type sequence). In one
example, such a variant includes an N-terminal truncation, at least
one point mutation (such as one or more of those shown in Table 1),
or combinations thereof, such as changes that decrease mitogenicity
of FGF1. Mutated FGF1 proteins include FGF1 chimeras (e.g.,
FGF1/FGF19 chimeras). Specific exemplary FGF1 mutant proteins are
shown in SEQ ID NOS: 6-13, 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 113, 114, 115, 116, 117,
118, 119, 120, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,
227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 and 238.
[0262] Fibroblast Growth Factor 19 (FGF19): OMIM 603891. Includes
FGF19 nucleic acid molecules and proteins. FGF19 regulates bile
acid synthesis and has effects on glucose and lipid metabolism.
FGF19 sequences are publically available, for example from the
GenBank.RTM. sequence database (e.g., Accession Nos. NP_005108.1
and AAQ88669.1 provide exemplary FGF19 protein sequences, while
Accession Nos. AY358302.1 and NM_005117.2 provide exemplary FGF19
nucleic acid sequences). One of ordinary skill in the art can
identify additional FGF19 nucleic acid and protein sequences,
including FGF19 variants.
[0263] Fibroblast Growth Factor 21 (FGF21): OMIM 609436. Includes
FGF21 nucleic acid molecules and proteins. FGF21 stimulates glucose
updated in adipocytes. FGF21 sequences are publically available,
for example from the GenBank.RTM. sequence database (e.g.,
Accession Nos. AAQ89444.1, NP_061986, and AAH49592.1 provide
exemplary FGF21 protein sequences, while Accession Nos. AY359086.1
and BC049592 provide exemplary FGF21 nucleic acid sequences). One
of ordinary skill in the art can identify additional FGF21 nucleic
acid and protein sequences, including FGF21 variants.
[0264] Fibroblast Growth Factor Receptor 1c (FGFR1c) binding domain
or protein: A peptide sequence that binds selectively to FGFR1c
(such as human FGFR1c, e.g., GeneBank Accession No. NP_001167536.1
or NP_056934.2), but not to other proteins. FGFR1c is a cofactor
for FGF21 activity. Such a binding domain can include one or more
monomers (wherein the monomers can be the same or different
sequences), thereby generating a multimer (such as a dimer). In
specific examples, such a domain/protein is not an antibody.
Exemplary FGFR1c-binding proteins can be found in SEQ ID NOS: 147,
148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,
161, 162, 163, 164, 165, 166, 167 and portions of 168, 169, 170 and
171, or a multimer thereof such as SEQ ID NO: 190, as well as U.S.
Pat. No. 8,372,952, U.S. Publication No. 2013/0197191, and Smith et
al., PLoS One 8:e61432, 2013, all herein incorporated by reference.
Thus, reference to a FGFR1c-binding protein multimer, includes
proteins made using two or more peptides having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to one or more of SEQ ID NO:
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167, and 190.
[0265] A FGFR1c binding protein "specifically binds" to FGFR1c when
the dissociation constant (K.sub.D) is at least about
1.times.10.sup.-7 M, at least about 1.5.times.10.sup.-7, at least
about 2.times.10.sup.-7, at least about 2.5.times.10.sup.-7, at
least about 3.times.10.sup.-7, at least about at least about
5.times.10.sup.-7 M, at least about 1.times.10.sup.-8 M, at least
about 5.times.10.sup.-8, at least about 1.times.10.sup.-9, at least
about 5.times.10.sup.-9, at least about 1.times.10.sup.-10, or at
least about 5.times.10.sup.-10 M. In one embodiment, K.sub.D is
measured by a radiolabeled antigen binding assay (RIA) performed
with the FGFR1c-binding protein and FGFR1c. In another example,
K.sub.D is measured using an ELISA assay.
[0266] Fibroblast Growth Factor Receptor 1c (FGFR1c): Also known as
FGFR1 isoform 2. Includes FGFR1c nucleic acid molecules and
proteins. FGFR1c and .beta.-Klotho can associate with FGF21 to form
a signaling complex. FGFR1c sequences are publically available, for
example from the GenBank.RTM. sequence database (e.g., Accession
Nos. NP_001167536.1 and NP_056934.2 provide exemplary FGFR1c
protein sequences). One of ordinary skill in the art can identify
additional FGFR1c nucleic acid and protein sequences, including
FGFR1c variants.
[0267] Fibroblast Growth Factor Receptor 4 (FGFR4): OMIM 134935.
Includes FGFR4 nucleic acid molecules and proteins. FGFR4 can bind
to some FGF proteins, including FGF1. FGFR4 sequences are
publically available, for example from the GenBank.RTM. sequence
database (e.g., Accession Nos. NM_002011 and AAB25788.1 provide
exemplary FGFR4 protein sequences, while Accession Nos. NM_002002
and L03840.1 provide exemplary FGFR4 nucleic acid sequences). One
of ordinary skill in the art can identify additional FGFR4 nucleic
acid and protein sequences, including FGFR4 variants.
[0268] Host cells: Cells in which a vector can be propagated and
its DNA expressed. The cell may be prokaryotic or eukaryotic. The
term also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used. Thus, host cells can be transgenic, in that they include
nucleic acid molecules that have been introduced into the cell,
such as a nucleic acid molecule encoding a mutant FGF1 protein
disclosed herein.
[0269] Isolated: An "isolated" biological component (such as a
mutated FGF1 protein or nucleic acid molecule) has been
substantially separated, produced apart from, or purified away from
other biological components in the cell of the organism in which
the component naturally occurs, such as other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acids molecules
and proteins which have been "isolated" thus include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acid molecules and proteins prepared by
recombinant expression in a host cell as well as chemically
synthesized nucleic acids. A purified or isolated cell, protein, or
nucleic acid molecule can be at least 70%, at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% pure.
[0270] Linker: A moiety or group of moieties that joins or connects
two or more discrete separate peptide or proteins, such as monomer
domains, for example to generate a chimeric protein. In one example
a linker is a substantially linear moiety.
[0271] Exemplary linkers that can be used to generate the chimeric
proteins provided herein include but are not limited to: peptides,
nucleic acid molecules, peptide nucleic acids, and optionally
substituted alkylene moieties that have one or more oxygen atoms
incorporated in the carbon backbone. A linker can be a portion of a
native sequence, a variant thereof, or a synthetic sequence.
Linkers can include naturally occurring amino acids, non-naturally
occurring amino acids, or a combination of both. In one example a
linker is composed of at least 5, at least 10, at least 15 or at
least 20 amino acids, such as 5 to 10, 5 to 20, or 5 to 50 amino
acids. In one example the linker is a poly alanine.
[0272] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects (such as cats, dogs, cows, and pigs) and rodents (such as
mice and rats).
[0273] Metabolic disorder/disease: A disease or disorder that
results from the disruption of the normal mammalian process of
metabolism. Includes metabolic syndrome.
[0274] Examples include but are not limited to: (1) glucose
utilization disorders and the sequelae associated therewith,
including diabetes mellitus (Type I and Type-2), gestational
diabetes, hyperglycemia, insulin resistance, abnormal glucose
metabolism, "pre-diabetes" (Impaired Fasting Glucose (IFG) or
Impaired Glucose Tolerance (IGT)), and other physiological
disorders associated with, or that result from, the hyperglycemic
condition, including, for example, histopathological changes such
as pancreatic .beta.-cell destruction; (2) dyslipidemias and their
sequelae such as, for example, atherosclerosis, coronary artery
disease, cerebrovascular disorders and the like; (3) other
conditions which may be associated with the metabolic syndrome,
such as obesity and elevated body mass (including the co-morbid
conditions thereof such as, but not limited to, nonalcoholic fatty
liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and
polycystic ovarian syndrome (PCOS)), and also include thromboses,
hypercoagulable and prothrombotic states (arterial and venous),
hypertension, cardiovascular disease, stroke and heart failure; (4)
disorders or conditions in which inflammatory reactions are
involved, including atherosclerosis, chronic inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), asthma,
lupus erythematosus, arthritis, or other inflammatory rheumatic
disorders; (5) disorders of cell cycle or cell differentiation
processes such as adipose cell tumors, lipomatous carcinomas
including, for example, liposarcomas, solid tumors, and neoplasms;
(6) neurodegenerative diseases and/or demyelinating disorders of
the central and peripheral nervous systems and/or neurological
diseases involving neuroinfiammatory processes and/or other
peripheral neuropathies, including Alzheimer's disease, multiple
sclerosis, Parkinson's disease, progressive multifocal
leukoencephalopathy and Guillian-Barre syndrome; (7) skin and
dermatological disorders and/or disorders of wound healing
processes, including erythemato-squamous dermatoses; and (8) other
disorders such as syndrome X, osteoarthritis, and acute respiratory
distress syndrome. Other examples are provided in WO 2014/085365
(herein incorporated by reference).
[0275] In specific examples, the metabolic disease includes one or
more of (such as at least 2 or at least 3 of): diabetes (such as
type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent
autoimmune diabetes (LAD), or maturity onset diabetes of the young
(MODY)), polycystic ovary syndrome (PCOS), metabolic syndrome
(MetS), obesity, non-alcoholic steatohepatitis (NASH),
non-alcoholic fatty liver disease (NAFLD), dyslipidemia (e.g.,
hyperlipidemia), and cardiovascular diseases (e.g.,
hypertension).
[0276] N-terminal portion: A region of a protein sequence that
includes a contiguous stretch of amino acids that begins at or near
the N-terminal residue of the protein. An N-terminal portion of the
protein can be defined by a contiguous stretch of amino acids
(e.g., a number of amino acid residues).
[0277] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence (such as a
mutated FGF1 coding sequence). Generally, operably linked DNA
sequences are contiguous and, where necessary to join two protein
coding regions, in the same reading frame.
[0278] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the disclosed mutated FGF1 proteins and FGFR1c-binding protein
multimers (or nucleic acid molecules encoding such) herein
disclosed.
[0279] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0280] Promoter: Ann array of nucleic acid control sequences which
direct transcription of a nucleic acid. A promoter includes
necessary nucleic acid sequences near the start site of
transcription, such as, in the case of a polymerase II type
promoter, a TATA element. A promoter also optionally includes
distal enhancer or repressor elements which can be located as much
as several thousand base pairs from the start site of
transcription.
[0281] Recombinant: A recombinant nucleic acid molecule is one that
has a sequence that is not naturally occurring (e.g., a mutated
FGF1 or chimeric protein) or has a sequence that is made by an
artificial combination of two otherwise separated segments of
sequence. This artificial combination can be accomplished by
routine methods, such as chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acids, such as by
genetic engineering techniques. Similarly, a recombinant protein is
one encoded for by a recombinant nucleic acid molecule. Similarly,
a recombinant or transgenic cell is one that contains a recombinant
nucleic acid molecule and expresses a recombinant protein.
[0282] Sequence identity of amino acid sequences: The similarity
between amino acid (or nucleotide) sequences is expressed in terms
of the similarity between the sequences, otherwise referred to as
sequence identity. Sequence identity is frequently measured in
terms of percentage identity (or similarity or homology); the
higher the percentage, the more similar the two sequences are.
Homologs or variants of a polypeptide will possess a relatively
high degree of sequence identity when aligned using standard
methods.
[0283] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and
Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and
Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989;
Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson
and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul
et al., Nature Genet. 6:119, 1994, presents a detailed
consideration of sequence alignment methods and homology
calculations.
[0284] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. A description of how to determine
sequence identity using this program is available on the NCBI
website on the internet.
[0285] Homologs and variants of the mutated FGF1 proteins and
coding sequences disclosed herein are typically characterized by
possession of at least about 80%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity counted over the full length alignment with the amino acid
sequence using the NCBI Blast 2.0, gapped blastp set to default
parameters. For comparisons of amino acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). When
aligning short peptides (fewer than around 30 amino acids), the
alignment should be performed using the Blast 2 sequences function,
employing the PAM30 matrix set to default parameters (open gap 9,
extension gap 1 penalties). Proteins with even greater similarity
to the reference sequences will show increasing percentage
identities when assessed by this method, such as at least 95%, at
least 98%, or at least 99% sequence identity. When less than the
entire sequence is being compared for sequence identity, homologs
and variants will typically possess at least 80% sequence identity
over short windows of 10-20 amino acids, and may possess sequence
identities of at least 85% or at least 90% or at least 95%
depending on their similarity to the reference sequence. Methods
for determining sequence identity over such short windows are
available at the NCBI website on the internet. One of skill in the
art will appreciate that these sequence identity ranges are
provided for guidance only; it is entirely possible that strongly
significant homologs could be obtained that fall outside of the
ranges provided.
[0286] Thus, a mutant FGF1 protein disclosed herein can have at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 95%, at least 96%, at least 97%, at least 98% or at least
99% sequence identity to SEQ ID NO: 5, but is not SEQ ID NO: 5
(which in some examples has one or more, such as 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 of the mutations or truncations shown in Tables 1
and 2). In addition, exemplary mutated FGF1 proteins have at least
80%, at least 85%, at least 90%, at least 92%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238, as well as such sequences
schematically shown in FIGS. 23-26 (e.g., at least 80%, at least
85%, at least 90%, at least 92%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO: 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, or
188), and retain the ability to reduce blood glucose levels in
vivo.
[0287] Similarly, exemplary mutated FGF1 coding sequences in some
examples have at least 70%, at least 80%, at least 85%, at least
90%, at least 92%, at least 95%, at least 98%, or at least 99%
sequence identity to SEQ ID NO: 18.
[0288] Similarly, exemplary .beta.-Klotho-binding domain sequences
that can be used in the mutant FGF1 chimeras disclosed herein in
some examples have at least 70%, at least 80%, at least 85%, at
least 90%, at least 92%, at least 95%, at least 97%, at least 98%,
or at least 99% sequence identity to SEQ ID NO: 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146 or
.beta.-Klotho-binding portions of SEQ ID NO: 168, 169, 170 or
171.
[0289] Similarly, exemplary FGFR1c binding sequences that can be
used in the mutant FGF1 chimeras disclosed herein in some examples
have at least 70%, at least 80%, at least 85%, at least 90%, at
least 92%, at least 95%, at least 97%, at least 98%, or at least
99% sequence identity to SEQ ID NO: 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,
166, 167, or FGFR1c-binding portions of SEQ ID NO: 168, 169, 170,
171, or multimers such as SEQ ID NO: 190.
[0290] Subject: Any mammal, such as humans, non-human primates,
pigs, sheep, cows, dogs, cats, rodents and the like which is to be
the recipient of the particular treatment, such as treatment with a
mutated FGF1 protein or chimera (or corresponding nucleic acid
molecule) provided herein. In two non-limiting examples, a subject
is a human subject or a murine subject. In some examples, the
subject has one or more metabolic diseases, such as diabetes (e.g.,
type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent
autoimmune diabetes (LAD), or maturity onset diabetes of the young
(MODY)), polycystic ovary syndrome (PCOS), metabolic syndrome
(MetS), obesity, non-alcoholic steatohepatitis (NASH),
non-alcoholic fatty liver disease (NAFLD), dyslipidemia (e.g.,
hyperlipidemia), cardiovascular disease (e.g., hypertension), or
combinations thereof. In some examples, the subject has elevated
blood glucose.
[0291] Transduced and Transformed: A virus or vector "transduces" a
cell when it transfers nucleic acid into the cell. A cell is
"transformed" or "transfected" by a nucleic acid transduced into
the cell when the DNA becomes stably replicated by the cell, either
by incorporation of the nucleic acid into the cellular genome, or
by episomal replication.
[0292] Numerous methods of transfection are known to those skilled
in the art, such as: chemical methods (e.g., calcium-phosphate
transfection), physical methods (e.g., electroporation,
microinjection, particle bombardment), fusion (e.g., liposomes),
receptor-mediated endocytosis (e.g., DNA-protein complexes, viral
envelope/capsid-DNA complexes) and by biological infection by
viruses such as recombinant viruses {Wolff, J. A., ed, Gene
Therapeutics, Birkhauser, Boston, USA (1994)}. In the case of
infection by retroviruses, the infecting retrovirus particles are
absorbed by the target cells, resulting in reverse transcription of
the retroviral RNA genome and integration of the resulting provirus
into the cellular DNA.
[0293] Transgene: An exogenous gene supplied by a vector. In one
example, a transgene includes a mutated FGF1 coding sequence (which
may be part of a chimera).
[0294] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in the
host cell, such as an origin of replication. A vector may also
include one or more mutated FGF1 coding sequences (which may be
part of a chimera) and/or selectable marker genes and other genetic
elements known in the art. A vector can transduce, transform or
infect a cell, thereby causing the cell to express nucleic acids
and/or proteins other than those native to the cell. A vector
optionally includes materials to aid in achieving entry of the
nucleic acid into the cell, such as a viral particle, liposome,
protein coating or the like.
Overview
[0295] Provided herein are mutated FGF1 proteins that can include
an N-terminal deletion, one or more point mutations (such as amino
acid substitutions, deletions, additions, or combinations thereof),
or combinations of N-terminal deletions and point mutations. Such
mutated FGF1 proteins can be part of a chimeric protein, such as a
C-terminal portion of FGF21 or 19 (e.g., SEQ ID NO: 86 or 100,
respectively), a .beta.-Klotho binding protein (e.g., SEQ ID NOS:
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, and 187), or
an FGFR1c binding protein (e.g., see SEQ ID NOS: 188 and 189), or
both a .beta.-Klotho binding protein and an FGFR1c binding protein
(e.g., linked directly or indirectly to any of SEQ ID NOS: 168,
169, 170 or 171). Thus, when referring to a mutated FGF1 protein(s)
herein, such reference also includes reference to mutated
FGF1/FGF21, mutated FGF1/FGF19 chimeras, mutated
FGF1/.beta.-Klotho-binding chimeras, mutated FGF1/FGF1Rc-binding
chimeras, or mutated FGF1/.beta.-Klotho-binding/FGF1Rc-binding
chimeras.
[0296] Also provided are methods of using FGF1 mutant proteins or
FGF1Rc-binding protein multimers (or their nucleic acid coding
sequences) to lower glucose, for example to treat a metabolic
disease. In some examples such methods include administering a
therapeutically effective amount of a mutated mature FGF21 protein
or FGF1Rc-binding protein multimer to the mammal, or a nucleic acid
molecule encoding the mutated mature FGF21 protein or
FGF1Rc-binding protein multimer or a vector comprising the nucleic
acid molecule, thereby reducing the blood glucose, treating the one
or more metabolic diseases, or combinations thereof. Exemplary
metabolic diseases that can be treated with the disclosed methods
include but are not limited to: type 2 diabetes, non-type 2
diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS),
metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis
(NASH), non-alcoholic fatty liver disease (NAFLD), dyslipidemia
(e.g., hyperlipidemia), cardiovascular diseases (e.g.,
hypertension), latent autoimmune diabetes (LAD), or maturity onset
diabetes of the young (MODY).
[0297] In some examples, mutations in FGF1 reduce the mitogenicity
of mature wild-type FGF1 (e.g., SEQ ID NO: 5), such as a reduction
of at least 20%, at least 50%, at least 75% or at least 90%. For
example, mutated FGF1 can be mutated to decrease binding affinity
for heparin and/or heparan sulfate compared to an FGF1 protein
without the modification (e.g., a native or wild-type FGF1
protein). Methods of measuring mitogenicity are known in the art.
In one example, the method provided in Example 2 is used.
[0298] In some examples, the mutant FGF1 protein is a truncated
version of the mature protein (e.g., SEQ ID NO: 5), which can
include for example deletion of at least 5, at least 6, at least 9,
at least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, or at least 20 consecutive N-terminal amino acids, such
as the N-terminal 5 to 10, 5 to 13, 5, 6, 7, 8, 9, 10, 11, 12, or
13 amino acids of mature FGF1. In some examples, such an
N-terminally deleted FGF1 protein has reduced mitogenic activity as
compared to wild-type mature FGF1 protein.
[0299] In some examples, one or more of the deleted N-terminal
amino acids are replaced with corresponding amino acids from FGF21
(e.g., see SEQ ID NO: 20), such as at least 1, at least 2, at least
3, at least 4, at least 5, at least 10, at least 15, or at least 20
amino acids from FGF21, such as 1-5, 1-4, 2-4, 4-6, 4-9, 3-10, 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 corresponding amino acids from FGF21.
An example of an FGF1 mutated protein with an N-terminal deletion
having four corresponding N-terminal amino acids from FGF21 is
shown in SEQ ID NO: 21 and 222. An example of an FGF1 mutated
protein with an N-terminal deletion having 16 N-terminal amino
acids from FGF21 is shown in SEQ ID NO: 221. One skilled in the art
will appreciate that amino acids from other FGFs besides FGF21 can
be used, including those having low affinity for FGFR4, including
FGF3, FGF5, FGF7, FGF9 and FGF10. The N-terminal residues of FGF1
include an FGFR4 binding site, and FGFR4 signaling is associated
with mitogenic activity. In contrast, FGF21 has low affinity for
FGFR4. Thus, replacing the FGFR4 binding residues of FGF1 with
those from FGF21 can be used to reduce mitogenicity of the
resulting FGF1 mutant protein.
[0300] In some examples, mutations in FGF1 increase the
thermostability of mature or truncated FGF1 (e.g., SEQ ID NO: 5),
such as an increase of at least 20%, at least 50%, at least 75% or
at least 90% compared to native FGF1. Exemplary mutations that can
be used to increase the thermostability of mutated FGF1 include but
are not limited to one or more of: K12V, C117V, C117P, C117T,
C117S, C117A and P134V (referred to as M1 mutations), L44F, C83T,
C83S, C83A C83V, C117V, C117P, C117T, C117S, C117A and F132W
(referred to as M2 mutations), and L44F, M67I, L73V, V109L, L111I,
C117V, C117P, C117T, C117S, C117A A103G, R119G, R119V,
.DELTA.104-106, and .DELTA.120-122 (referred to as M3 deletions),
wherein the numbering refers to SEQ ID NO: 5 (e.g., see Xia et al.,
PLoS One. 7:e48210, 2012). For example, mutated FGF1 can be mutated
to increase the thermostability of the protein compared to an FGF1
protein without the modification (e.g., SEQ ID NO: 5). Methods of
measuring thermostability are known in the art. In one example, the
method provided in Xia et al., PLoS One. 7:e48210, 2012 is
used.
[0301] In some examples, the mutant FGF1 protein is a mutated
version of the mature protein (e.g., SEQ ID NO: 5), such as one
containing at least 1, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 11, at least 12,
at least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24 or at least 25 amino acid substitutions, such
as 1-20, 1-10, 4-8, 5-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, or 37 amino acid substitutions (such as
those shown in Table 1). In some examples, the mutant FGF1 protein
includes deletion of one or more amino acids, such as deletion of
1-10, 4-8, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 amino acid deletions. In some examples, the
mutant FGF1 protein includes a combination of amino acid
substitutions and deletions, such as at least 1 substitution and at
least 1 deletion, such as 1 to 10 substitutions with 1 to 10
deletions.
[0302] Exemplary mutations are shown in Table 1 below, with amino
acids referenced to either SEQ ID NO: 2 or 5. One skilled in the
art will recognize that these mutations can be used singly, or in
combination (such as 1-20, 1-10, 4-8, 5-25, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41
of these amino acid substitutions and/or deletions). In addition,
such mutant FGF1 proteins are part of a chimeric protein, such as
with FGF19, FGF21, a protein that selectively binds to
.beta.-Klotho, or a protein that selectively binds to FGFR1c.
TABLE-US-00001 TABLE 1 Exemplary FGF1 mutations Location of Point
Location of Point Mutation Position Mutation Position in SEQ ID NO:
2 Mutation Citation in SEQ ID NO: 5 K24 K9T K9 K25 K10T K10 K27
K12V K12 L29 L14A L14 Y30 Y15F, Y15A, Y15V Y15 C31 C16V, C16A,
C16T, C16S C16 H36 H21Y H21 R50 R35E, R35V R35 Q55 Q40P Q40 L59
L44F L44 L61 L46V L46 S62 S47I S47 E64 E49Q, E49A E49 Y70 Y55F,
Y55S, Y55A Y55 M82 M67I M67 L88 L73V L73 C98 C83T, C83S, C83A C83V
C83 E102 E87V, E87A, E87S, E87T E87 H108 H93G, H93A H93 Y109 Y94V,
Y94F, Y94A Y94 N110 N95V, N95A, N95S, N95T N95 H117 H102Y H102 A118
A103G A103 EKN 119-121 .DELTA.104-106 EKN (104-106) F123 F108Y F108
V124 V109L V109 L126 L111I L111 K127 K112D, K112E, K112Q K112 K128
K113Q, K113E, K113D K113 C132 C117V, C117P, C117T, C117 C117S,
C117A K133 K118N, K118E, K118V K118 R134 R119G, R119V, R119E R119
GPR 135-137 .DELTA.120-122 GPR (120-122) F147 F132W F132 L148
L133A, L133S L133 P149 P134V P134 L150 L135A, L135S L135
[0303] In some examples, the mutant FGF1 protein includes mutations
at one or more of the following positions: K9, K10, K12, L14, Y15,
C16, H21, R35, Q40, L44, L46, S47, E49, Y55, M67, L73, C83, L86,
E87, H93, Y94, N95, H102, A103, E104, K105, N106, F108, V109, L111,
K112, K113, C117, K118, R119, G120, P121, R122, F132, L133, P134,
L135, such as one or more of K9, K10, K12, K112, K113, such as 1 to
5, 2 to 5, 3 to 6, 3 to 8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or all 42 of these
positions. In one example, K9 and K10 are replaced with DQ (as in
the mutated nuclear localization sequence) or with equivalent
residues from FGF21 (or another FGF that does not bind to FGFR4)
(wherein the numbering refers to SEQ ID NO: 5).
[0304] In some examples, the mutant FGF1 protein includes mutations
at 1, 2, 3 or 4 of the following positions: Y15, E87, Y94, and N95
(wherein the numbering refers to SEQ ID NO: 5), such as one or more
of Y15F, Y15A, Y15V, E87V, E87A, E87S, E87T, N95V, N95A, N95S,
N95T, Y94V, Y94F, and Y94A (such as 1, 2, 3 or 4 of these
mutations). For example, E87 or N95 can be replaced with a
non-charged amino acid. In addition, Y15 and Y94 can be replaced
with an amino acid that destabilizes the hydrophobic interactions.
In some examples, the mutant FGF1 protein includes mutations on
either side of Y15, E87, Y94, and N95, such as one or more of L14,
C16, H93, and T96, such as mutations at 1, 2, 3, or 4 of these
positions.
[0305] In some examples, the mutant FGF1 protein includes mutations
at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the following positions: Y15,
C16, E87, H93, Y94, and N95 (wherein the numbering refers to SEQ ID
NO: 5), such as one or more of Y15F, Y15A, Y15V, E87V, E87A, E87S,
E87T, H93A, N95V, N95A, N95S, N95T, Y94V, Y94F, and Y94A (such as
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these mutations).
[0306] In some examples, the mutant FGF1 protein includes mutations
at one or more of the following positions: C16, C83, and C117
(wherein the numbering refers to SEQ ID NO: 5), such as one or more
of C16V, C16A, C16T, C16S, C83T, C83S, C83A C83V, C117V, C117P,
C117T, C117S, and C117A (such as 1, 2, or 3 of these
mutations).
[0307] In some examples, the mutant FGF1 protein includes mutations
at only one or two of the following positions: E87, Y94, and N95
(wherein the numbering refers to SEQ ID NO: 5), such as one or two
of E87V, E87A, E87S, E87T, Y94V, Y94F, Y94A, N95V, N95A, N95S, and
N95T.
[0308] In some examples, the mutant FGF1 protein includes mutations
at 1, 2, or 3 of the following positions: K12, C83, and C117
(wherein the numbering refers to SEQ ID NO: 5), such as one or more
of K12V, K12C, C83T, C83S, C83A, C83V, C117V, C117P, C117T, C117S,
and C117A (such as 1, 2, or 3 of these mutations, such as K12V,
C83T, and C117V).
[0309] FIG. 31 shows specific examples of positions that can be
mutated in FGF1 to alter its activity. For example, residues that
interact with the FGF1 receptor include Y15, E87, Y94 and N95.
Thus, in some examples, 1, 2, 3, or 4 of these positions are
mutated, for example the amino acid at position 87 and/or 95 of SEQ
ID NO: 5 can be changed to a V, A, S or T. In some examples, the
amino acid at position 15 and/or 95 of SEQ ID NO: 5 can be changed
to a V, A, or F. In some examples, combinations of these changes
are made.
[0310] FIG. 31 also shows that K12 of FGF1 is predicted to be at
the receptor interface. Thus, K12 of SEQ ID NO: 5 can be mutated,
for example to a V or C. FIG. 31 also shows that amino acids K112,
K113, and K118 are part of the heparin binding site, and thus can
be mutated, for example to a E, Q, N, V or D, such as a N, E or V
at position K118, and a D, E or Q at positions K112 and K113. FIG.
31 also shows that amino acid R35 of SEQ ID NO: 5 that forms a salt
bridge with the D2 domain of the FGF receptor, and thus can be
mutated, for example to an E or V.
[0311] In some examples, the mutant FGF1 protein includes one or
more of K12V, L46V, R35E, R35V, E87V, N95V, K118N, K118E, C117V,
and P134V (wherein the numbering refers to SEQ ID NO: 5). In some
examples, the point mutation includes replacing amino acid sequence
ILFLPLPV (amino acids 145-152 of SEQ ID NO: 2 and 4) to AAALPLPV
(SEQ ID NO: 14), ILALPLPV (SEQ ID NO: 15), ILFAPLPV (SEQ ID NO:
16), or ILFLPAPA (SEQ ID NO: 17). In some examples, such an FGF1
protein with one or more point mutations has reduced mitogenic
activity as compared to wild-type mature FGF1 protein. In some
examples, the mutant FGF1 protein includes R35E (wherein the
numbering refers to SEQ ID NO: 5).
[0312] In some examples, the mutant FGF1 protein includes at least
120 consecutive amino acids from amino acids 5-141 of FGF1 (e.g.,
of SEQ ID NO: 2 or 4), (which in some examples can include further
deletion of N-terminal amino acids 1-20 and/or point mutations,
such as substitutions, deletions, or additions). In some examples,
the mutant FGF1 protein includes at least 120 or at least 130
consecutive amino acids from amino acids 5-141 of FGF1, such as at
least 120 consecutive amino acids from amino acids 5-141 of SEQ ID
NO: 2 or 4 or at least 120 consecutive amino acids from SEQ ID NO:
5.
[0313] In some examples, the mutant FGF1 protein includes both an
N-terminal truncation and point mutations. Specific exemplary FGF1
mutant proteins are shown in SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12,
13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 113, 114,
115, 116, 117, 118, 119, 120, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 and 238. In some examples,
the FGF1 mutant includes an N-terminal deletion, but retains a
methionine at the N-terminal position. In some examples, the FGF1
mutant is 120-140 or 125-140 amino acids in length.
[0314] In some examples, the FGF1 mutant protein is part of a
chimeric protein. For example, one end of the mutant FGF1 mutant
protein can be joined directly or indirectly to the end of FGF19 or
FGF21, such as a C-terminal region of FGF 19 or FGF21. In some
examples, the mutated FGF1 portion of the chimera is at the
N-terminus of the chimera, and the FGF19 or FGF21 portion is the
C-terminus of the chimera. However, this can be reversed, such that
the mutated FGF1 portion of the chimera is the C-terminus of the
chimera, and the FGF19 or FGF21 portion is the N-terminus of the
chimera. For example, at least 10, at least 20, at least 30, at
least 40, at least 41, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, at
least 50 or at least 60 C-terminal amino acids of FGF19 or FGF21
(such as the C-terminal 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42,
41, 40, 35, 30, 25, 20, 15 or 10 amino acids) can be part of the
chimera. Examples of C-terminal fragments of FGF21 and FGF19 that
can be used are shown in SEQ ID NOS: 86 and 100, respectively. In
some examples, the mutant FGF1 and FGF21 or FGF19 portion are
linked indirectly through the use of a linker, such as one composed
of at least 5, at least 10, at least 15 or at least 20 amino acids.
In one example the linker is a poly alanine.
[0315] In some examples, the FGF1 mutant protein is part of a
chimeric protein with a 3-Klotho-binding protein. For example, one
end of the mutant FGF1 mutant protein can be joined directly or
indirectly to the end of a .beta.-Klotho-binding protein (see for
example FIGS. 23-26). In some examples, the mutated FGF1 portion of
the chimera is at the N-terminus of the chimera, and the
.beta.-Klotho-binding protein portion is the C-terminus of the
chimera (e.g., see FIGS. 23B-23D, 23G-23I and 25B-25D, 25G-25I,
respectively). However, this can be reversed, such that the mutated
FGF1 portion of the chimera is the C-terminus of the chimera, and
the .beta.-Klotho binding protein portion is the N-terminus of the
chimera (e.g., see FIGS. 24B-24D, 24F-24H and 26B-26D, 26F-26H).
Examples of .beta.-Klotho-binding proteins that can be used are
shown in SEQ ID NOS: 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145 and 146 and .beta.-Klotho-binding portions of SEQ ID
NOS: 168, 169, 170, and 171. In some examples, the mutant FGF1 and
.beta.-Klotho-binding protein portion are linked indirectly through
the use of a linker, such as one composed of at least 5, at least
10, at least 15 or at least 20 amino acids. In one example the
linker is a poly alanine.
[0316] In some examples, the FGF1 mutant protein is part of a
chimeric protein with an FGFR1c-binding protein. For example, one
end of the mutant FGF1 mutant protein can be joined directly or
indirectly to the end of an FGFR1c-binding protein. In some
examples, the mutated FGF1 portion of the chimera is at the
N-terminus of the chimera, and the FGFR1c-binding protein portion
is the C-terminus of the chimera (e.g., see FIG. 23J). However,
this can be reversed, such that the mutated FGF1 portion of the
chimera is the C-terminus of the chimera, and the FGFR1c-binding
protein portion is the N-terminus of the chimera (e.g., see FIG.
241). Examples of FGFR1c-binding proteins that can be used are
shown in SEQ ID NOS: 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167 and
FGFR1c-binding portions of 168, 169, 170 and 171. In some examples,
the mutant FGF1 and FGFR1c-binding protein portion are linked
indirectly through the use of a linker, such as one composed of at
least 5, at least 10, at least 15 or at least 20 amino acids. In
one example the linker is a poly alanine.
[0317] In some examples, the FGF1 mutant protein is part of a
chimeric protein with both an FGFR1c-binding protein and a
.beta.-Klotho-binding protein, in any order. For example, one end
of the mutant FGF1 mutant protein can be joined directly or
indirectly to the end of an FGFR1c-binding/.beta.-Klotho-binding or
.beta.-Klotho-binding/FGFR1c-binding chimeric protein. In some
examples, the mutated FGF1 portion of the chimera is at the
N-terminus of the chimera, and the
FGFR1c-binding/.beta.-Klotho-binding or
.beta.-Klotho-binding/FGFR1c-binding chimeric protein portion is
the C-terminus of the chimera (e.g., see FIG. 23K). However, this
can be reversed, such that the mutated FGF1 portion of the chimera
is the C-terminus of the chimera, and the
FGFR1c-binding/.beta.-Klotho-binding or
.beta.-Klotho-binding/FGFR1c-binding chimeric protein portion is
the N-terminus of the chimera (e.g., see FIG. 24J). In one example
the FGFR1c-binding/.beta.-Klotho-binding or
.beta.-Klotho-binding/FGFR1c-binding chimeric protein is any one of
those shown in SEQ ID NOS: 168, 169, 170, and 171. In some
examples, the mutant FGF1 and FGFR1c-binding/.beta.-Klotho-binding
or .beta.-Klotho-binding/FGFR1c-binding chimeric protein portion
are linked indirectly through the use of a linker, such as one
composed of at least 5, at least 10, at least 15 or at least 20
amino acids. In one example the linker is a poly alanine.
[0318] In some examples, the FGF1 mutant protein or chimera
including such includes at least 80% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238. Thus, the FGF1 mutant protein can have at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11,
12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186,
187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,
227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238 (but
is not a native FGF1 sequence, such as SEQ ID NO: 5). In some
examples, the FGF1 mutant protein includes or consists of SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 and 238. The disclosure encompasses variants of the
disclosed FGF1 mutant proteins, such as SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238
having 1 to 8, 2 to 10, 1 to 5, 1 to 6, or 5 to 10 mutations, such
as conservative amino acid substitutions.
[0319] In some examples, a mutant FGF1/FGF21 chimera protein
includes at least 80% sequence identity to SEQ ID NO: 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 219, 221, 222, or 223. Thus,
the mutant FGF1/FGF21 chimeric protein can have at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 219, 221, 222, or 223. In some examples, the mutant
FGF1/FGF21 chimera protein includes or consists of SEQ ID NO: 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 219, 221, 222, or 223.
The disclosure encompasses variants of the disclosed mutant
FGF1/FGF21 chimera proteins, such as SEQ ID NO: 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 219, 221, 222, or 223 having 1-8
mutations, such as conservative amino acid substitutions.
[0320] In some examples, a mutant FGF1/FGF19 chimera protein
includes at least 80% sequence identity to SEQ ID NO: 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 220, or 224.
Thus, the mutant FGF1/FGF19 chimeric protein can have at least 90%,
at least 95%, at least 96%, at least 97%, at least 98% or at least
99% sequence identity to SEQ ID NO: 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 220, or 224. In some examples, the
mutant FGF1/FGF19 chimera protein includes or consists of any of
SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 220, or 224. The disclosure encompasses variants of the
disclosed mutant FGF1/FGF19 chimera proteins, such as SEQ ID NO:
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 220, or
224 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, such as
conservative amino acid substitutions.
[0321] In some examples, a mutant FGF1/.beta.-Klotho-binding
protein chimera includes at least 80% sequence identity to SEQ ID
NO: 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, or 187.
Thus, the mutant FGF1/.beta.-Klotho chimeric protein can have at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% sequence identity to SEQ ID NO: 1173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, or 187. In some examples,
the mutant FGF1/.beta.-Klotho chimera protein includes or consists
of SEQ ID NO: 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, or 187. The disclosure encompasses variants of the disclosed
mutant FGF1/.beta.-Klotho chimera proteins, such as SEQ ID NO: 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, or 187 having 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, such as conservative amino
acid substitutions.
[0322] In some examples, a mutant FGF1/FGF1Rc-binding protein
chimera includes at least 80% sequence identity to any of SEQ ID
NOS: 188-189. Thus, the mutant FGF1/FGF1Rc chimeric protein can
have at least 90%, at least 95%, at least 96%, at least 97%, at
least 98% or at least 99% sequence identity to SEQ ID NO: 188 or
189. In some examples, the mutant FGF1/FGF1Rc chimera protein
includes or consists of any of SEQ ID NOS: 188-189. The disclosure
encompasses variants of the disclosed mutant FGF1/FGF1Rc chimera
proteins, such as SEQ ID NO: 188 or 189 having 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 mutations, such as conservative amino acid
substitutions.
[0323] In one example the FGFR1c-binding/.beta.-Klotho-binding or
.beta.-Klotho-binding/FGFR1c-binding protein portion of a chimera
includes at least 80% sequence identity to SEQ ID NO: 168, 169, 170
or 171, such as at least 90%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% sequence identity to SEQ ID NO:
168, 169, 170 or 171.
[0324] In one example an FGFR1c-binding protein multimer includes
at least one monomer having 80% sequence identity to the
FGFR1c-binding portion of SEQ ID NO: 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,
166, 167, 168, 169, or 170, such as at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to the FGFR1c portion of SEQ ID NO: 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166, 167, 168, 169, or 170. In one example an
FGFR1c-binding protein dimer includes at least 80% sequence
identity SEQ ID NO: 190, such as at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO: 190.
[0325] Also provided are isolated nucleic acid molecules encoding
the disclosed mutated FGF1 proteins and chimeras, such as a nucleic
acid molecule encoding a protein having at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237, or 238 (but is not a native FGF1
sequence). One exemplary coding sequence is shown in SEQ ID NO: 18;
thus, the disclosure provides sequences having at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, or at least
99% sequence identity to any of SEQ ID NO: 18. Vectors and cells
that include such nucleic acid molecules are also provided. For
example, such nucleic acid molecules can be expressed in a host
cell, such as a bacterium or yeast cell (e.g., E. coli), thereby
permitting expression of the mutated FGF1 protein. The resulting
mutated FGF1 protein can be purified from the cell.
[0326] Methods of using the disclosed mutated FGF1 proteins and
chimeras (or nucleic acid molecules encoding such), as well as the
FGFR1c-binding protein multimers, are provided. As discussed
herein, the mutated mature FGF1 protein can include a deletion of
at least six contiguous N-terminal amino acids, at least one point
mutation, or combinations thereof. For example, such methods
include administering a therapeutically effective amount of a
disclosed mutated FGF1 protein or chimeric protein including the
mutant FGF1 mutant protein, or FGFR1c-binding protein multimer,
(such as at least 0.01, at least 0.1 mg/kg, or at least 0.5 mg/kg)
(or nucleic acid molecules encoding such) to reduce blood glucose
in a mammal, such as a decrease of at least 5%, at least 10%, at
least 25% or at least 50%, for example as compared to
administration of no mutant FGF1 mutant protein or FGFR1c-binding
protein multimer (e.g., administration of PBS).
[0327] In one example, the method is a method of reducing fed and
fasting blood glucose, improving insulin sensitivity and glucose
tolerance, reducing systemic chronic inflammation, ameliorating
hepatic steatosis in a mammal, reducing triglycerides, decreasing
insulin resistance, reducing hyperinsulinemia, increasing glucose
tolerance, reducing hyperglycemia, reducing food intake, or
combinations thereof. Such a method can include administering a
therapeutically effective amount of a disclosed mutated FGF1
protein or chimeric protein including the mutant FGF1 mutant
protein, or FGFR1c-binding protein multimer, (such as at least 0.5
mg/kg) (or nucleic acid molecules encoding such) to reduce fed and
fasting blood glucose, improve insulin sensitivity and glucose
tolerance, reduce systemic chronic inflammation, ameliorate hepatic
steatosis in a mammal, reduce food intake, or combinations
thereof.
[0328] In one example, the method is a method of treating a
metabolic disease (such as metabolic syndrome, diabetes, or
obesity) in a mammal. Such a method can include administering a
therapeutically effective amount of a disclosed mutated FGF1
protein or chimeric protein including the mutant FGF1 mutant
protein, or FGFR1c-binding protein multimer, (such as at least 0.5
mg/kg) (or nucleic acid molecules encoding such) to treat the
metabolic disease.
[0329] In some examples, the mammal, such as a human, cat or dog,
has diabetes. Methods of administration are routine, and can
include subcutaneous, intraperitoneal, intramuscular, or
intravenous injection.
[0330] In some examples, use of the FGF1 mutants or chimeric
proteins including a mutant FGF1 mutant protein, or FGFR1c-binding
protein multimer, disclosed herein does not lead to (or
significantly reduces, such as a reduction of at least 20%, at
least 50%, at least 75%, or at least 90%) the adverse side effects
observed with thiazolidinediones (TZDs) therapeutic insulin
sensitizers, including weight gain, increased liver steatosis and
bone fractures (e.g., reduced affects on bone mineral density,
trabecular bone architecture and cortical bone thickness).
[0331] Provided are methods of reducing fed and fasting blood
glucose, improving insulin sensitivity and glucose tolerance,
reducing systemic chronic inflammation, ameliorating hepatic
steatosis, reducing food intake, or combinations thereof, in a
mammal. Such methods can include administering a therapeutically
effective amount of a FGF1 mutant and/or FGFR1c-binding protein
multimer disclosed herein, including those that further include a
.beta.-Klotho-binding peptide and/or FGFR1c-binding peptide, to the
mammal, or a nucleic acid molecule encoding the FGF1 mutant or
multimer or a vector comprising the nucleic acid molecule, thereby
reducing fed and fasting blood glucose, improving insulin
sensitivity and glucose tolerance, reducing systemic chronic
inflammation, ameliorating hepatic steatosis, reduce one or more
non-HDL lipid levels, reduce food intake, or combinations thereof,
in a mammal. In some examples, the fed and fasting blood glucose is
reduced in the treated subject by at least 10%, at least 20%, at
least 30%, at least 50%, at least 75%, or at least 90% as compared
to an absence of administration of the FGF1 mutant and/or
FGFR1c-binding protein multimer. In some examples, insulin
sensitivity and glucose tolerance is increased in the treated
subject by at least 10%, at least 20%, at least 30%, at least 50%,
at least 75%, or at least 90% as compared to an absence of
administration of the FGF1 mutant and/or FGFR1c-binding protein
multimer. In some examples, systemic chronic inflammation is
reduced in the treated subject by at least 10%, at least 20%, at
least 30%, at least 50%, at least 75%, or at least 90% as compared
to an absence of administration of the FGF1 mutant and/or
FGFR1c-binding protein multimer. In some examples, hepatic
steatosis is reduced in the treated subject by at least 10%, at
least 20%, at least 30%, at least 50%, at least 75%, or at least
90% as compared to an absence of administration of the FGF1 mutant
and/or FGFR1c-binding protein multimer. In some examples, one or
more lipids (such as a non-HDL, for example IDL, LDL and/or VLDL)
are reduced in the treated subject by at least 10%, at least 20%,
at least 30%, at least 50%, at least 75%, or at least 90% as
compared to an absence of administration of the FGF1 mutant and/or
FGFR1c-binding multimer In some examples, triglyceride and or
cholesterol levels are reduced with the FGF1 mutant and/or
FGFR1c-binding protein multimer by at least 10%, at least 20%, at
least 30%, at least 50%, at least 75%, or at least 90% as compared
to an absence of administration of the FGF1 mutant and/or
FGFR1c-binding protein multimer. In some examples, the amount of
food intake is reduced in the treated subject by at least 10%, at
least 20%, at least 30%, at least 50%, at least 75%, or at least
90% as compared to an absence of administration of the FGF1 mutant
and/or FGFR1c-binding protein multimer (such as within 12 hours,
within 24 hours, or within 48 hours of the treatment, such as
within 12 to 24 hours, within 12 to 36 hours, or within 24 to 48
hours). In some examples, combinations of these reductions are
achieved.
Mutated FGF1 Proteins
[0332] The present disclosure provides mutated FGF1 proteins that
can include an N-terminal deletion, one or more point mutations
(such as amino acid substitutions, deletions, additions, or
combinations thereof), or combinations of N-terminal deletions and
point mutations. Such proteins and corresponding coding sequences
can be used in the methods provided herein. In some examples, the
disclosed FGF1 mutant proteins have reduced mitogenicity compared
to mature native FGF1 (e.g., SEQ ID NO: 5), such as a reduction of
at least 20%, at least 50%, at least 75% or at least 90%. For
example, mutated FGF1 can be mutated to decrease binding affinity
for heparin and/or heparan sulfate compared to a native FGF1
protein without the modification. Methods of measuring mitogenicity
are known in the art.
[0333] In some examples, the mutant FGF1 protein is a truncated
version of the mature protein (e.g., SEQ ID NO: 5), which can
include for example deletion of at least 5, at least 6, at least 9,
at least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, or at least 20 consecutive N-terminal amino acids. Thus,
in some examples, the mutant FGF1 protein is a truncated version of
the mature protein (e.g., SEQ ID NO: 5), such a deletion of the
N-terminal 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 amino acids shown in SEQ ID NO: 5. Examples of N-terminally
truncated FGF1 proteins are shown in SEQ ID NOS: 6, 7, 8, 9, 21,
24, 25, 26, 27, 32, 33, 34, 35, 36, 37, 38, 39, 44, 45, 46, 47, 48,
49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 71, 72, 74, 75,
76, 77, 79, 80, 81, 82, 194, 195, 197, 198, 202, 203, 205, 206,
214, 215, 216, 217, 221, 222, 225, 228, 232, and 238. In some
examples, the FGF1 mutant includes an N-terminal deletion, but
retains a methionine at the N-terminal position. In some examples,
such an N-terminally deleted FGF1 protein has reduced mitogenic
activity as compared to wild-type mature FGF1 protein.
[0334] In some examples, one or more of the deleted N-terminal
amino acids are replaced with corresponding amino acids from FGF21
(e.g., see SEQ ID NO: 20), such as at least 1, at least 2, at least
3, at least 4, at least 5, at least 10, at least 15, or at least 20
amino acids from FGF21, such as 1-5, 1-4, 2-4, 4-6, 4-9, 3-10, 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 corresponding amino acids from FGF21.
An example of an FGF1 mutated protein with an N-terminal deletion
having four corresponding N-terminal amino acids from FGF21 is
shown in SEQ ID NO: 21. The N-terminal residues of FGF1 include an
FGFR4 binding site, and FGFR4 signaling is associated with
mitogenic activity. In contrast, FGF21 has low affinity for FGFR4.
Thus, replacing the FGFR4 binding residues of FGF1 with those from
FGF21 (or from another FGF having low affinity for FGFR4, including
FGF3, FGF5, FGF7, FGF9 and FGF10) can be used to reduce
mitogenicity of the resulting FGF1 mutant protein.
[0335] Thus, in some examples, the mutant FGF1 protein includes at
least 120 consecutive amino acids from amino acids 5-141 or 5-155
of FGF1 (e.g., of SEQ ID NO: 2 or 4), (which in some examples can
include further deletion of N-terminal amino acids 1-20 and/or
point mutations, such as substitutions, deletions, or additions).
In some examples, the mutant FGF1 protein includes at least 120
consecutive amino acids from amino acids 1-140 of FGF1 (e.g., of
SEQ ID NO: 5), (which in some examples can include further deletion
of N-terminal amino acids 1-20 and/or point mutations, such as
substitutions, deletions, or additions). Thus, in some examples,
the mutant FGF1 protein includes at least 120 consecutive amino
acids from amino acids 5-141 of FGF1, such as at least 120, at
least 121, at least 122, at least 123, at least 124, at least 125,
at least 126, at least 127, at least 128, at least 129, at least
130, at least 131, at least 132, at least 133, at least 134, at
least 135, at least 136, at least 137, at least 138, at least 139,
or at least 140 consecutive amino acids from amino acids 5-141 of
SEQ ID NO: 2 or 4 (such as 120-130, 120-135, 130-135, 130-140, or
120-140 consecutive amino acids from amino acids 5-141 of SEQ ID
NO: 2 or 4). In some examples, the mutant FGF1 protein includes at
least 120 or at least 130 consecutive amino acids from amino acids
5-141 of FGF1, such as at least 120 consecutive amino acids from
amino acids 5-141 of SEQ ID NO: 2 or 4 or at least 120 consecutive
amino acids from SEQ ID NO: 5. Thus, in some examples, the mutant
FGF1 protein includes at least 120, at least 121, at least 122, at
least 123, at least 124, at least 125, at least 126, at least 127,
at least 128, at least 129, at least 130, at least 131, at least
132, at least 133, at least 134, at least 135, at least 136, at
least 137, at least 138, at least 139, or at least 140 consecutive
amino acids from SEQ ID NO: 5 (such as 120-130, 120-135, or 120-140
consecutive amino acids from SEQ ID NO: 5). Examples of least 120
consecutive amino acids from amino acids 5 to 141 of FGF1 that can
be used to generate a mutant FGF1 protein includes but are not
limited to amino acids 4 to 140 of SEQ ID NO: 5 and the protein
sequence shown in any of SEQ ID NOs: 6, 7, 8, and 9.
[0336] In some examples, the mutant FGF1 protein is a mutated
version of the mature protein (e.g., SEQ ID NO: 5), or a N-terminal
truncation of the mature protein (e.g., SEQ ID NOS: 7, 8, 9), such
as one containing at least 1, at least 4, at least 5, at least 6,
at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, or at least 20 amino acid
substitutions, such as 1-20, 1-10, 4-8, 5-12, 5-10, 5-25, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid
substitutions. For example, point mutations can be introduced into
an FGF1 sequence to decrease mitogenicity, increase stability,
decrease binding affinity for heparin and/or heparan sulfate
(compared to the portion of a native FGF1 protein without the
modification), or combinations thereof. Specific exemplary point
mutations that can be used are shown above in Table 1, and
exemplary combinations are provided in FIGS. 1, 3A-3D, 4A-4B,
5A-5B, 6A-6B, 20, and 27-30.
[0337] In some examples, the mutant FGF1 protein includes mutations
(such as a substitution or deletion) at one or more of the
following positions K9, K10, K12, L14, Y15, C16, H21, R35, Q40,
L44, L46, S47, E49, Y55, M67, L73, C83, L86, E87, H93, Y94, N95,
H102, A103, E104, K105, N106, F108, V109, L111, K112, K113, C117,
K118, R119, G120, P121, R122, F132, L133, P134, L135, such as one
or more of K9, K10, K12, K112, K113, such as 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41
or all 42 of these positions. In some examples the mutant FGF1
protein has as one or more of K9T, K10T, K12V, L14A, Y15F, Y15A,
Y15V, C16V, C16A, C16T, C16S, H21Y, R35E, R35V, Q40P, L44F, L46V,
S47I, E49Q, E49A, Y55F, Y55S, Y55A, M67I, L73V, C83T, C83S, C83A
C83V, E87V, E87A, E87S, E87T, H93G, H93A, Y94V, Y94F, Y94A, N95V,
N95A, N95S, N95T, H102Y, A103G, .DELTA.104-106, F108Y, V109L,
L111I, K112D, K112E, K112Q, K113Q, K113E, K113D, C117V, C117P,
C117T, C117S, C117A, K118N, K118E, K118V, R119G, R119V, R119E,
.DELTA.120-122, F132W, L133A, L133S, P134V, L135A, L135S, (wherein
the numbering refers to SEQ ID NO: 5), such as 1 to 5, 1 to 10, 2
to 5, 2 to 10, 2 to 20, 5 to 10, 5 to 40, or 5 to 20 of these
mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 of these mutations.
[0338] In some examples, the mutant FGF1 protein includes one or
more (such as 2, 3, 4, 5 or 6) of K12V, R35E, R35V, L46V, E87V,
N95V, C117V/A, K118N, K118E/V, and P134V (wherein the numbering
refers to SEQ ID NO: 5). In some examples, the point mutation
includes replacing amino acid sequence ILFLPLPV (amino acids
145-152 of SEQ ID NO: 2 and 4) to AAALPLPV (SEQ ID NO: 14),
ILALPLPV (SEQ ID NO: 15), ILFAPLPV (SEQ ID NO: 16), or ILFLPAPA
(SEQ ID NO: 17). In some examples, such an FGF1 protein with one or
more point mutations has reduced mitogenic activity as compared to
wild-type mature FGF1 protein. In some examples, the mutant FGF1
protein includes R35E, (wherein the numbering refers to SEQ ID NO:
5). Examples of FGF1 mutant proteins containing point mutations
include but are not limited to the protein sequence shown in SEQ ID
NOS: 10, 11, 12, 13, 22, 23, 28, 29, 30, 31, 40, 41, 42, 43, 42,
53, 54, 55, 56, 67, 68, 69, 70, 73, 78, 83, 84, 113, 114, 115, 116,
117, 118, 119, 120, 191, 192, 193, 196, 199, 200, 201, 204, 207,
208, 209, 210, 211, 212, 213, 218, 226, 227, 229, 230, 231, 232,
233, 234, 235, 236, and 237. In some examples, mutations in FGF1
increase the thermostability of mature or truncated native FGF1.
For example, mutations can be made at one or more of the following
positions. Exemplary mutations that can be used to increase the
thermostability of mutated FGF1 include but are not limited to one
or more of: K12, C117, P134, L44, C83, F132, M67, L73, V109, L111,
A103, R119, .DELTA.104-106, and .DELTA.120-122, Q40, H93, S47,
wherein the numbering refers to SEQ ID NO: 5 (e.g., see Xia et al.,
PLoS One. 7:e48210, 2012). In some examples, thermostability of
FGF1 is increased by using one or more of the following mutations:
Q40P and S47I or Q40P, S47I, and H93G (or any other combination of
these mutations).
[0339] In some examples, the FGF1 mutant protein is part of a
chimeric protein. For example, any mutant FGF1 protein provided
herein can be joined directly or indirectly to the end of a
.beta.-Klotho-binding protein, an FGFR1c binding protein, both a
.beta.-Klotho-binding protein and an FGFR1c binding protein, FGF19,
or FGF21, such as a C-terminal region of FGF 19 or FGF21. For
example, at least 10, at least 20, at least 30, at least 40, at
least 41, at least 42, at least 43, at least 44, at least 45, at
least 46, at least 47, at least 48, at least 49, at least 50 or at
least 60 C-terminal amino acids of FGF19 or FGF21 (such as the
C-terminal 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35,
30, 25, 20, 15 or 10 amino acids) can be part of the chimera.
Examples of C-terminal fragments of FGF21 and FGF19 that can be
used are shown in SEQ ID NOS: 86 and 100, respectively. Examples of
.beta.-Klotho-binding proteins that can be used are shown in SEQ ID
NOS: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 and
146. Examples of FGFR1c-binding proteins that can be used are shown
in SEQ ID NOS: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. Examples
of .beta.-Klotho-binding/FGFR1c-binding protein chimeras that can
be directly or indirectly attached to a mutant FGF1 protein are
shown in SEQ ID NOS: 168, 169, 170, and 171.
[0340] In some examples, the mutant FGF1 protein includes both an
N-terminal truncation and point mutations. Specific exemplary FGF1
mutant proteins are shown in SEQ ID NOS: 6-13, 21-84, 113-120,
191-218 and 225-238. In some examples, the FGF1 mutant protein
includes at least 80% sequence identity to any of SEQ ID NOS: 6-13,
21-84, 113-120, 191-218 and 225-238. Thus, the FGF1 mutant protein
can have at least 90%, at least 95%, at least 96%, at least 97%, at
least 98% or at least 99% sequence identity to any of SEQ ID NOS:
6-13, 21-84, 113-120, 191-218 and 225-238. In some examples, the
FGF1 mutant protein includes or consists of any of SEQ ID NOS:
6-13, 21-84, 113-120, 191-218 and 225-238. The disclosure
encompasses variants of the disclosed FGF1 mutant proteins, such as
any of SEQ ID NOS: 6-13, 21-84, and 113-120, 191-218 and 225-238
having 1 to 20, 1 to 15, 1 to 10, 1 to 8, 2 to 10, 1 to 5, 1 to 6,
2 to 12, 3 to 12, 5 to 12, or 5 to 10 mutations, such as
conservative amino acid substitutions. Such mutant FGF1 proteins
can be used to generate an FGF1 mutant chimera.
[0341] In some examples, the mutant FGF1 protein has at its
N-terminus a methionine. In some examples, the mutant FGF1 protein
is at least 120 amino acids in length, such as at least 125, at
least 130, at least 135, at least 140, at least 145, at least 150,
at least 155, at least 160, or at least 175 amino acids in length,
such as 120-160, 125-160, 130-160, 150-160, 130-200, 130-180,
130-170, or 120-160 amino acids in length.
[0342] Exemplary N-terminally truncated FGF1 sequences and FGF1
point mutations that can be used to generate an FGF1 mutant protein
are shown in Tables 1 and 2 (as well as those provided in any of
SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178,
179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194,
195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,
208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,
234, 235, 236, 237 and 238). One skilled in the art will appreciate
that any N-terminal truncation in Table 2 (as well as those
provided in any of SEQ ID NOS: 6, 7, 8, 9, 21, 24, 25, 26, 27, 32,
33, 34, 35, 36, 37, 38, 39, 44, 45, 46, 47, 48, 49, 50, 51, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 71, 72, 74, 75, 76, 77, 79, 80, 81,
82, 194, 195, 197, 198, 202, 203, 205, 206, 214, 215, 216, 217,
221, 222, 225, 228, 232, and 238) can be combined with any FGF1
point mutation in Table 1 or Table 2, to generate an FGF1 mutant
protein, and that such an FGF1 mutant protein can be used directly
or be used as part of a mutant FGF1/.beta.-Klotho-binding protein
chimera, mutant FGF1/FGFR1c-binding protein chimera, mutant
FGF1/.beta.-Klotho-binding protein/FGFR1c-binding protein chimera,
mutant FGF1/FGF21 or mutant FGF1/FGF19 chimera. In addition,
mutations can be made to the sequences shown in the Table, such as
one or more of the mutations discussed herein (such as 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid substitutions, such as conservative amino acid substitutions,
deletions, or additions).
TABLE-US-00002 TABLE 2 Exemplary mutations that can be used to
generate an FGF1 mutant protein FGF1 Point Mutations FGF1 Fragments
FNLPPGNYKK PVLLYCSNGG PPGNYK KPKLLYCSNG HFLRILPDGT VDGTRDRSDQ
GHFLRILPDG TVDGTRDRSD HIQLQLSAES VGEVYIKSTE QHIQLQLSAE SVGEVYIKST
TGQYLAMDTD GLLYGSQTPN ETGQYLAMDT DGLLYGSQTP EECLFLERLE ENHYVTYISK
NEECLFLERL EENHYNTYIS KHAEKNWFVG LKKNGSCKRG KKHAEKNWFVGLKKNGSCKR
PRTHYGQKAI LFLPLPVSSD GPRTHYGQKA ILFLPLPVSSD (SEQ ID NO: 10) (SEQ
ID NO: 6) FNLPPGNYKK PVLLYCSNGG KPKLLYCSNGG HFLRILPDGT HFLRILPDGT
VDGTRDRSDQ VDGTRDRSDQ HIQLQLSAES HIQLQVSAES VGEVYIKSTE VGEVYIKSTE
TGQYLAMDTD TGQYLAMDTDGLLYGSQTPN GLLYGSQTPN EECLFLERLE EECLFLVRLE
ENHYVTYISK ENHYNTYISK KHAEKNWFVG KHAEKNWFVG LKKNGSCKRG LKKNGSCKRG
PRTHYGQKAI PRTHYGQKAI LFLVLPVSSD LFLPLPVSSD (SEQ ID NO: 11) (SEQ ID
NO: 7) NYKK PKLLYCSNGG LYCSNGG HFLRILPDGT HFLRILPDGT VDGTRDRSDQ
HIQLQLSAES VDGTRDRSDQ HIQLQLSAES VGEVYIKSTE TGQYLAMDTD VGEVYIKSTE
TGQYLAMDTD GLLYGSQTPN EECLFLERLE GLLYGSQTPN EECLFLERLE ENHYNTYISK
KHAEKNWFVG ENHYNTYISK KHAEKNWFVG LKKNGSCKRG PRTHYGQKAI LKKNGSCNRG
PRTHYGQKAI LFLPLPVSSD LFLPLPVSSD (SEQ ID NO: 8) (SEQ ID NO: 12)
NYKK PKLLYCSNGG KLLYCSNGG HFLRILPDGT HFLRILPDGT VDGTRDRSDQ
HIQLQLSAES VDGTRDRSDQ HIQLQLSAES VGEVYIKSTE TGQYLAMDTD VGEVYIKSTE
TGQYLAMDTD GLLYGSQTPN EECLFLERLE GLLYGSQTPN EECLFLERLE ENHYNTYISK
KHAEKNWFVG ENHYNTYISK KHAEKNWFVG LKKNGSCKRG PRTHYGQKAI LKKNGSCERG
PRTHYGQKAI LFLPLPVSSD LFLPLPVSSD (SEQ ID NO: 9) (SEQ ID NO: 13)
GGQVKPKLLYCSNG GHFLRILPDG TVDGTRDRSD QHIQLQLSAE SVGEVYIKST
ETGQYLAMDT DGLLYGSQTP NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR
GPRTHYGQKA ILFLPLPVSSD (SEQ ID NO: 21)
[0343] Exemplary mutant FGF1 proteins are provided in SEQ ID NOS:
6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 113, 114, 115, 116, 117, 118, 119, 120, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 and 238,
mutant FGF1/FGF21 chimeras in SEQ ID NOS: 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 219, 221, 222 and 223, mutant FGF1/FGF19
chimeras in SEQ ID NOS: 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 220 and 224, mutant FGF1/.beta.-Klotho-binding
protein chimeras in SEQ ID NOS: 173, 174, 175, 177, 178, 179, 181,
182, 183, 185, 186, and 187, and mutant FGF1/FGFR1c-binding protein
chimeras in SEQ ID NOS: 188 and 189. One skilled in the art will
recognize that minor variations can be made to these sequences,
without adversely affecting the function of the protein (such as
its ability to reduce blood glucose). For example, variants of the
mutant FGF1 proteins include those having at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238 (but are not a native
FGF1 sequence, e.g., SEQ ID NO: 5), but retain the ability to treat
a metabolic disease, or decrease blood glucose in a mammal (such as
a mammal with type II diabetes). Thus, variants of SEQ ID NO: 6, 7,
8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182,
183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237 or 238 retaining at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% sequence identity are of
use in the disclosed methods.
FGF1
[0344] Mature forms of FGF1 (such as SEQ ID NO: 2 or 4) can be
mutated to control (e.g., reduce) the mitogenicity of the protein
(for example by mutating the nuclear localization sequence (NLS) or
the heparan sulfate binding region or both) and to provide
glucose-lowering ability to the protein. Mutations can also be
introduced into a wild-type mature FGF1 sequence that affects the
stability and receptor binding selectivity of the protein.
[0345] Exemplary full-length FGF1 proteins are shown in SEQ ID NOS:
2 (human) and 4 (mouse). In some examples, FGF1 includes SEQ ID NO:
2 or 4, but without the N-terminal methionine (thus resulting in a
154 aa FGF1 protein). In addition, the mature/active form of FGF1
is one where a portion of the N-terminus is removed, such as the
N-terminal 15, 16, 20, or 21 amino acids from SEQ ID NO: 2 or 4.
Thus, in some examples the active form of FGF1 comprises or
consists of amino acids 16-155 or 22-155 of SEQ ID NO: 2 or 4
(e.g., see SEQ ID NO: 5). In some examples, the mature form of FGF1
that can be mutated includes SEQ ID NO: 5 with a methionine added
to the N-terminus (wherein such a sequence can be mutated as
discussed herein). Thus, the mutated mature FGF1 protein can
include an N-terminal truncation.
[0346] Mutations can be introduced into a wild-type FGF1 (such as
SEQ ID NO: 2, 4, or 5). In some examples, multiple types of
mutations disclosed herein are made to the FGF1 protein. Although
mutations below are noted by a particular amino acid for example in
SEQ ID NO: 2, 4 or 5, one skilled in the art will appreciate that
the corresponding amino acid can be mutated in any FGF1 sequence.
For example, Q40 of SEQ ID NO: 5 corresponds to Q55 of SEQ ID NO: 2
and 4.
[0347] In one example, mutations are made to the N-terminal region
of FGF1 (such as SEQ ID NO: 2, 4 or 5), such as deletion of the
first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30 amino acids of SEQ ID NO: 2 or 4 (such as deletion of at
least the first 14 amino acids of SEQ ID NO: 2 or 4, such as
deletion of at least the first 15, at least 16, at least 20, at
least 25, or at least 29 amino acids of SEQ ID NO: 2 or 4),
deletion of the first 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 amino acids of SEQ ID NO: 5 (e.g., see SEQ ID NOS: 7, 8 and 9
and FIG. 1).
[0348] Mutations can be made to FGF1 (such as SEQ ID NO: 2, 4 or 5)
to reduce its mitogenic activity. In some examples, such mutations
reduce mitogenic activity by at least 50%, at least 60%, at least
70%, at least 75%, at least 80%, at least 90%, at least 92%, at
least 95%, at least 98%, at least 99%, or even complete elimination
of detectable mitogenic activity, as compared to a native FGF1
protein without the mutation. Methods of measuring mitogenic
activity are known in the art, such as thymidine incorporation into
DNA in serum-starved cells (e.g., NIH 3T3 cells) stimulated with
the mutated FGF1, methylthiazoletetrazolium (MTT) assay (for
example by stimulating serum-starved cells with mutated FGF1 for 24
hr then measuring viable cells), cell number quantification or BrdU
incorporation. In some examples, the assay provided by Fu et al.,
World J. Gastroenterol. 10:3590-6, 2004; Klingenberg et al., J.
Biol. Chem. 274:18081-6, 1999; Shen et al., Protein Expr Purif
81:119-25, 2011, or Zou et al., Chin. Med. J. 121:424-429, 2008 is
used to measure mitogenic activity. Examples of such mutations
include, but are not limited to K12V, R35E, L46V, E87V, N95V,
K12V/N95V (e.g., see SEQ ID NO: 10, which can also include a
methionine on its N-terminus), and Lys12Val/Pro134Val,
Lys12Val/Leu46Val/Glu87Val/Asn95Val/Pro134Val (e.g., see SEQ ID NO:
11, which can also include a methionine on its N-terminus) (wherein
the numbering refers to the sequence shown SEQ ID NO: 5). In some
examples, a portion of contiguous N-terminal residues are removed,
such as amino acids 1-9 of SEQ ID NO: 5, to produce a non-mitogenic
form of FGF1. An example is shown in SEQ ID NO: 9.
[0349] Mutations that reduce the heparan binding affinity (such as
a reduction of at least 10%, at least 20%, at least 50%, or at
least 75%, e.g., as compared to a native FGF1 protein without the
mutation), can also be used to reduce mitogenic activity, for
example by substituting heparan binding residues from a paracrine
FGFs into FGF1. In some examples, mitogenicity is reduced or
eliminated by deleting the N-terminal region of FGF1 (such as the
region that binds FGF4) and replacing some or all of the amino
acids deleted with corresponding residues from FGF21.
[0350] Mutations can also be introduced into one or both nuclear
localization sites (NLS1, amino acids 24-27 of SEQ ID NO: 2 and
NLS2, amino acids 115-128 of SEQ ID NO: 4) of FGF1, for example to
reduce mitogenicity, as compared to a native FGF1 protein without
the mutation. Examples of NLS mutations that can be made to FGF1
include but are not limited to: deleting or mutating all or a part
of NLS1 (such as deleting or mutating the lysines), deleting or
mutating the lysines in NLS2 such as .sup.115KK . . . .sup.127KK .
. . , or combinations thereof (wherein the numbering refers to the
sequence shown SEQ ID NO: 2). For example, one or more of 24K, 25K,
27K, 115K, 127K or 128K (wherein the numbering refers to the
sequence shown SEQ ID NO: 2) or can be mutated (for example changed
to an alanine or deleted). Particular examples of such mutations
that can be made to the heparan binding site in the NLS2 (KKN . . .
KR) domain are shown in SEQ ID NOS: 12 and 13 (K118N or K118E,
respectively, wherein numbering refers to SEQ ID NO: 5).
[0351] Mutations can be introduced into the phosphorylation site of
FGF1, for example to create a constitutively active or inactive
mutant to affect nuclear signaling.
[0352] In some examples, mutations are introduced into the FGF1
nuclear export sequence, for example to decrease the amount of FGF1
in the nucleus and reduce its mitogenicity as measured by thymidine
incorporation assays in cultured cells (e.g., see Nilsen et al., J.
Biol. Chem. 282(36):26245-56, 2007). Mutations to the nuclear
export sequence decrease FGF1-induced proliferation (e.g., see
Nilsen et al., J. Biol. Chem. 282(36):26245-56, 2007). Methods of
measuring FGF1 degradation are known in the art, such as measuring
[.sup.35S]Methionine-labeled FGF1 or immunoblotting for
steady-state levels of FGF1 in the presence or absence of
proteasome inhibitors. In one example, the assay provided by Nilsen
et al., J. Biol. Chem. 282(36):26245-56, 2007 or Zakrzewska et al.,
J. Biol. Chem. 284:25388-403, 2009 is used to measure FGF1
degradation.
[0353] The FGF1 nuclear export sequence includes amino acids
145-152 of SEQ ID NO: 2 and 4 or amino acids 130-137 of SEQ ID NO:
5. Examples of FGF1 nuclear export sequence mutations that can be
made to include but are not limited to changing the sequence
ILFLPLPV (amino acids 145-152 of SEQ ID NO: 2 and 4) to AAALPLPV
(SEQ ID NO: 14), ILALPLPV (SEQ ID NO: 15), ILFAPLPV (SEQ ID NO:
16), or ILFLPAPA (SEQ ID NO: 17).
[0354] In one example, mutations are introduced to improve
stability of FGF1. In some examples, the sequence NYKKPKL (amino
acids 22-28 of SEQ ID NO: 2) is not altered, and in some examples
ensures for structural integrity of FGF1 and increases interaction
with the FGF1 receptor. Methods of measuring FGF1 stability are
known in the art, such as measuring denaturation of FGF1 or mutants
by fluorescence and circular dichroism in the absence and presence
of a 5-fold molar excess of heparin in the presence of 1.5 M urea
or isothermal equilibrium denaturation by guanidine hydrochloride.
In one example, the assay provided by Dubey et al., J. Mol. Biol.
371:256-268, 2007 is used to measure FGF1 stability. Examples of
mutations that can be used to increase stability of the protein
include, but are not limited to, one or more of Q40P, S47I and H93G
(wherein the numbering refers to the sequence shown SEQ ID NO:
5).
[0355] In one example, mutations are introduced to improve the
thermostability of FGF1, such as an increase of at least 10%, at
least 20%, at least 50%, or at least 75%, as compared to a native
FGF1 protein without the mutation (e.g., see Xia et al., PLoS One.
2012; 7(11):e48210 and Zakrzewska, J Biol Chem. 284:25388-25403,
2009). In one example, mutations are introduced to increase
protease resistance of FGF1 (e.g., see Kobielak et al., Protein
Pept Lett. 21(5):434-43, 2014). Other mutations that can be made to
FGF1 include those mutations provided in Lin et al., J Biol Chem.
271(10):5305-8, 1996).
[0356] In some examples, the mutant FGF1 protein or chimera is
PEGylated at one or more positions, such as at N95 (for example see
methods of Niu et al., J. Chromatog. 1327:66-72, 2014, herein
incorporated by reference). Pegylation consists of covalently
linking a polyethylene glycol group to surface residues and/or the
N-terminal amino group. N95 is known to be involved in receptor
binding, thus is on the surface of the folded protein. As mutations
to surface exposed residues could potentially generate immunogenic
sequences, pegylation is an alternative method to abrogate a
specific interaction. Pegylation is an option for any surface
exposed site implicated in the receptor binding and/or proteolytic
degradation. Pegylation can "cover" functional amino acids, e.g.
N95, as well as increase serum stability.
[0357] In some examples, the mutant FGF1 protein or chimera
includes an immunoglobin FC domain (for example see Czajkowsky et
al., EMBO Mol. Med. 4:1015-28, 2012, herein incorporated by
reference). The conserved FC fragment of an antibody can be
incorporated either n-terminal or c-terminal of the mutant FGF1
protein or chimera, and can enhance stability of the protein and
therefore serum half-life. The FC domain can also be used as a
means to purify the proteins on protein A or Protein G sepharose
beads. This makes the FGF1 mutants having heparin binding mutations
easier to purify.
Variant Sequences
[0358] Variant FGF1 proteins, including variants of the sequences
shown in Tables 1 and 2, and variants of SEQ ID NOS: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, and
238, can contain one or more mutations, such as a single insertion,
a single deletion, a single substitution. In some examples, the
mutant FGF1 protein includes 1-20 insertions, 1-20 deletions, 1-20
substitutions, or any combination thereof (e.g., single insertion
together with 1-19 substitutions). In some examples, the disclosure
provides a variant of any disclosed mutant FGF1 protein having 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 amino acid changes. In some examples, SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238,
includes 1-8 insertions, 1-15 deletions, 1-10 substitutions, or any
combination thereof (e.g., 1-15, 1-4, or 1-5 amino acid deletions
together with 1-10, 1-5 or 1-7 amino acid substitutions). In some
examples, the disclosure provides a variant of any of SEQ ID NOS:
6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,
182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,
210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,
236, 237 and 238, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 amino acid changes. In one example, such variant peptides are
produced by manipulating the nucleotide sequence encoding a peptide
using standard procedures such as site-directed mutagenesis or PCR.
Such variants can also be chemically synthesized. Similar changes
can be made to the FGFR1c dimer of SEQ ID NO: 190 (1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid
changes).
[0359] One type of modification or mutation includes the
substitution of amino acids for amino acid residues having a
similar biochemical property, that is, a conservative substitution
(such as 1-4, 1-8, 1-10, or 1-20 conservative substitutions).
Typically, conservative substitutions have little to no impact on
the activity of a resulting peptide. For example, a conservative
substitution is an amino acid substitution in SEQ ID NO: 6, 7, 8,
9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183,
185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198,
199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,
212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,
225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or
238, that does not substantially affect the ability of the peptide
to decrease blood glucose in a mammal. An alanine scan can be used
to identify which amino acid residues in a mutant FGF1 protein,
such as SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177,
178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237 or 238, can tolerate an amino acid
substitution. In one example, the blood glucose lowering activity
of FGF1, or any of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238, is not altered by
more than 25%, for example not more than 20%, for example not more
than 10%, when an alanine, or other conservative amino acid, is
substituted for 1-4, 1-8, 1-10, or 1-20 native amino acids.
Examples of amino acids which may be substituted for an original
amino acid in a protein and which are regarded as conservative
substitutions include: Ser for Ala; Lys for Arg; Gln or His for
Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for
Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu;
Arg or Gln for Lys; Leu or Be for Met; Met, Leu or Tyr for Phe; Thr
for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or
Leu for Val.
[0360] More substantial changes can be made by using substitutions
that are less conservative, e.g., selecting residues that differ
more significantly in their effect on maintaining: (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation; (b)
the charge or hydrophobicity of the polypeptide at the target site;
or (c) the bulk of the side chain. The substitutions that in
general are expected to produce the greatest changes in polypeptide
function are those in which: (a) a hydrophilic residue, e.g.,
serine or threonine, is substituted for (or by) a hydrophobic
residue, e.g., leucine, isoleucine, phenylalanine, valine or
alanine; (b) a cysteine or proline is substituted for (or by) any
other residue; (c) a residue having an electropositive side chain,
e.g., lysine, arginine, or histidine, is substituted for (or by) an
electronegative residue, e.g., glutamic acid or aspartic acid; or
(d) a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine.
The effects of these amino acid substitutions (or other deletions
or additions) can be assessed by analyzing the function of the
mutant FGF1 protein, such as any of SEQ ID NOS: 6, 7, 8, 9, 10, 11,
12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186,
187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,
227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238, by
analyzing the ability of the variant protein to decrease blood
glucose in a mammal.
Generation of Proteins
[0361] Isolation and purification of recombinantly expressed
mutated FGF1 proteins can be carried out by conventional means,
such as preparative chromatography and immunological separations.
Once expressed, mutated FGF1 proteins can be purified according to
standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, and the
like (see, generally, R. Scopes, Protein Purification,
Springer-Verlag, N.Y., 1982). Substantially pure compositions of at
least about 90 to 95% homogeneity are disclosed herein, and 98 to
99% or more homogeneity can be used for pharmaceutical
purposes.
[0362] In addition to recombinant methods, mutated FGF1 proteins
disclosed herein can also be constructed in whole or in part using
standard peptide synthesis. In one example, mutated FGF1 proteins
are synthesized by condensation of the amino and carboxyl termini
of shorter fragments. Methods of forming peptide bonds by
activation of a carboxyl terminal end (such as by the use of the
coupling reagent N, N'-dicylohexylcarbodimide) are well known in
the art.
Mutated FGF1 and FGFR1c-Binding Protein Multimer Nucleic Acid
Molecules and Vectors
[0363] Nucleic acid molecules encoding a mutated FGF1 protein are
encompassed by this disclosure. Based on the genetic code, nucleic
acid sequences coding for any mutated FGF1 sequence, such as those
generated using the sequences shown in Tables 1 and 2, can be
routinely generated. Similarly, mutant FGF1/.beta.-Klotho-binding,
mutant FGF1/FGFR1c-binding, mutant
FGF1/.beta.-Klotho-binding/FGFR1c-binding, mutant FGF1/FGF21 or
mutant FGF1/FGF19 chimeras can be generated using routine methods
based on the amino acid sequences provided herein. In some
examples, such a sequence is optimized for expression in a host
cell, such as a host cell used to express the mutant FGF1 protein.
Also provided are nucleic acid molecules encoding an FGFR1c-binding
protein multimer, such as those encoding multimers of any of SEQ ID
NOS: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, or 190, as well as
cells and vectors including such nucleic acids.
[0364] In one example, a nucleic acid sequence codes for a mutant
FGF1 protein (or chimera including such protein) having at least
60%, at least 70%, at least 75%, at least 80%, at least 90%, at
least 92%, at least 95%, at least 96%, at least 97%, at least 99%
or at least 99% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11,
12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186,
187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,
227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238, can
readily be produced by one of skill in the art, using the amino
acid sequences provided herein, and the genetic code. In addition,
one of skill can readily construct a variety of clones containing
functionally equivalent nucleic acids, such as nucleic acids which
differ in sequence but which encode the same mutant FGF1 protein
sequence. In one example, a mutant FGF1 nucleic acid sequence has
at least 70%, at least 80%, at least 85%, at least 90%, at least
92%, at least 95%, at least 98%, or at least 99% sequence identity
to SEQ ID NO: 18.
[0365] In one example, a nucleic acid sequence codes for a
FGFR1c-binding protein multimer made using peptide sequences having
at least 60%, at least 70%, at least 75%, at least 80%, at least
90%, at least 92%, at least 95%, at least 96%, at least 97%, at
least 99% or at least 99% sequence identity to SEQ ID NO: 147, 148,
149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164, 165, 166, 167, or 190.
[0366] Nucleic acid molecules include DNA, cDNA and RNA sequences
which encode a mutated FGF1 peptide. Silent mutations in the coding
sequence result from the degeneracy (i.e., redundancy) of the
genetic code, whereby more than one codon can encode the same amino
acid residue. Thus, for example, leucine can be encoded by CTT,
CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA,
TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic
acid can be encoded by GAT or GAC; cysteine can be encoded by TGT
or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine
can be encoded by CAA or CAG; tyrosine can be encoded by TAT or
TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables
showing the standard genetic code can be found in various sources
(see, for example, Stryer, 1988, Biochemistry, 3.sup.rd Edition,
W.H. 5 Freeman and Co., NY).
[0367] Codon preferences and codon usage tables for a particular
species can be used to engineer isolated nucleic acid molecules
encoding a FGFR1c-binding protein multimer or a mutated FGF1
protein (such as one encoding a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) that take advantage of the codon usage
preferences of that particular species. For example, the
FGFR1c-binding protein multimers and mutated FGF1 proteins
disclosed herein can be designed to have codons that are
preferentially used by a particular organism of interest.
[0368] A nucleic acid encoding a FGFR1c-binding protein multimer or
a mutant FGF1 protein (such as one encoding a protein generated
using the sequences shown in Tables 1 and 2, the sequences in any
of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding a
protein having at least 90%, at least 95%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238) can be cloned or amplified by
in vitro methods, such as the polymerase chain reaction (PCR), the
ligase chain reaction (LCR), the transcription-based amplification
system (TAS), the self-sustained sequence replication system (3SR)
and the Q.beta. replicase amplification system (QB). A wide variety
of cloning and in vitro amplification methodologies are well known
to persons skilled in the art. In addition, nucleic acids encoding
sequences encoding a FGFR1c-binding protein multimer or a mutant
FGF1 protein (such as one encoding a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) can be prepared by cloning techniques.
Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through cloning
are found in Sambrook et al. (ed.), Molecular Cloning: A Laboratory
Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold
Spring, Harbor, N.Y., 1989, and Ausubel et al., (1987) in "Current
Protocols in Molecular Biology," John Wiley and Sons, New York,
N.Y.
[0369] Nucleic acid sequences encoding a FGFR1c-binding protein
multimer or a mutated FGF1 protein (such as one encoding a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) can be prepared by
any suitable method including, for example, cloning of appropriate
sequences or by direct chemical synthesis by methods such as the
phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99,
1979; the phosphodiester method of Brown et al., Meth. Enzymol.
68:109-151, 1979; the diethylphosphoramidite method of Beaucage et
al., Tetra. Lett. 22:1859-1862, 1981; the solid phase
phosphoramidite triester method described by Beaucage &
Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using
an automated synthesizer as described in, for example,
Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984;
and, the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis produces a single stranded oligonucleotide. This can be
converted into double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill would recognize
that while chemical synthesis of DNA is generally limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
[0370] In one example, a mutant FGF1 protein (such as a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) is prepared by
inserting the cDNA which encodes the mutant FGF1 protein into a
vector. The insertion can be made so that the mutant FGF1 protein
is read in frame so that the mutant FGF1 protein is produced.
Similar methods can be used for a FGFR1c-binding protein
multimer.
[0371] The mutated FGF1 protein nucleic acid coding sequence (such
as one encoding a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
can be inserted into an expression vector including, but not
limited to a plasmid, virus or other vehicle that can be
manipulated to allow insertion or incorporation of sequences and
can be expressed in either prokaryotes or eukaryotes. Hosts can
include microbial, yeast, insect, plant and mammalian organisms.
Methods of expressing DNA sequences having eukaryotic or viral
sequences in prokaryotes are well known in the art. Biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host are known in the art. The vector can encode a
selectable marker, such as a thymidine kinase gene. Similar methods
can be used for a FGFR1c-binding protein multimer.
[0372] Nucleic acid sequences encoding a FGFR1c-binding protein
multimer or a mutated FGF1 protein (such as one encoding a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) can be operatively
linked to expression control sequences. An expression control
sequence operatively linked to a FGFR1c-binding protein multimer or
mutated FGF1 protein coding sequence is ligated such that
expression of the FGFR1c-binding protein multimer or mutant FGF1
protein coding sequence is achieved under conditions compatible
with the expression control sequences. The expression control
sequences include, but are not limited to appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a FGFR1c-binding protein multimer or mutated FGF1
protein-encoding gene, splicing signal for introns, maintenance of
the correct reading frame of that gene to permit proper translation
of mRNA, and stop codons.
[0373] In one embodiment, vectors are used for expression in yeast
such as S. cerevisiae, P. pastoris, or Kluyveromyces lactis.
Several promoters are known to be of use in yeast expression
systems such as the constitutive promoters plasma membrane
H.sup.+-ATPase (PMA1), glyceraldehyde-3-phosphate dehydrogenase
(GPD), phosphoglycerate kinase-1 (PGK1), alcohol dehydrogenase-1
(ADH1), and pleiotropic drug-resistant pump (PDR5). In addition,
many inducible promoters are of use, such as GAL1-10 (induced by
galactose), PHO5 (induced by low extracellular inorganic
phosphate), and tandem heat shock HSE elements (induced by
temperature elevation to 37.degree. C.). Promoters that direct
variable expression in response to a titratable inducer include the
methionine-responsive MET3 and MET25 promoters and copper-dependent
CUP1 promoters. Any of these promoters may be cloned into multicopy
(20 or single copy (CEN) plasmids to give an additional level of
control in expression level. The plasmids can include nutritional
markers (such as URA3, ADE3, HIS1, and others) for selection in
yeast and antibiotic resistance (AMP) for propagation in bacteria.
Plasmids for expression on K. lactis are known, such as pKLAC1.
Thus, in one example, after amplification in bacteria, plasmids can
be introduced into the corresponding yeast auxotrophs by methods
similar to bacterial transformation. The nucleic acid molecules
encoding a FGFR1c-binding protein multimer or a mutated FGF1
protein (such as one encoding a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) can also be designed to express in insect
cells.
[0374] A FGFR1c-binding protein multimer or mutated FGF1 protein
(such as a protein generated using the sequences shown in Tables 1
and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and
191-238 or those encoding a protein having at least 90%, at least
95%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
can be expressed in a variety of yeast strains. For example, seven
pleiotropic drug-resistant transporters, YOR1, SNQ2, PDR5, YCF1,
PDR10, PDR11, and PDR15, together with their activating
transcription factors, PDR1 and PDR3, have been simultaneously
deleted in yeast host cells, rendering the resultant strain
sensitive to drugs. Yeast strains with altered lipid composition of
the plasma membrane, such as the erg6 mutant defective in
ergosterol biosynthesis, can also be utilized. Proteins that are
highly sensitive to proteolysis can be expressed in a yeast cell
lacking the master vacuolar endopeptidase Pep4, which controls the
activation of other vacuolar hydrolases. Heterologous expression in
strains carrying temperature-sensitive (ts) alleles of genes can be
employed if the corresponding null mutant is inviable.
[0375] Viral vectors can also be prepared that encode a
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
one encoding a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238).
Exemplary viral vectors include polyoma, SV40, adenovirus, vaccinia
virus, adeno-associated virus, herpes viruses including HSV and
EBV, Sindbis viruses, alphaviruses and retroviruses of avian,
murine, and human origin. Baculovirus (Autographa californica
multinuclear polyhedrosis virus; AcMNPV) vectors are also known in
the art, and may be obtained from commercial sources. Other
suitable vectors include retrovirus vectors, orthopox vectors,
avipox vectors, fowlpox vectors, capripox vectors, suipox vectors,
adenoviral vectors, herpes virus vectors, alpha virus vectors,
baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors
and poliovirus vectors. Specific exemplary vectors are poxvirus
vectors such as vaccinia virus, fowlpox virus and a highly
attenuated vaccinia virus (MVA), adenovirus, baculovirus and the
like. Pox viruses of use include orthopox, suipox, avipox, and
capripox virus. Orthopox include vaccinia, ectromelia, and raccoon
pox. One example of an orthopox of use is vaccinia. Avipox includes
fowlpox, canary pox and pigeon pox. Capripox include goatpox and
sheeppox. In one example, the suipox is swinepox. Other viral
vectors that can be used include other DNA viruses such as herpes
virus and adenoviruses, and RNA viruses such as retroviruses and
polio.
[0376] Viral vectors that encode a FGFR1c-binding protein multimer
or a mutated FGF1 protein (such as one encoding a protein generated
using the sequences shown in Tables 1 and 2, the sequences in any
of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding a
protein having at least 90%, at least 95%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238) can include at least one
expression control element operationally linked to the nucleic acid
sequence encoding the FGFR1c-binding protein multimer or mutated
FGF1 protein. The expression control elements are inserted in the
vector to control and regulate the expression of the nucleic acid
sequence. Examples of expression control elements of use in these
vectors includes, but is not limited to, lac system, operator and
promoter regions of phage lambda, yeast promoters and promoters
derived from polyoma, adenovirus, retrovirus or SV40. Additional
operational elements include, but are not limited to, leader
sequence, termination codons, polyadenylation signals and any other
sequences necessary for the appropriate transcription and
subsequent translation of the nucleic acid sequence encoding the
mutated FGF1 protein in the host system. The expression vector can
contain additional elements necessary for the transfer and
subsequent replication of the expression vector containing the
nucleic acid sequence in the host system. Examples of such elements
include, but are not limited to, origins of replication and
selectable markers. It will further be understood by one skilled in
the art that such vectors are easily constructed using conventional
methods (Ausubel et al., (1987) in "Current Protocols in Molecular
Biology," John Wiley and Sons, New York, N.Y.) and are commercially
available.
[0377] Basic techniques for preparing recombinant DNA viruses
containing a heterologous DNA sequence encoding the FGFR1c-binding
protein multimer or mutated FGF1 protein (such as one encoding a
protein generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) are known. Such
techniques involve, for example, homologous recombination between
the viral DNA sequences flanking the DNA sequence in a donor
plasmid and homologous sequences present in the parental virus. The
vector can be constructed for example by steps known in the art,
such as by using a unique restriction endonuclease site that is
naturally present or artificially inserted in the parental viral
vector to insert the heterologous DNA.
[0378] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors can be used.
Eukaryotic cells can also be co-transformed with polynucleotide
sequences encoding a FGFR1c-binding protein multimer or a mutated
FGF1 protein (such as one encoding a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238), and a second foreign DNA molecule encoding a
selectable phenotype, such as the herpes simplex thymidine kinase
gene. Another method is to use a eukaryotic viral vector, such as
simian virus 40 (SV40) or bovine papilloma virus, to transiently
infect or transform eukaryotic cells and express the protein (see
for example, Eukaryotic Viral Vectors, Cold Spring Harbor
Laboratory, Gluzman ed., 1982). One of skill in the art can readily
use an expression systems such as plasmids and vectors of use in
producing mutated FGF1 proteins in cells including higher
eukaryotic cells such as the COS, CHO, HeLa and myeloma cell
lines.
Cells Expressing Mutated FGF1 Proteins or FGFR1c-Binding Protein
Multimers
[0379] A nucleic acid molecule encoding a mutated FGF1 protein
disclosed herein (or chimeric protein including a mutant FGF1), or
an FGFR1c-binding protein multimer disclosed herein, can be used to
transform cells and make transformed cells. Thus, cells expressing
a FGFR1c-binding protein multimer (such as a FGFR1c-binding protein
multimer made using peptides having at least 60%, at least 70%, at
least 75%, at least 80%, at least 90%, at least 92%, at least 95%,
at least 96%, at least 97%, at least 99%, at least 99%, or 100%
sequence identity to SEQ ID NO: 147, 148, 149, 150, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, or 190) or a mutated FGF1 protein (such as a protein generated
using the sequences shown in Tables 1 and 2, the sequences in any
of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding a
protein having at least 90%, at least 95%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238), are disclosed. Cells
expressing a mutated FGF1 protein disclosed herein, or expressing
an FGFR1c-binding protein multimer, can be eukaryotic or
prokaryotic. Examples of such cells include, but are not limited to
bacteria, archea, plant, fungal, yeast, insect, and mammalian
cells, such as Lactobacillus, Lactococcus, Bacillus (such as B.
subtilis), Escherichia (such as E. coli), Clostridium,
Saccharomyces or Pichia (such as S. cerevisiae or P. pastoris),
Kluyveromyces lactis, Salmonella typhimurium, SF9 cells, C129
cells, 293 cells, Neurospora, and immortalized mammalian myeloid
and lymphoid cell lines.
[0380] Cells expressing a mutated FGF1 protein or an FGFR1c-binding
protein multimer are transformed or recombinant cells. Such cells
can include at least one exogenous nucleic acid molecule that
encodes a mutated FGF1 protein, for example one encoding a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238. Such cells can
include at least one exogenous nucleic acid molecule that encodes
an FGFR1c-binding protein multimer, such as one encoding a protein
made using two or more peptides having at least 90%, at least 95%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity to SEQ ID NO: 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166, 167, or 190. It is understood that all progeny may
not be identical to the parental cell since there may be mutations
that occur during replication. Methods of stable transfer, meaning
that the foreign DNA is continuously maintained in the host cell,
are known in the art.
[0381] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known. Where the
host is prokaryotic, such as E. coli, competent cells which are
capable of DNA uptake can be prepared from cells harvested after
exponential growth phase and subsequently treated by the CaCl.sub.2
method using procedures well known in the art. Alternatively,
MgCl.sub.2 or RbCl can be used. Transformation can also be
performed after forming a protoplast of the host cell if desired,
or by electroporation. Techniques for the propagation of mammalian
cells in culture are well-known (see, Jakoby and Pastan (eds),
1979, Cell Culture. Methods in Enzymology, volume 58, Academic
Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly
used mammalian host cell lines are VERO and HeLa cells, CHO cells,
and WI38, BHK, and COS cell lines, although cell lines may be used,
such as cells designed to provide higher expression desirable
glycosylation patterns, or other features. Techniques for the
transformation of yeast cells, such as polyethylene glycol
transformation, protoplast transformation and gene guns are also
known in the art.
Pharmaceutical Compositions that Include Mutated FGF1 Molecules
and/or FGFR1c-Binding Protein Multimers
[0382] Pharmaceutical compositions that include a mutated FGF1
protein (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
or a nucleic acid encoding these proteins, can be formulated with
an appropriate pharmaceutically acceptable carrier, depending upon
the particular mode of administration chosen. Similarly, the
disclosure provides pharmaceutical compositions that include one or
more FGFR1c-binding protein multimers, such as multimers of SEQ ID
NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, or 167, such as SEQ ID NO:
190 (or sequences having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 147, 148, 149, 150, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, or 190).
[0383] In some embodiments, the pharmaceutical composition consists
essentially of an FGFR1c-binding protein multimer or a mutated FGF1
protein (such as a protein generated using the sequences shown in
Table 1, the sequences in any of SEQ ID NOS: 21-84, or a protein
having at least 90%, at least 95%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
any of SEQ ID NOS: 6-13, 21-84, 87-98, 101-112, 173-175, 177-179,
181-183, 185-189, and 191-238) (or a nucleic acid encoding such a
protein) and a pharmaceutically acceptable carrier. In these
embodiments, additional therapeutically effective agents are not
included in the compositions.
[0384] In other embodiments, the pharmaceutical composition
includes an FGFR1c-binding protein multimer or a mutated FGF1
protein (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
(or a nucleic acid encoding such a protein) and a pharmaceutically
acceptable carrier. Additional therapeutic agents, such as agents
for the treatment of diabetes, can be included. Thus, the
pharmaceutical compositions can include a therapeutically effective
amount of another agent. Examples of such agents include, without
limitation, anti-apoptotic substances such as the Nemo-Binding
Domain and compounds that induce proliferation such as cyclin
dependent kinase (CDK)-6, CDK-4 and cyclin D1. Other active agents
can be utilized, such as antidiabetic agents for example,
metformin, sulphonylureas (e.g., glibenclamide, tolbutamide,
glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g.,
rosiglitazone, pioglitazone), peroxisome proliferator-activated
receptor (PPAR)-gamma-agonists (such as C1262570, aleglitazar,
farglitazar, muraglitazar, tesaglitazar, and TZD) and PPAR-.gamma.
antagonists, PPAR-gamma/alpha modulators (such as KRP 297),
alpha-glucosidase inhibitors (e.g., acarbose, voglibose),
dipeptidyl peptidase (DPP)-IV inhibitors (such as LAF237, MK-431),
alpha2-antagonists, agents for lowering blood sugar,
cholesterol-absorption inhibitors,
3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors
(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1
analogues (e.g. exendin-4) or amylin. Additional examples include
immunomodulatory factors such as anti-CD3 mAb, growth factors such
as HGF, VEGF, PDGF, lactogens, and PTHrP. In some examples, the
pharmaceutical compositions containing a mutated FGF1 protein can
further include a therapeutically effective amount of other FGFs,
such as FGF21, FGF19, or both, heparin, or combinations
thereof.
[0385] The pharmaceutically acceptable carriers and excipients
useful in this disclosure are conventional. See, e.g., Remington:
The Science and Practice of Pharmacy, The University of the
Sciences in Philadelphia, Editor, Lippincott, Williams, &
Wilkins, Philadelphia, Pa., 21.sup.st Edition (2005). For instance,
parenteral formulations usually include injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, pH buffering agents, or the like, for
example sodium acetate or sorbitan monolaurate. Excipients that can
be included are, for instance, other proteins, such as human serum
albumin or plasma preparations.
[0386] In some embodiments, an FGFR1c-binding protein multimer or a
mutated FGF1 protein (such as a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) is included in a controlled release
formulation, for example, a microencapsulated formulation. Various
types of biodegradable and biocompatible polymers, methods can be
used, and methods of encapsulating a variety of synthetic
compounds, proteins and nucleic acids, have been well described in
the art (see, for example, U.S. Patent Publication Nos.
2007/0148074; 2007/0092575; and 2006/0246139; U.S. Pat. Nos.
4,522,811; 5,753,234; and 7,081,489; PCT Publication No.
WO/2006/052285; Benita, Microencapsulation: Methods and Industrial
Applications, 2.sup.nd ed., CRC Press, 2006).
[0387] In other embodiments, an FGFR1c-binding protein multimer or
a mutated FGF1 protein (such as a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) is included in a nanodispersion system.
Nanodispersion systems and methods for producing such
nanodispersions are well known to one of skill in the art. See,
e.g., U.S. Pat. No. 6,780,324; U.S. Pat. Publication No.
2009/0175953. For example, a nanodispersion system includes a
biologically active agent and a dispersing agent (such as a
polymer, copolymer, or low molecular weight surfactant). Exemplary
polymers or copolymers include polyvinylpyrrolidone (PVP),
poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid
(PLGA), poly(ethylene glycol). Exemplary low molecular weight
surfactants include sodium dodecyl sulfate, hexadecyl pyridinium
chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers,
poly(oxyethylene) alkyl esters, and combinations thereof. In one
example, the nanodispersion system includes PVP and ODP or a
variant thereof (such as 80/20 w/w). In some examples, the
nanodispersion is prepared using the solvent evaporation method,
see for example, Kanaze et al., Drug Dev. Indus. Pharm. 36:292-301,
2010; Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006. With
regard to the administration of nucleic acids, one approach to
administration of nucleic acids is direct treatment with plasmid
DNA, such as with a mammalian expression plasmid. As described
above, the nucleotide sequence encoding an FGFR1c-binding protein
multimer or a mutated FGF1 protein (such as a protein generated
using the sequences shown in Tables 1 and 2, the sequences in any
of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding a
protein having at least 90%, at least 95%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238) can be placed under the
control of a promoter to increase expression of the protein.
[0388] Many types of release delivery systems are available and
known. Examples include polymer based systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems, such as lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; silastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which an FGFR1c-binding protein multimer or a mutated FGF1 protein
(such as a protein generated using the sequences shown in Tables 1
and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and
191-238 or those encoding a protein having at least 90%, at least
95%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238),
or polynucleotide encoding this protein, is contained in a form
within a matrix such as those described in U.S. Pat. Nos.
4,452,775; 4,667,014; 4,748,034; 5,239,660; and 6,218,371 and (b)
diffusional systems in which an active component permeates at a
controlled rate from a polymer such as described in U.S. Pat. Nos.
3,832,253 and 3,854,480. In addition, pump-based hardware delivery
systems can be used, some of which are adapted for
implantation.
[0389] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic conditions, such as
diabetes. Long-term release, as used herein, means that the implant
is constructed and arranged to deliver therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well known to those of
ordinary skill in the art and include some of the release systems
described above. These systems have been described for use with
nucleic acids (see U.S. Pat. No. 6,218,371). For use in vivo,
nucleic acids and peptides are preferably relatively resistant to
degradation (such as via endo- and exo-nucleases). Thus,
modifications of the disclosed mutated FGF1 proteins, such as the
inclusion of a C-terminal amide, can be used.
[0390] The dosage form of the pharmaceutical composition can be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical, inhalation, oral and
suppository formulations can be employed. Topical preparations can
include eye drops, ointments, sprays, patches and the like.
Inhalation preparations can be liquid (e.g., solutions or
suspensions) and include mists, sprays and the like. Oral
formulations can be liquid (e.g., syrups, solutions or
suspensions), or solid (e.g., powders, pills, tablets, or
capsules). Suppository preparations can also be solid, gel, or in a
suspension form. For solid compositions, conventional non-toxic
solid carriers can include pharmaceutical grades of mannitol,
lactose, cellulose, starch, or magnesium stearate. Actual methods
of preparing such dosage forms are known, or will be apparent, to
those skilled in the art.
[0391] The pharmaceutical compositions that include an
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) can be
formulated in unit dosage form, suitable for individual
administration of precise dosages. In one non-limiting example, a
unit dosage contains from about 1 mg to about 1 g of an
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), such as about
10 mg to about 100 mg, about 50 mg to about 500 mg, about 100 mg to
about 900 mg, about 250 mg to about 750 mg, or about 400 mg to
about 600 mg. In other examples, a therapeutically effective amount
of an FGFR1c-binding protein multimer or a mutated FGF1 protein
(such as a protein generated using the sequences shown in Tables 1
and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and
191-238 or those encoding a protein having at least 90%, at least
95%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
is about 0.01 mg/kg to about 50 mg/kg, for example, about 0.5 mg/kg
to about 25 mg/kg or about 1 mg/kg to about 10 mg/kg. In other
examples, a therapeutically effective amount of an FGFR1c-binding
protein multimer or a mutated FGF1 protein (such as a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) is about 1 mg/kg to
about 5 mg/kg, for example about 2 mg/kg. In a particular example,
a therapeutically effective amount of an FGFR1c-binding protein
multimer or a mutated FGF1 protein (such as a protein generated
using the sequences shown in Tables 1 and 2, the sequences in any
of SEQ ID NOS: 21-84, 113-120 and 191-238 or those encoding a
protein having at least 90%, at least 95%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, 174, 175,
177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,
219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237 or 238) includes about 1 mg/kg to
about 10 mg/kg, such as about 2 mg/kg.
Treatment Using Mutated FGF1 or FGFR1c-Binding Protein
Multimers
[0392] The disclosed FGFR1c-binding protein multimers (such as a
protein made using two or more peptides having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 147, 148, 149,
150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165, 166, 167, or 190) and mutated FGF1 proteins and
chimeras (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238),
or nucleic acids encoding such proteins, can be administered to a
subject, for example to treat a metabolic disease, for example by
reducing fed and fasting blood glucose, improving insulin
sensitivity and glucose tolerance, reducing systemic chronic
inflammation, ameliorating hepatic steatosis in a mammal, reducing
food intake, or combinations thereof.
[0393] The compositions of this disclosure that include an
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) (or nucleic
acids encoding these molecules) can be administered to humans or
other animals by any means, including orally, intravenously,
intramuscularly, intraperitoneally, intranasally, intradermally,
intrathecally, subcutaneously, via inhalation or via suppository.
In one non-limiting example, the composition is administered via
injection. In some examples, site-specific administration of the
composition can be used, for example by administering an
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) (or a nucleic
acid encoding these molecules) to pancreas tissue (for example by
using a pump, or by implantation of a slow release form at the site
of the pancreas). The particular mode of administration and the
dosage regimen will be selected by the attending clinician, taking
into account the particulars of the case (e.g. the subject, the
disease, the disease state involved, the particular treatment, and
whether the treatment is prophylactic). Treatment can involve daily
or multi-daily or less than daily (such as weekly or monthly etc.)
doses over a period of a few days to months, or even years. For
example, a therapeutically effective amount of an FGFR1c-binding
protein multimer or a mutated FGF1 protein (such as a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) can be administered
in a single dose, twice daily, weekly, or in several doses, for
example daily, or during a course of treatment. In a particular
non-limiting example, treatment involves once daily dose or twice
daily dose.
[0394] The amount of an FGFR1c-binding protein multimer or mutated
FGF1 protein (such as a protein generated using the sequences shown
in Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84,
113-120 and 191-238 or those encoding a protein having at least
90%, at least 95%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6,
7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179, 181,
182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,
210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,
236, 237 or 238) administered can be dependent on the subject being
treated, the severity of the affliction, and the manner of
administration, and is best left to the judgment of the prescribing
clinician. Within these bounds, the formulation to be administered
will contain a quantity of the FGFR1c-binding protein multimer or
mutated FGF1 protein in amounts effective to achieve the desired
effect in the subject being treated. A therapeutically effective
amount of an FGFR1c-binding protein multimer or mutated FGF1
protein (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238)
can be the amount of the mutant FGF1protein or FGFR1c-binding
protein multimer, or a nucleic acid encoding these molecules that
is necessary to treat diabetes or reduce blood glucose levels (for
example a reduction of at least 5%, at least 10% or at least 20%,
for example relative to no administration of the mutant FGF1 or
FGFR1c-binding protein multimer).
[0395] When a viral vector is utilized for administration of an
nucleic acid encoding an FGFR1c-binding protein multimer or a
mutated FGF1 protein (such as a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238), the recipient can receive a dosage of each
recombinant virus in the composition in the range of from about
10.sup.5 to about 10.sup.10 plaque forming units/mg mammal,
although a lower or higher dose can be administered. Examples of
methods for administering the composition into mammals include, but
are not limited to, exposure of cells to the recombinant virus ex
vivo, or injection of the composition into the affected tissue or
intravenous, subcutaneous, intradermal or intramuscular
administration of the virus. Alternatively the recombinant viral
vector or combination of recombinant viral vectors may be
administered locally by direct injection into the pancreases in a
pharmaceutically acceptable carrier.
[0396] Generally, the quantity of recombinant viral vector,
carrying the nucleic acid sequence of an FGFR1c-binding protein
multimer or the mutated FGF1 protein to be administered (such as a
protein generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238) is based on the
titer of virus particles. An exemplary range to be administered is
10.sup.5 to 10.sup.10 virus particles per mammal, such as a
human.
[0397] In some examples, an FGFR1c-binding protein multimer or
mutated FGF1 protein (such as a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238), or a nucleic acid encoding the
FGFR1c-binding protein multimer or the mutated FGF1 protein, is
administered in combination (such as sequentially or simultaneously
or contemporaneously) with one or more other agents, such as those
useful in the treatment of diabetes or insulin resistance.
[0398] Anti-diabetic agents are generally categorized into six
classes: biguanides (e.g., metformin); thiazolidinediones
(including rosiglitazone (Avandia.RTM.), pioglitazone (Actos.RTM.),
rivoglitazone, and troglitazone); sulfonylureas; inhibitors of
carbohydrate absorption; fatty acid oxidase inhibitors and
anti-lipolytic drugs; and weight-loss agents. Any of these agents
can also be used in the methods disclosed herein. The anti-diabetic
agents include those agents disclosed in Diabetes Care,
22(4):623-634. One class of anti-diabetic agents of use is the
sulfonylureas, which are believed to increase secretion of insulin,
decrease hepatic glucogenesis, and increase insulin receptor
sensitivity. Another class of anti-diabetic agents use the
biguanide antihyperglycemics, which decrease hepatic glucose
production and intestinal absorption, and increase peripheral
glucose uptake and utilization, without inducing
hyperinsulinemia.
[0399] In some examples, an FGFR1c-binding protein multimer or
mutated FGF1 protein (such as a protein generated using the
sequences shown in Tables 1 and 2, the sequences in any of SEQ ID
NOS: 21-84, 113-120 and 191-238 or those encoding a protein having
at least 90%, at least 95%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID
NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 173, 174, 175, 177, 178, 179,
181, 182, 183, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237 or 238) can be administered in combination with
effective doses of anti-diabetic agents (such as biguanides,
thiazolidinediones, or incretins) and/or lipid lowering compounds
(such as statins or fibrates). The term "administration in
combination" or "co-administration" refers to both concurrent and
sequential administration of the active agents. Administration of
an FGFR1c-binding protein multimer or mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 2388) or a nucleic
acid encoding such an FGFR1c-binding protein multimer or a mutant
FGF1 protein, may also be in combination with lifestyle
modifications, such as increased physical activity, low fat diet,
low sugar diet, and smoking cessation.
[0400] Additional agents that can be used in combination with the
disclosed FGFR1c-binding protein multimers and mutated FGF1
proteins include, without limitation, anti-apoptotic substances
such as the Nemo-Binding Domain and compounds that induce
proliferation such as cyclin dependent kinase (CDK)-6, CDK-4 and
Cyclin D1. Other active agents can be utilized, such as
antidiabetic agents for example, metformin, sulphonylureas (e.g.,
glibenclamide, tolbutamide, glimepiride), nateglinide, repaglinide,
thiazolidinediones (e.g., rosiglitazone, pioglitazone), peroxisome
proliferator-activated receptor (PPAR)-gamma-agonists (such as
C1262570) and antagonists, PPAR-gamma/alpha modulators (such as KRP
297), alpha-glucosidase inhibitors (e.g., acarbose, voglibose),
Dipeptidyl peptidase (DPP)-IV inhibitors (such as LAF237, MK-431),
alpha2-antagonists, agents for lowering blood sugar,
cholesterol-absorption inhibitors,
3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors
(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1
analogues (e.g., exendin-4) or amylin. In some embodiments the
agent is an immunomodulatory factor such as anti-CD3 mAb, growth
factors such as HGF, vascular endothelial growth factor (VEGF),
platelet derived growth factor (PDGF), lactogens, or parathyroid
hormone related protein (PTHrP). In one example, the mutated FGF1
protein is administered in combination with a therapeutically
effective amount of another FGF, such as FGF21, FGF19, or both,
heparin, or combinations thereof.
[0401] In some embodiments, methods are provided for treating
diabetes or pre-diabetes in a subject by administering a
therapeutically effective amount of a composition including an
FGFR1c-binding protein multimer or a mutated FGF1 protein (such as
a protein generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238), or a nucleic
acid encoding the FGFR1c-binding protein multimer or the mutated
FGF1 protein, to the subject. The subject can have diabetes type I
or diabetes type II. The subject can be any mammalian subject,
including human subjects and veterinary subjects such as cats and
dogs. The subject can be a child or an adult. The subject can also
be administered insulin. The method can include measuring blood
glucose levels.
[0402] In some examples, the method includes selecting a subject
with diabetes, such as type I or type II diabetes, or a subject at
risk for diabetes, such as a subject with pre-diabetes. These
subjects can be selected for treatment with the disclosed
FGFR1c-binding protein multimer or mutated FGF1 proteins (such as a
protein g generated using the sequences shown in Tables 1 and 2,
the sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or
those encoding a protein having at least 90%, at least 95%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188,
189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237 or 238) or nucleic acid
molecules encoding such.
[0403] In some examples, a subject with diabetes may be clinically
diagnosed by a fasting plasma glucose (FPG) concentration of
greater than or equal to 7.0 millimole per liter (mmol/L) (126
milligram per deciliter (mg/dL)), or a plasma glucose concentration
of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two
hours after an oral glucose tolerance test (OGTT) with a 75 gram
(g) load, or in a patient with classic symptoms of hyperglycemia or
hyperglycemic crisis, a random plasma glucose concentration of
greater than or equal to 11.1 mmol/L (200 mg/dL), or HbA1c levels
of greater than or equal to 6.5%. In other examples, a subject with
pre-diabetes may be diagnosed by impaired glucose tolerance (IGT).
An OGTT two-hour plasma glucose of greater than or equal to 140
mg/dL and less than 200 mg/dL (7.8-11.0 mM), or a fasting plasma
glucose (FPG) concentration of greater than or equal to 100 mg/dL
and less than 125 mg/dL (5.6-6.9 mmol/L), or HbA1c levels of
greater than or equal to 5.7% and less than 6.4% (5.7-6.4%) is
considered to be IGT, and indicates that a subject has
pre-diabetes. Additional information can be found in Standards of
Medical Care in Diabetes--2010 (American Diabetes Association,
Diabetes Care 33:S11-61, 2010).
[0404] In some examples, the subject treated with the disclosed
compositions and methods has HbA1C of greater than 6.5% or greater
than 7%.
[0405] In some examples, treating diabetes includes one or more of
increasing glucose tolerance (such as an increase of at least 5%,
at least 10%, at least 20%, or at least 50%, for example relative
to no administration of the FGFR1c-binding protein multimer or
mutant FGF1), decreasing insulin resistance (for example,
decreasing plasma glucose levels, decreasing plasma insulin levels,
or a combination thereof, such as decreases of at least 5%, at
least 10%, at least 20%, or at least 50%, for example relative to
no administration of the FGFR1c-binding protein multimer or mutant
FGF1), decreasing serum triglycerides (such as a decrease of at
least 10%, at least 20%, or at least 50%, for example relative to
no administration of the FGFR1c-binding protein multimer or mutant
FGF1), decreasing free fatty acid levels (such as a decrease of at
least 5%, at least 10%, at least 20%, or at least 50%, for example
relative to no administration of the FGFR1c-binding protein
multimer or mutant FGF1), and decreasing HbA1c levels in the
subject (such as a decrease of at least 0.5%, at least 1%, at least
1.5%, at least 2%, or at least 5% for example relative to no
administration of the FGFR1c-binding protein multimer or mutant
FGF1). In some embodiments, the disclosed methods include measuring
glucose tolerance, insulin resistance, plasma glucose levels,
plasma insulin levels, serum triglycerides, free fatty acids,
and/or HbA1c levels in a subject.
[0406] In some examples, administration of an FGFR1c-binding
protein multimer or a mutated FGF1 protein (such as a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238), or nucleic acid
molecule encoding such, treats a metabolic disease, such as
diabetes (such as type II diabetes) or pre-diabetes, by decreasing
of HbA1C, such as a reduction of at least 0.5%, at least 1%, or at
least 1.5%, such as a decrease of 0.5% to 0.8%, 0.5% to 1%, 1 to
1.5% or 0.5% to 2%. In some examples the target for HbA1C is less
than about 6.5%, such as about 4-6%, 4-6.4%, or 4-6.2%. In some
examples, such target levels are achieved within about 26 weeks,
within about 40 weeks, or within about 52 weeks. Methods of
measuring HbA1C are routine, and the disclosure is not limited to
particular methods. Exemplary methods include HPLC, immunoassays,
and boronate affinity chromatography.
[0407] In some examples, administration of an FGFR1c-binding
protein multimer or a mutated FGF1 protein (such as a protein
generated using the sequences shown in Tables 1 and 2, the
sequences in any of SEQ ID NOS: 21-84, 113-120 and 191-238 or those
encoding a protein having at least 90%, at least 95%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 or 238), or nucleic acid
molecule encoding such, treats diabetes or pre-diabetes by
increasing glucose tolerance, for example, by decreasing blood
glucose levels (such as two-hour plasma glucose in an OGTT or FPG)
in a subject. In some examples, the method includes decreasing
blood glucose by at least 5% (such as at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, or more) as
compared with a control (such as no administration of any of
insulin, an FGFR1c-binding protein multimer or a mutated FGF1
protein (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238),
or a nucleic acid molecule encoding such). In particular examples,
a decrease in blood glucose level is determined relative to the
starting blood glucose level of the subject (for example, prior to
treatment with an FGFR1c-binding protein multimer or a mutated FGF1
protein (such as a protein generated using the sequences shown in
Tables 1 and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120
and 191-238 or those encoding a protein having at least 90%, at
least 95%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9,
10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238),
or nucleic acid molecule encoding such). In other examples,
decreasing blood glucose levels of a subject includes reduction of
blood glucose from a starting point (for example greater than about
126 mg/dL FPG or greater than about 200 mg/dL OGTT two-hour plasma
glucose) to a target level (for example, FPG of less than 126 mg/dL
or OGTT two-hour plasma glucose of less than 200 mg/dL). In some
examples, a target FPG may be less than 100 mg/dL. In other
examples, a target OGTT two-hour plasma glucose may be less than
140 mg/dL. Methods to measure blood glucose levels in a subject
(for example, in a blood sample from a subject) are routine.
[0408] In other embodiments, the disclosed methods include
comparing one or more indicator of diabetes (such as glucose
tolerance, triglyceride levels, free fatty acid levels, or HbA1c
levels) to a control (such as no administration of any of insulin,
any FGFR1c-binding protein multimer or any mutated FGF1 protein
(such as a protein generated using the sequences shown in Tables 1
and 2, the sequences in any of SEQ ID NOS: 21-84, 113-120 and
191-238 or those encoding a protein having at least 90%, at least
95%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to SEQ ID NO: 6, 7, 8, 9, 10,
11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238),
or a nucleic acid molecule encoding such), wherein an increase or
decrease in the particular indicator relative to the control (as
discussed above) indicates effective treatment of diabetes. The
control can be any suitable control against which to compare the
indicator of diabetes in a subject. In some embodiments, the
control is a sample obtained from a healthy subject (such as a
subject without diabetes). In some embodiments, the control is a
historical control or standard reference value or range of values
(such as a previously tested control sample, such as a group of
subjects with diabetes, or group of samples from subjects that do
not have diabetes). In further examples, the control is a reference
value, such as a standard value obtained from a population of
normal individuals that is used by those of skill in the art.
Similar to a control population, the value of the sample from the
subject can be compared to the mean reference value or to a range
of reference values (such as the high and low values in the
reference group or the 95% confidence interval). In other examples,
the control is the subject (or group of subjects) treated with
placebo compared to the same subject (or group of subjects) treated
with the therapeutic compound in a cross-over study. In further
examples, the control is the subject (or group of subjects) prior
to treatment.
[0409] The disclosure is illustrated by the following non-limiting
Examples.
Example 1
Preparation of Mutated FGF1 Proteins
[0410] Mutated FGF1 proteins can be made using known methods (e.g.,
see Xia et al., PLoS One. 7(11):e48210, 2012). An example is
provided below.
[0411] Briefly, a nucleic acid sequence encoding an FGF1 mutant
protein (e.g., any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173,
174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237 and 238) can be fused
downstream of an enterokinase (EK) recognition sequence (Asp4Lys)
preceded by a flexible 20 amino acid linker (derived from the S-tag
sequence of pBAC-3) and an N-terminal (His).sub.6 tag. The
resulting expressed fusion protein utilizes the (His).sub.6 tag for
efficient purification and can be subsequently processed by EK
digestion to yield the mutant FGF1 protein.
[0412] The mutant FGF1 protein can be expressed from an E. coli
host after induction with isopropyl-.beta.-D-thio-galactoside. The
expressed protein can be purified utilizing sequential column
chromatography on Ni-nitrilotriacetic acid (NTA) affinity resin
followed by ToyoPearl HW-40S size exclusion chromatography. The
purified protein can be digested with EK to remove the N-terminal
(His).sub.6 tag, 20 amino acid linker, and (Asp4Lys) EK recognition
sequence. A subsequent second Ni-NTA chromatographic step can be
utilized to remove the released N-terminal mutant FGF1 protein
(along with any uncleaved fusion protein). Final purification can
be performed using HiLoad Superdex 75 size exclusion chromatography
equilibrated to 50 mM Na.sub.2PO.sub.4, 100 mM NaCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 0.1 mM ethylenediaminetetraacetic acid
(EDTA), 5 mM L-Methionine, pH at 6.5 ("PBX" buffer); L-Methionine
can be included in PBX buffer to limit oxidization of reactive
thiols and other potential oxidative degradation.
[0413] In some examples, the enterokinase is not used, and instead,
an FGF1 mutant protein (such as one that includes an N-terminal
methionine) can be made and purified using heparin affinity
chromatography.
[0414] For storage and use, the purified mutant FGF1 protein can be
sterile filtered through a 0.22 micron filter, purged with N.sub.2,
snap frozen in dry ice and stored at -80.degree. C. prior to use.
The purity of the mutant FGF 1protein can be assessed by both
Coomassie Brilliant Blue and Silver Stain Plus (BIO-RAD
Laboratories, Inc., Hercules Calif.) stained sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS PAGE). Mutant FGF1 proteins
can be prepared in the absence of heparin. Prior to IV bolus,
heparin, or PBS, can be added to the protein.
Example 2
N-Terminally Truncated FGF1 Reduces Blood Glucose in Ob/Ob Mice
[0415] We have shown that administration of mature rFGF1 to ob/ob
mice can lower blood glucose and have reduced adverse effects as
compared to those observed with thiazolidinediones (TZDs).
[0416] To dissociate the mitogenic effects of rFGF1 from its
glucose lowering activities, an FGF1 ligand was generated that
lacks the first 24 residues from the N-terminus,
rFGF1.sup..DELTA.NT (SEQ ID NO: 7). Based on the crystal structures
of FGF1-FGFR complexes, the truncation was predicted to reduce the
binding affinity of FGF1 for selected FGFRs including FGFR4, and
hence the ligand's mitogenicity.
Animals
[0417] Mice were housed in a temperature-controlled environment
with a 12-hour light/12-hour dark cycle and handled according to
institutional guidelines complying with U.S. legislation. Male
ob/ob mice (B6.V-Lep.sup.ob/J, Jackson laboratories) and male
C57BL/6J mice received a standard or high fat diet (MI laboratory
rodent diet 5001, Harlan Teklad; high fat (60%) diet F3282,
Bio-Serv) and acidified water ad libitum. STZ-induced diabetic mice
on the C57BL/6J background were purchased from Jackson
laboratories. 0.1 mg/ml solutions in PBS of mouse FGF1 (Prospec,
Ness Ziona, Israel), human FGF1 (Prospec, Ness Ziona, Israel),
mouse FGF2 (Prospec, Ness Ziona, Israel), mouse FGF9 (Prospec, Ness
Ziona, Israel), and mouse FGF10 (R&D systems) were injected as
described.
Serum Analysis
[0418] Blood was collected by tail bleeding either in the ad
libitum fed state or following overnight fasting. Free fatty acids
(Wako), triglycerides (Thermo) and cholesterol (Thermo) were
measured using enzymatic colorimetric methods following the
manufacturer's instructions. Serum insulin levels were measured
using an Ultra Sensitive Insulin ELISA kit (Crystal Chem). Plasma
adipokine and cytokine levels were measured using Milliplex.TM. MAP
and Bio-Plex Pro.TM. kits (Millipore and Bio-Rad).
Metabolic Studies
[0419] Glucose tolerance tests (GTT) were conducted after o/n
fasting. Mice were injected i.p. with 1 g of glucose per/kg
bodyweight and blood glucose was monitored at 0, 15, 30, 60, and
120 min using a OneTouch Ultra glucometer (Lifescan Inc). Insulin
tolerance tests (ITT) were conducted after 3 h fasting. Mice were
injected i.p. with 2 U of insulin/kg bodyweight (Humulin R; Eli
Lilly) and blood glucose was monitored at 0, 15, 30, 60, and 90 min
using a OneTouch Ultra glucometer (Lifescan Inc). Real-time
metabolic analyses were conducted in a Comprehensive Lab Animal
Monitoring System (Columbus Instruments). CO.sub.2 production,
O.sub.2 consumption, RQ (relative rates of carbohydrate versus fat
oxidation), and ambulatory counts were determined for six
consecutive days and nights, with at least 24 h for adaptation
before data recording. Total body composition analysis was
performed using an EchoMRI-100.TM. (Echo Medical Systems, LLC)
Purification of FGF and FGFR Proteins
[0420] Human FGF1 (M1 to D155; SEQ ID NO: 2) and N-terminally
truncated human FGF1 (FGF1.sup..DELTA.NT; K25 to D155; SEQ ID NO:
7) were expressed in Escherichia coli cells and purified from the
soluble bacterial cell lysate fraction by heparin affinity, ion
exchange, and size exclusion chromatographies. The minimal
ligand-binding domain of human FGFR1c (D142 to R365), FGFR2b (A140
to E369), FGFR2c (N149 to E368), FGFR3c (D147 to E365), and FGFR4
(Q144 to D355) was refolded in vitro from bacterial inclusion
bodies and purified by published protocols.
[0421] Mice received a standard diet (ob/ob and db/db mice) or high
fat diet (C57/BL6 mice, 60% fat, F3282, Bio-Serv) and acidified
water ad libitum. Blood glucose levels were monitored either in the
ad libitum fed state or following overnight fasting after injection
of recombinant FGF1 or rFGF1.sup..DELTA.NT (in PBS, Prospec,
Israel) using the specified delivery route and dosage. Glucose
tolerance tests (GTT) and Insulin tolerance tests (ITT) were
conducted after overnight and 5 hour fasting, respectively. Glucose
(1 g/kg i.p.) or insulin (0.5 U insulin/kg i.p.) was injected and
blood glucose monitored. Serum analyses were performed on blood
collected by tail bleeding either in the ad libitum fed state or
following overnight fasting.
[0422] Remarkably, parenteral delivery of rFGF1.sup..DELTA.NT
lowered blood glucose levels to the same extent as rFGF1 in both
genetic- and diet-induced mouse models of diabetes (FIGS. 2A and
2B). rFGF1.sup..DELTA.NT also retained the feeding suppression
effects observed with rFGF1 (FIG. 2C). Therefore, the synthetic
effects of exogenous rFGF1 on physiology, such as glucose
homeostasis and feeding behavior, differ from and are independent
of its classical role as a growth factor and mitogen.
Example 3
FGF1 Mutant Proteins Reduce Blood Glucose Levels in Diabetic
Mice
[0423] FGF1 mutants shown in Table 3 were tested as described in
Example 2.
[0424] As shown in Table 3, up to 12 N-terminal amino acids can be
deleted from FGF1 without significantly affecting activity, while
an FGF1 mutant lacking 14 N-terminal amino acids failed to lower
glucose in diabetic mice. Mutations that increase the thermal
stability of FGF1 were generally well tolerated, however glucose
lowering activity was lost in a mutant with high thermal stability.
Furthermore, mutations to the putative heparan sulfate binding site
had minimal effect to the glucose lowering actions of FGF1.
Notably, the effects of N-terminal deletions and selected
stabilizing mutations appeared additive.
TABLE-US-00003 FGF1 (SEQ ID NO:) Relative activity wt FGF1 (5) xxx
Reduced mitogenic mutants FGF1.sup..DELTA.NT(10-140.alpha..alpha.)
(7) xxxx FGF1.sup..DELTA.NT2(14-140.alpha..alpha.) (8) inactive
FGF1.sup..DELTA.NT3(12-140.alpha..alpha.) (9) xxx (shorter
duration) FGF1(1-140.alpha..alpha.) K12V, N95V (10) xxxx
FGF1(1-140.alpha..alpha.) K12V, L46V, E87V, inactive N95V, P134V
(11) FGF1(7-140.alpha..alpha.) K118N (12) inactive
FGF1(7-140.alpha..alpha.) K118E (13) inactive Stabilizing Mutants
FGF1(1-140.alpha..alpha.)K12V, P134V, C117V (22) xxx
FGF1(1-140.alpha..alpha.) L44F, C83T, C117V, xxx F132W (28)
FGF1(1-140.alpha..alpha.) L44F, M67I, L73V, xxx (shorter duration)
V109L, L111I, C117V, A103G, R119G, .DELTA.104-106, .DELTA.120-122
(40) FGF1(1-140.alpha..alpha.) K12V, N95V, C117V (54) xxx (longer
duration) FGF1(1-140.alpha..alpha.) K12V, L46V, E87V, inactive
N95V, P134V, C117V (212) Heparan binding site mutations
FGF1.sup..DELTA.HBS K112D, K113Q, K118V (113) xx
FGF1.sup..DELTA.HBS (1-140.alpha..alpha.) K112D, xx K113Q, K118V
(226) Combination mutations FGF.sup..DELTA.NT1C
(10-140.alpha..alpha.) K12V, N95V, xxxx (longer duration) C117V
(225) FGF1 (1-140.alpha..alpha.) K12V, Q40P, S47I, xx H93G, N95V
(227) FGF1.sup..DELTA.NT1(10-140.alpha..alpha.) K12V, Q40P, xx
S47I, H93G, N95V (228) FGF1 (1-140.alpha..alpha.) K12V, L44F, C83T,
xxx N95V, C117V, F132W (229) Chimeras
wtFGF1.sup..DELTA.HBS-FGF21.sup.C-tail (219) xx
wtFGF1.sup..DELTA.HBS-FGF19.sup.C-tail (220) xx
Example 4
Effect on Intracellular Signaling with FGF1 Mutants
[0425] Peptides M1, M2, M3, M4, and M5 (see SEQ ID NO: 22, 28, 40,
54 and 212, respectively); KN (SEQ ID NO: 10), KLE (SEQ ID NO: 11),
and FGF1 (SEQ ID NO: 5) were generated as described in Example 1.
The NT truncations, peptides NT1 (SEQ ID NO: 7), NT2 (SEQ ID NO:
8), and NT3 (SEQ ID NO: 9), were prepared without the His tag and
enterokinase cleavage, and purified with heparin affinity and ion
exchange chromatography. Peptides (10 ng/ml) were incubated with
serum-starved HEK293 cells for 15 minutes. Total cell lysates were
subject to Western blotting with antibodies specific for pAkt, Akt,
pERK and ERK.
[0426] As shown in FIG. 8, the thermostable M3 analog shows reduced
ERK signaling, similar to that seen with the M5 analog, correlating
with the reduced glucose lowering activity seen in ob/ob mice.
[0427] As shown in FIG. 9, deletion of 9 (NT1) or 11 (NT3)
N-terminal residues of FGF1 does not significantly affecting FGFR
downstream signaling, while deletion of 13 (NT2) residues severely
compromises ERK phosphorylation. The introduction of the point
mutations K12V, N95V reduced ERK phosphorylation, while
incorporating the additional mutations L46V, E87V and P134V totally
abrogates ERK signaling.
[0428] As shown in FIG. 10, deletion of 9 amino acids from the
N-terminus of FGF1 (NT1, FGF1.sup..DELTA.NT) induces an .about.100
fold reduction in FGFR signaling, as seen in the reduced
phosphorylation of downstream ERK and AKT pathways.
Example 5
Effect on Blood Glucose with FGF1 Mutants
[0429] Peptides M1, M2, M3 (see SEQ ID NOS: 22, 28, and 40,
respectively), FGF1 (SEQ ID NO: 5), NT1 (SEQ ID NO: 7) and NT2 (SEQ
ID NO: 8) were generated as described in Example 4. Peptides (0.5
mg/kg) or PBS (control) were injected SQ into 5 mo old C57BL/6J
ob/ob mice fed normal chow. Blood glucose levels were subsequently
determined.
[0430] As shown in FIGS. 11A and 11B, peptides M1 and M2 lowered
glucose as well as wild-type FGF1. Thus, FGF1 analogs can be
designed with increased thermostability, and improved
pharmacokinetic properties, while still having desired effects on
lowing blood glucose. Thus, the FGF1 portion of the FGF2/FGF1
chimeras provided herein can include these mutations (e.g., one or
more of K12V, C117V, P134V, L44F, C83T, and F132W).
[0431] As shown in FIG. 12 peptide FGF1.sup..DELTA.NT (NT1)
significantly lowered glucose, while FGF1.sup..DELTA.NT2 (NT2) lost
its ability to significantly lowered glucose. Thus, FGF1 can be
N-terminally truncated (such as the first 9 amino acids, but not
more than 13 amino acids), while still having desired effects on
lowing blood glucose. Thus, the FGF1 portion of the FGF2/FGF1
chimeras provided herein can include such a truncation.
Example 6
Glucose Lowering Correlates with FGFR Signaling
[0432] Peptides FGF1 (SEQ ID NO: 5), FGF1.sup..DELTA.NT (SEQ ID NO:
7) and NT2 (SEQ ID NO: 8) were generated as described in Example 4.
Peptides (10 ng/ml) were incubated with serum-starved HEK293 cells
for 15 minutes. Total cell lysates were subject to Western blotting
with antibodies specific for pAkt, Akt, pERK and ERK.
[0433] As shown in FIG. 13, comparable activation of the downstream
signaling effectors ERK and AKT is seen with FGF1 and two
independent preparations of FGF1.sup..DELTA.NT that lacks the
N-terminal 9 amino acids (SEQ ID NO: 7). In contrast, the deletion
of an additional 4 N-terminal amino acids markedly reduces both ERK
and AKT phosphorylation. These in vitro FGFR-mediated signaling
results correlate with the in vivo glucose lowering effect observed
in FIG. 12, supporting the hypothesis that the glucose-lowering
activity is mediated through an FGF receptor.
Example 7
Effect on Blood Glucose with FGF1 Mutants
[0434] Peptides FGF1-KLE (SEQ ID NO: 11) or FGF1-KN (SEQ ID NO: 10)
were generated as described in Example 1. Peptides (0.5 mg/kg) were
injected SQ into 5 mo old C57BL/6J ob/ob mice fed normal chow.
Blood glucose levels were subsequently determined 0 to 120 hours
later.
[0435] As shown in FIG. 14, the FGF1-KN mutant retained the ability
to lower glucose for 120 hrs despite a marked reduction in its
mitogenic activity. In contrast, the mitogenically dead FGF1-KLE
failed to lower glucose. These results indicate that the
mitogenicity and glucose-lowering activity can be independently
affected through targeted mutations. Thus, the FGF1 portion of the
FGF2/FGF1 chimeras provided herein can include the mutations in the
KN mutant (e.g., one or more of K12V and N95V) to reduce its
mitogenicity without significantly compromising its ability to
lower blood glucose levels.
Example 8
Dose-Response Effects on Blood Glucose with FGF1 Mutants
[0436] Peptides rFGF1.sup..DELTA.NT (SEQ ID NO: 7) and rFGF1 (SEQ
ID NO: 5) were generated as described in Example 4 (generated with
an N-terminal methionine and purified with heparin affinity and ion
exchange chromatography). Peptides (0.016 to 10 ng/ml) or PBS were
incubated with serum-starved HEK293 cells for 15 minutes. Total
cell lysates were subject to Western blotting with antibodies
specific for pFRS2.alpha., pAkt, Akt, pERK and ERK. Peptides (0.5
mg/kg) were injected SQ into high fat diet (HFD) fed diet-induced
obesity (DIO) mice or into 12 week old C57BL/6J ob/ob mice (0 to
0.5 mg/kg) fed normal chow. Blood glucose levels were subsequently
determined.
[0437] As shown in FIG. 15A, deletion of 9 N-terminal amino acids
of FGF1 significantly reduces FGFR downstream signaling, including
phosphorylation of ERK and AKT. Dose dependent phosphorylation of
the FGFR substrate FRS2a, confirms that both FGF1 and
FGF1.sup..DELTA.NT are capable of activating FGF receptors.
[0438] As shown in FIG. 15B, food intake in DIO mice after
receiving, rFGF1 or rFGF1.sup..DELTA.NT was significantly reduced,
as compared to mice that received PBS alone. The similarity in the
extent of the transient reduction in food intake between rFGF1 and
rFGF1.sup..DELTA.NT further supports the conclusion that both
proteins achieve their in vivo glucose lowering effects by
signaling through an FGF receptor.
[0439] As shown in FIG. 15C, an essentially identical dose-response
curve was observed for the glucose lowering effects of rFGF1 and
rFGF1.sup..DELTA.NT (NT1) in ob/ob mice. Given the significant
reduction in mitogenicity of rFGF1.sup..DELTA.NT, these results
demonstrate that the glucose lowering and mitogenic activities of
FGF1 can be dissociated.
Example 9
Effect of N-Terminal FGF1 Truncations on Blood Glucose Levels
[0440] Peptides NT1 (SEQ ID NO: 7), NT2 (SEQ ID NO: 8), and NT3
(SEQ ID NO: 9) were generated as described in Example 4. Peptides
(0.5 mg/kg) were injected SQ into 5 mo old C57BL/6J ob/ob mice fed
normal chow, or peptides (0 to 0.5 mg/kg) were injected SQ into 12
week old ob/ob mice fed normal chow. Blood glucose levels were
subsequently determined (0 hr, 16 hrs, or 24 hrs).
[0441] As shown in FIG. 16 if the N-terminus is truncated at 14
amino acids, glucose lowering ability is dramatically decreased
(NT2). Thus, FGF1 can be N-terminally truncated (such as the first
9, 10, or 11 amino acids), while maintaining the desired effects on
lowering blood glucose. Thus, the FGF1 mutants provided herein can
include such a truncation.
[0442] In another experiment, NT1 (SEQ ID NO: 7) (0.5 mg/kg) was
injected SQ into 8 month old HFD-fed wildtype (FGFR1 f/f, open
bars) or adipose-specific FGFR1 knockout (R1 KO, aP2-Cre; FGFR1
f/f, filled bars) mice Blood glucose levels were subsequently
determined (0 hr, 12 hrs, or 24 hrs).
[0443] As shown in FIGS. 17A and 17B, rFGF1.sup..DELTA.NT (NT1)
(SEQ ID NO: 7) lowers blood glucose levels in HFD-fed wildtype mice
(control) but has no effect on FGFR1 KO (mutant) mice. FIG. 17A
reports the changes in blood glucose, while FIG. 17B reports the
data normalized to starting glucose levels at 100%. These results
demonstrate that expression of FGFR1 in adipose tissue is required
for rFGF1.sup..DELTA.NT mediated glucose lowering.
[0444] As shown in FIGS. 18A and 18B mouse rFGF1 (amino acids 1-15
of SEQ ID NO: 4) lowers blood glucose levels in HFD-fed wildtype
mice (FGFR1 f/f mice, filled bars) but has no effect on aP2-Cre;
FGFR1 f/f (FGFR1 KO, speckled bars) mice. FIG. 17A reports the
changes in blood glucose, while FIG. 17B reports the data
normalized to starting glucose levels at 100%. These results
demonstrate that expression of FGFR1 in adipose tissue is required
for rFGF1 mediated glucose lowering.
Example 10
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0445] Peptides K118E (SEQ ID NO: 13), K118N (SEQ ID NO: 12), FGF1
(SEQ ID NO: 5), and KKK (SEQ ID NO: 114) were generated as
described in Example 1, while FGF1.sup..DELTA.NT (NT1) (SEQ ID NO:
7) was expressed with an N-terminal methionine and purified using
heparin affinity and ion exchange chromatography. Peptides (0.5
mg/kg) or PBS were injected SQ into 7 months HFD-fed C57BL/6J mice.
Blood glucose levels were subsequently determined (0 to 120 hours).
These mice are diet-induced obese (DIO) mice.
[0446] As shown in FIG. 19, mutation of the single lysine, K118, to
either Asn (K118N (SEQ ID NO: 12)) or Glu (K118E (SEQ ID NO: 13)),
is sufficient to abrogate glucose lowering activity in DIO
mice.
[0447] As shown in FIGS. 21 and 22, mutating selected amino acids
implicated in the heparin binding site of FGF1, namely amino acids
K112, K113, and K118, resulted in a mutated FGF1 sequence that
could lower blood glucose levels in ob/ob mice. Thus, the FGF1
mutants provided herein can include mutations at all three of K112,
K113, and K118, such as a K112D, K113Q, and K118V substitution.
However, while the mutation of K118 to the hydrophobic residue
valine was tolerated, mutations involving a charge reversal (K118E)
or to a polar residue (K118N) are not tolerated.
Example 11
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0448] Peptides KN (SEQ ID NO: 10), KKK (Salk_010, SEQ ID NO: 226),
FGF1 (SEQ ID NO: 5), and KLE (Salk_011, SEQ ID NO: 11) were
generated as described in Example 1, while FGF1.sup..DELTA.NT (NT1)
(SEQ ID NO: 7) and FGF1.sup..DELTA.NTKN (Salk_009, SEQ ID NO: 225)
were expressed with an N-terminal methionine and purified using
heparin affinity and ion exchange chromatographies. Peptides (0.5
mg/kg) or PBS were injected SQ into 7 months HFD-fed C57BL/6J mice.
Blood glucose levels were subsequently determined (0 to 120
hours).
[0449] As shown in FIGS. 32A and 32B, combining the mutations K12V
and N95V with the deletion of the N-terminal residues resulted in a
mutated FGF1 sequence (SEQ ID NO: 225) that could lower blood
glucose levels in ob/ob mice. The combined mutations of K12V N95V
with the stabilizing mutations Q40P S47I H93G (SEQ ID NO: 11) also
lowered blood glucose levels in ob/ob mice. Transient reductions in
food intake were observed with selected FGF1 analogs (FIGS. 32C and
32D). Furthermore, a single injection of SEQ ID NO: 225 was able to
sustain the low glucose levels for more than 7 days.
Example 12
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0450] Peptides FGF1 (SEQ ID NO: 5), and Salk_013 (SEQ ID NO: 31)
were generated as described in Example 1, while Salk_012 (SEQ ID
NO: 79) was expressed with an N-terminal methionine and purified
using heparin affinity and ion exchange chromatographies. Peptides
(0.5 mg/kg) or PBS were injected SQ into 7 months HFD-fed C57BL/6J
mice. Blood glucose levels were subsequently determined (0 to 96
hours).
[0451] As shown in FIG. 33A, combining the mutations K12V N95V with
the stabilizing mutations Q40P S47I H93G and the deletion of the
N-terminal residues resulted in a mutated FGF1 sequence (Salk_012)
that could lower blood glucose levels in ob/ob mice. The combined
mutations of K12V N95V with the stabilizing mutations L44F, C83T,
C117V, and F132W (Salk_013) also lowered blood glucose levels in
ob/ob mice. Salk_012 and Salk_013 has minimal effects on food
intake (FIG. 33B).
Example 13
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0452] Peptides Salk_014 (SEQ ID NO: 230), Salk_024 (SEQ ID NO:
84), Salk_025 (SEQ ID NO: 208), and Salk_026 (SEQ ID NO: 209), were
generated as described in Example 1, while Salk_023 (SEQ ID NO: 38)
was expressed with an N-terminal methionine and purified using
heparin affinity and ion exchange chromatographies. Peptides (0.5
mg/kg) or PBS were injected SQ into 7 months HFD-fed C57BL/6J mice.
Blood glucose levels were subsequently determined (0 to 72
hours).
[0453] As shown in FIG. 34A, FGF1 with the stabilizing point
mutation C117V alone, or in combination with additional mutations
reduces blood glucose in ob/ob mice for more than 72 hours. Various
transient effects on food intake in the 24 hours after injection
were observed (FIG. 34B).
Example 14
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0454] Peptides Salk_014 (SEQ ID NO: 230), Salk_022 (SEQ ID NO:
119), and Salk_027 (SEQ ID NO: 207) were generated as described in
Example 1. Peptides (0.5 mg/kg) or PBS were injected SQ into 7
months HFD-fed C57BL/6J mice. Blood glucose levels were
subsequently determined (0 to 24 hours).
[0455] As shown in FIG. 35A, FGF1 with the stabilizing point
mutation C117V alone, or in combination with additional mutations
that affect the ability to bind to heparan sulfate proteoglycans
(HSPG), reduce blood glucose in ob/ob mice. The transient
suppression of food intake is lost in analogs incorporating
mutations that affect HSPG binding (FIG. 35B).
Example 15
Effect of FGF1 Point Mutations on Blood Glucose Lowering
[0456] Peptides Salk_014 (SEQ ID NO: 230), was generated as
described in Example 1, while Salk_032 (SEQ ID NO: 215) was
expressed with an N-terminal methionine and purified using heparin
affinity and ion exchange chromatographies. Peptides (0.5 mg/kg) or
PBS were injected SQ into 7 months HFD-fed C57BL/6J mice. Blood
glucose levels were subsequently determined (0 to 24 hours).
[0457] As shown in FIG. 36A, the K12V N95V mutations, the
stabilizing cysteine mutations C12T, C83S C117V, and the deletion
of the N-terminal residues can be combined to generate an FGF1
analog that is able to robustly reduce blood glucose levels with
only minor effects on feeding (FIG. 36B), indicating that these
effects can be differentiated.
Example 16
Effect of FGF1-FGF19 Chimeric Proteins on Blood Glucose
Lowering
[0458] Peptides Salk_014 (SEQ ID NO: 230), and Salk_019 (SEQ ID NO:
224) were generated as described in Example 1. Peptides (Salk_014;
0.5 mg/kg, Salk_019; indicated doses) or PBS were injected SQ into
7 months HFD-fed C57BL/6J mice. Blood glucose levels were
subsequently determined (0 to 24 hours).
[0459] As shown in FIGS. 37A and 37B, the chimeric protein (SEQ ID
NO: 224) generated by fusing the .beta.-klotho binding domain of
FGF19 (SEQ ID NO: 100) to the C-terminus of FGF1 failed to affect
blood glucose or to suppress food intake, even at a 5 fold higher
concentration (2.5 mg/kg compared to 0.5 mg/kg). Given that a
chimeric protein composed of FGF1 and the .beta.-klotho binding
region of FGF21 was active in lowering blood glucose, these results
indicate that this analog is being targeted to FGFR4-.beta.-klotho
receptor complex that does not affect blood glucose levels.
Example 17
Effect of FGF1-FGF21 Chimeric Proteins on Blood Glucose
Lowering
[0460] Peptides FGF1 (SEQ ID NO: 5), FGF1.sup..DELTA.NT (SEQ ID NO:
7), FGF21 (SEQ ID 20) and FGF1-FGF21 chimera (wherein the FGF1
portion includes K112D, K113Q, and K118V mutations, thus the
chimera is SEQ ID NO: 114+SEQ ID NO: 86) were generated as
described in Example 1. Peptides (0.5 mg/kg,) or PBS were injected
SQ into 7 months HFD-fed C57BL/6J mice. Blood glucose levels were
subsequently determined (0 to 48 hours).
[0461] As shown in FIG. 38, wildtype FGF21 weakly lowers glucose
compared to wildtype FGF1, and FGF1.sup..DELTA.NT. In contrast, a
chimeric protein constructed from a mutant FGF1 fused to the
.beta.-klotho binding region of FGF21 (FGF1-FGF21.sup.C-tail) was
effective at lowering blood glucose.
[0462] In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples of
the disclosure and should not be taken as limiting the scope of the
invention. Rather, the scope of the disclosure is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
2381468DNAHomo sapiens 1atggctgaag gggaaatcac caccttcaca gccctgaccg
agaagtttaa tctgcctcca 60gggaattaca agaagcccaa actcctctac tgtagcaacg
ggggccactt cctgaggatc 120cttccggatg gcacagtgga tgggacaagg
gacaggagcg accagcacat tcagctgcag 180ctcagtgcgg aaagcgtggg
ggaggtgtat ataaagagta ccgagactgg ccagtacttg 240gccatggaca
ccgacgggct tttatacggc tcacagacac caaatgagga atgtttgttc
300ctggaaaggc tggaggagaa ccattacaac acctatatat ccaagaagca
tgcagagaag 360aattggtttg ttggcctcaa gaagaatggg agctgcaaac
gcggtcctcg gactcactat 420ggccagaaag caatcttgtt tctccccctg
ccagtctctt ctgattaa 4682155PRTHomo sapiens 2Met Ala Glu Gly Glu Ile
Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro Pro
Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly
Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50
55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr
Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr
Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn
His Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn
Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser Asp 145 150 155 3468DNAMus musculus 3atggctgaag
gggagatcac aaccttcgca gccctgaccg agaggttcaa cctgcctcta 60ggaaactaca
aaaagcccaa actgctctac tgcagcaacg ggggccactt cttgaggatc
120cttcctgatg gcaccgtgga tgggacaagg gacaggagcg accagcacat
tcagctgcag 180ctcagtgcgg aaagtgcggg cgaagtgtat ataaagggta
cggagaccgg ccagtacttg 240gccatggaca ccgaagggct tttatacggc
tcgcagacac caaatgagga atgtctgttc 300ctggaaaggc tggaagaaaa
ccattataac acttacacct ccaagaagca tgcggagaag 360aactggtttg
tgggcctcaa gaagaacggg agctgtaagc gcggtcctcg gactcactat
420ggccagaaag ccatcttgtt tctgcccctc ccggtgtctt ctgactag
4684155PRTMus musculus 4Met Ala Glu Gly Glu Ile Thr Thr Phe Ala Ala
Leu Thr Glu Arg Phe 1 5 10 15 Asn Leu Pro Leu Gly Asn Tyr Lys Lys
Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg
Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser
Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser Ala Gly
Glu Val Tyr Ile Lys Gly Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala
Met Asp Thr Glu Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90
95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr
100 105 110 Thr Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu
Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr
Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser
Asp 145 150 155 5140PRTHomo sapiens 5Phe Asn Leu Pro Pro Gly Asn
Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His
Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg
Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55
60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn
65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 6137PRTArtificial sequenceSynthetic
polypeptide 6Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys
Ser Asn Gly 1 5 10 15 Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr
Val Asp Gly Thr Arg 20 25 30 Asp Arg Ser Asp Gln His Ile Gln Leu
Gln Leu Ser Ala Glu Ser Val 35 40 45 Gly Glu Val Tyr Ile Lys Ser
Thr Glu Thr Gly Gln Tyr Leu Ala Met 50 55 60 Asp Thr Asp Gly Leu
Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys 65 70 75 80 Leu Phe Leu
Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser 85 90 95 Lys
Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly 100 105
110 Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
115 120 125 Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
7131PRTArtificial sequenceSynthetic polypeptide 7Lys Pro Lys Leu
Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro
Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30
Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35
40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu
Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu
Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys
Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn
Gly Ser Cys Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys
Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
8127PRTArtificial sequenceSynthetic polypeptide 8Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 1 5 10 15 Thr Val
Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 20 25 30
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 35
40 45 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln 50 55 60 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His 65 70 75 80 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val 85 90 95 Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro Arg Thr His Tyr 100 105 110 Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 115 120 125 9129PRTArtificial
sequenceSynthetic polypeptide 9Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 10140PRTArtificial
sequenceSynthetic polypeptide 10Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 11140PRTArtificial sequenceSynthetic
polypeptide 11Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Val Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Val Arg Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Val Leu Pro Val Ser Ser Asp 130 135
140 12134PRTArtificial sequenceSynthetic polypeptide 12Asn Tyr Lys
Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe 1 5 10 15 Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser 20 25
30 Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val
35 40 45 Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp
Thr Asp 50 55 60 Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu
Cys Leu Phe Leu 65 70 75 80 Glu Arg Leu Glu Glu Asn His Tyr Asn Thr
Tyr Ile Ser Lys Lys His 85 90 95 Ala Glu Lys Asn Trp Phe Val Gly
Leu Lys Lys Asn Gly Ser Cys Asn 100 105 110 Arg Gly Pro Arg Thr His
Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu Pro Val Ser
Ser Asp 130 13134PRTArtificial sequenceSynthetic polypeptide 13Asn
Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe 1 5 10
15 Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
20 25 30 Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly
Glu Val 35 40 45 Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp 50 55 60 Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn
Glu Glu Cys Leu Phe Leu 65 70 75 80 Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr Tyr Ile Ser Lys Lys His 85 90 95 Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys Lys Asn Gly Ser Cys Glu 100 105 110 Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu Pro
Val Ser Ser Asp 130 148PRTArtificial sequenceSynthetic polypeptide
14Ala Ala Ala Leu Pro Leu Pro Val 1 5 158PRTArtificial
sequenceSynthetic polypeptide 15Ile Leu Ala Leu Pro Leu Pro Val 1 5
168PRTArtificial sequenceSynthetic polypeptide 16Ile Leu Phe Ala
Pro Leu Pro Val 1 5 178PRTArtificial sequenceSynthetic polypeptide
17Ile Leu Phe Leu Pro Ala Pro Ala 1 5 18414DNAArtificial
sequenceSynthetic polynucleotide 18ccgccgggta actacaaaaa accgaaactg
ctgtattgca gcaacggcgg tcattttctg 60cgtattctgc cggatggcac cgtcgacggt
acgcgtgatc gcagtgacca gcacattcag 120ctgcaactga gcgcggaatc
tgtgggtgaa gtttatatca aatcaaccga aacgggccag 180tacctggcca
tggataccga cggcctgctg tacggttcgc aaacgccgaa tgaagaatgc
240ctgtttctgg aacgtctgga agaaaaccat tacaacacct acatcagtaa
aaaacacgcg 300gagaaaaact ggttcgttgg cctgaagaaa aacggttcct
gtaaacgcgg cccgcgcacc 360cattacggtc aaaaagccat tctgtttctg
ccgctgccgg tttcgtccga ctaa 41419939DNAHomo sapiens 19ctgtcagctg
aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc 60acctggacaa
ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc
120ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg
gttcgagcac 180tcaggactgt gggtttctgt gctggctggt ctgctgggag
cctgccaggc acaccccatc 240cctgactcca gtcctctcct gcaattcggg
ggccaagtcc ggcagcggta cctctacaca 300gatgatgccc agcagacaga
agcccacctg gagatcaggg aggatgggac ggtggggggc 360gctgctgacc
agagccccga aagtctcctg cagctgaaag ccttgaagcc gggagttatt
420caaatcttgg gagtcaagac atccaggttc ctgtgccagc ggccagatgg
ggccctgtat 480ggatcgctcc actttgaccc tgaggcctgc agcttccggg
agctgcttct tgaggacgga 540tacaatgttt accagtccga agcccacggc
ctcccgctgc acctgccagg gaacaagtcc 600ccacaccggg accctgcacc
ccgaggacca gctcgcttcc tgccactacc aggcctgccc 660cccgcactcc
cggagccacc cggaatcctg gccccccagc cccccgatgt gggctcctcg
720gaccctctga gcatggtggg accttcccag ggccgaagcc ccagctacgc
ttcctgaagc 780cagaggctgt ttactatgac atctcctctt tatttattag
gttatttatc ttatttattt 840ttttattttt cttacttgag ataataaaga
gttccagagg agaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaag 93920208PRTHomo sapiens 20Met 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 Gly Ala Cys Gln Ala His Pro Ile Pro Asp 20 25 30 Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu 35 40
45 Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu
50 55 60 Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu 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 Leu 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 21135PRTArtificial sequenceSynthetic
polypeptide 21Gly Gly Gln Val Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His 1 5 10 15 Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg 20 25 30 Ser Asp Gln His Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu 35 40 45 Val Tyr Ile Lys Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr 50 55 60
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe 65
70 75 80 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys 85 90 95 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
Asn Gly Ser Cys 100 105 110 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu 115 120 125 Pro Leu Pro Val Ser Ser Asp 130
135 22140PRTArtificial sequenceSynthetic polypeptide 22Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Val Leu Pro Val Ser Ser Asp 130 135 140 23140PRTArtificial
sequenceSynthetic polypeptide 23Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Val Leu Pro Val
Ser Ser Asp 130 135 140 24131PRTArtificial sequenceSynthetic
polypeptide 24Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Val Leu Pro Val
115 120 125 Ser Ser Asp 130 25129PRTArtificial sequenceSynthetic
polypeptide 25Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn His Tyr
Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe
Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr 100 105
110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Val Leu Pro Val Ser Ser
115 120 125 Asp 26131PRTArtificial sequenceSynthetic polypeptide
26Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1
5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln
His 20 25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val
Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp
Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu
Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr
Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly
Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His
Tyr Gly Gln Lys Ala Ile Leu Phe Leu Val Leu Pro Val 115 120 125 Ser
Ser Asp 130 27129PRTArtificial sequenceSynthetic polypeptide 27Val
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10
15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln
20 25 30 Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Val Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys
Asn Gly Ser Val Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Val Leu Pro Val Ser Ser 115 120 125 Asp
28140PRTArtificial sequenceSynthetic polypeptide 28Phe Asn Leu Pro
Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala 35
40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln
Thr Pro Asn 65 70 75 80 Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys
Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg
Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Trp Leu
Pro Leu Pro Val Ser Ser Asp 130 135 140 29140PRTArtificial
sequenceSynthetic polypeptide 29Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Trp Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 30140PRTArtificial sequenceSynthetic
polypeptide 30Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Thr
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Trp Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 31140PRTArtificial sequenceSynthetic polypeptide 31Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Trp
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 32131PRTArtificial
sequenceSynthetic polypeptide 32Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 33129PRTArtificial
sequenceSynthetic polypeptide 33Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 34131PRTArtificial
sequenceSynthetic polypeptide 34Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 35129PRTArtificial
sequenceSynthetic polypeptide 35Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 36131PRTArtificial
sequenceSynthetic polypeptide 36Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 37129PRTArtificial
sequenceSynthetic polypeptide 37Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly
Pro Arg Thr 100
105 110 His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val Ser
Ser 115 120 125 Asp 38131PRTArtificial sequenceSynthetic
polypeptide 38Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 39129PRTArtificial sequenceSynthetic
polypeptide 39Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn
Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn His Tyr
Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe
Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr 100 105
110 His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val Ser Ser
115 120 125 Asp 40134PRTArtificial sequenceSynthetic polypeptide
40Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1
5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val
Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln
Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Ile Asp Thr Asp Gly Leu Val
Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys
His Gly Trp Phe Leu Gly Ile Lys Lys Asn Gly 100 105 110 Ser Val Lys
Gly Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu
Pro Val Ser Ser Asp 130 41134PRTArtificial sequenceSynthetic
polypeptide 41Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Ile Asp Thr
Asp Gly Leu Val Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Gly Trp Phe Leu Gly Ile Lys Lys Asn Gly 100 105
110 Ser Val Lys Gly Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
115 120 125 Leu Pro Val Ser Ser Asp 130 42134PRTArtificial
sequenceSynthetic polypeptide 42Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Gly Trp Phe Leu Gly Ile Lys
Lys Asn Gly 100 105 110 Ser Val Lys Gly Thr His Tyr Gly Gln Lys Ala
Ile Leu Phe Leu Pro 115 120 125 Leu Pro Val Ser Ser Asp 130
43134PRTArtificial sequenceSynthetic polypeptide 43Phe Asn Leu Pro
Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala 35
40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
Tyr 50 55 60 Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly Ser Gln
Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Gly Trp Phe
Leu Gly Ile Lys Lys Asn Gly 100 105 110 Ser Val Lys Gly Thr His Tyr
Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu Pro Val Ser Ser
Asp 130 44125PRTArtificial sequenceSynthetic polypeptide 44Lys Pro
Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp
Gly Leu Val 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile
Ser Lys Lys His Gly Trp Phe 85 90 95 Leu Gly Ile Lys Lys Asn Gly
Ser Val Lys Gly Thr His Tyr Gly Gln 100 105 110 Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 115 120 125 45123PRTArtificial
sequenceSynthetic polypeptide 45Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Gly Trp Phe Leu
Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys Gly Thr His Tyr Gly
Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
115 120 46125PRTArtificial sequenceSynthetic polypeptide 46Lys Pro
Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp
Gly Leu Val 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile
Ser Lys Lys His Gly Trp Phe 85 90 95 Leu Gly Ile Lys Lys Asn Gly
Ser Val Lys Gly Thr His Tyr Gly Gln 100 105 110 Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 115 120 125 47123PRTArtificial
sequenceSynthetic polypeptide 47Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Gly Trp Phe Leu
Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys Gly Thr His Tyr Gly
Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
115 120 48125PRTArtificial sequenceSynthetic polypeptide 48Lys Pro
Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp
Gly Leu Val 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Gly Trp Phe 85 90 95 Leu Gly Ile Lys Lys Asn Gly
Ser Val Lys Gly Thr His Tyr Gly Gln 100 105 110 Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 115 120 125 49123PRTArtificial
sequenceSynthetic polypeptide 49Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Gly Trp Phe Leu
Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys Gly Thr His Tyr Gly
Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
115 120 50125PRTArtificial sequenceSynthetic polypeptide 50Val Pro
Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp
Gly Leu Val 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Gly Trp Phe 85 90 95 Leu Gly Ile Lys Lys Asn Gly
Ser Val Lys Gly Thr His Tyr Gly Gln 100 105 110 Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 115 120 125 51123PRTArtificial
sequenceSynthetic polypeptide 51Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Phe Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Gly Trp Phe Leu
Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys Gly Thr His Tyr Gly
Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
115 120 52140PRTArtificial sequenceSynthetic polypeptide 52Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys
Asn Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 53140PRTArtificial
sequenceSynthetic polypeptide 53Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Glu Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 54140PRTArtificial sequenceSynthetic
polypeptide 54Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val Thr 85 90
95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys
100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly
Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
130 135 140 55140PRTArtificial sequenceSynthetic polypeptide 55Phe
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10
15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp
20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu
Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu
Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr
Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg
Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His
Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser
Val Asn Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile
Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140
56140PRTArtificial sequenceSynthetic polypeptide 56Phe Asn Leu Pro
Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35
40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln
Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys
Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Glu Arg
Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu
Pro Leu Pro Val Ser Ser Asp 130 135 140 57131PRTArtificial
sequenceSynthetic polypeptide 57Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 58129PRTArtificial
sequenceSynthetic polypeptide 58Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 59131PRTArtificial
sequenceSynthetic polypeptide 59Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 60129PRTArtificial
sequenceSynthetic polypeptide 60Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 61131PRTArtificial
sequenceSynthetic polypeptide 61Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 62129PRTArtificial
sequenceSynthetic polypeptide 62Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 63131PRTArtificial
sequenceSynthetic polypeptide 63Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Asn
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 64129PRTArtificial
sequenceSynthetic polypeptide 64Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Glu Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 65131PRTArtificial
sequenceSynthetic polypeptide 65Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Asn
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 66129PRTArtificial
sequenceSynthetic polypeptide 66Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Glu Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 67140PRTArtificial
sequenceSynthetic polypeptide 67Phe Asn Leu Pro Pro Gly Asn Tyr Thr
Thr Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 68140PRTArtificial sequenceSynthetic
polypeptide 68Phe Asn Leu Pro Pro Gly Asn Tyr Thr Thr Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 69140PRTArtificial sequenceSynthetic polypeptide 69Phe Asn Leu
Pro Pro Gly Asn Tyr Thr Thr Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Asn
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 70140PRTArtificial
sequenceSynthetic polypeptide 70Phe Asn Leu Pro Pro Gly Asp Gln Asp
Gln Asn Gln Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 71131PRTArtificial
sequenceSynthetic polypeptide 71Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Pro His 20 25 30 Ile Gln Leu Gln Leu
Ile Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 72129PRTArtificial
sequenceSynthetic polypeptide 72Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Pro His Ile Gln 20 25 30 Leu Gln Leu Ile Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 73140PRTArtificial
sequenceSynthetic polypeptide 73Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Pro His Ile Gln Leu Gln Leu Ile Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 74131PRTArtificial sequenceSynthetic
polypeptide 74Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Pro His 20 25 30 Ile Gln Leu Gln Leu Ile Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 75129PRTArtificial sequenceSynthetic
polypeptide 75Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
Asp Pro His Ile Gln 20 25 30 Leu Gln Leu Ile Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn His Tyr
Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe
Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr 100 105
110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser
115 120 125 Asp 76131PRTArtificial sequenceSynthetic polypeptide
76Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1
5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Pro
His 20 25 30 Ile Gln Leu Gln Leu Ile Ala Glu Ser Val Gly Glu Val
Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp
Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu
Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn Gly Tyr Asn Thr
Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly
Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 100 105 110 Arg Thr His
Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser
Ser Asp 130 77129PRTArtificial sequenceSynthetic polypeptide 77Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10
15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Pro His Ile Gln
20 25 30 Leu Gln Leu Ile Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn Gly Tyr Asn Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser 115 120 125 Asp
78140PRTArtificial sequenceSynthetic polypeptide 78Phe Asn Leu Pro
Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Pro His Ile Gln Leu Gln Leu Ile Ala 35
40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln
Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn Gly Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys
Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg
Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu
Pro Leu Pro Val Ser Ser Asp 130 135 140 79131PRTArtificial
sequenceSynthetic polypeptide 79Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Pro His 20 25 30 Ile Gln Leu Gln Leu
Ile Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn Gly Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 80129PRTArtificial
sequenceSynthetic polypeptide 80Val Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Pro His Ile Gln 20 25 30 Leu Gln Leu Ile Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn Gly Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 81131PRTArtificial
sequenceSynthetic polypeptide 81Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Pro Val
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130 82129PRTArtificial
sequenceSynthetic polypeptide 82Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Pro Val Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser 115 120 125 Asp 83140PRTArtificial
sequenceSynthetic polypeptide 83Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Pro Val Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 84140PRTArtificial sequenceSynthetic
polypeptide 84Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Glu Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 85145PRTArtificial sequenceSynthetic polypeptide 85Pro Ala Leu
Pro Glu Asp Gly Gly Ala Ala Phe Pro Pro Gly His Phe 1 5 10 15 Lys
Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg 20 25
30 Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro
35 40 45 His Val Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val
Ser Ile 50 55 60 Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys
Glu Asp Gly Arg 65 70 75 80 Leu Leu Ala Ser Lys Cys Val Thr Glu Glu
Cys Phe Phe Phe Glu Arg 85 90 95 Leu Glu Ser Asn Asn Tyr Asn Thr
Tyr Arg Ser Arg Lys Tyr Ser Ser 100 105 110 Trp Tyr Val Ala Leu Lys
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys 115 120 125 Thr Gly Pro Gly
Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys 130 135 140 Ser 145
8642PRTArtificial sequenceSynthetic polypeptide 86Pro Gly Leu Pro
Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro 1 5 10 15 Gln Pro
Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro 20 25 30
Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 35 40 87177PRTArtificial
sequenceSynthetic polypeptide 87Phe Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu
Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg
Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Gly
Leu Pro Pro Ala Leu
Pro Glu 130 135 140 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
Gly Ser Ser Asp 145 150 155 160 Pro Leu Ser Met Val Gly Pro Ser Gln
Gly Arg Ser Pro Ser Tyr Ala 165 170 175 Ser 88168PRTArtificial
sequenceSynthetic polypeptide 88Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro Leu Pro Gly 115 120 125 Leu Pro Pro Ala Leu Pro Glu Pro Pro
Gly Ile Leu Ala Pro Gln Pro 130 135 140 Pro Asp Val Gly Ser Ser Asp
Pro Leu Ser Met Val Gly Pro Ser Gln 145 150 155 160 Gly Arg Ser Pro
Ser Tyr Ala Ser 165 89166PRTArtificial sequenceSynthetic
polypeptide 89Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser Gln Thr Pro Asn
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70 75 80 Asn His Tyr
Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp 85 90 95 Phe
Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr 100 105
110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Gly Leu Pro
115 120 125 Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp 130 135 140 Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro
Ser Gln Gly Arg 145 150 155 160 Ser Pro Ser Tyr Ala Ser 165
90168PRTArtificial sequenceSynthetic polypeptide 90Lys Pro Val Leu
Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro
Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30
Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35
40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu
Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu
Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys
Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn
Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys
Ala Ile Leu Phe Leu Val Leu Pro Gly 115 120 125 Leu Pro Pro Ala Leu
Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 130 135 140 Pro Asp Val
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln 145 150 155 160
Gly Arg Ser Pro Ser Tyr Ala Ser 165 91166PRTArtificial
sequenceSynthetic polypeptide 91Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30 Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35 40 45 Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly 50 55 60 Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu 65 70
75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly
Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Val
Leu Pro Gly Leu Pro 115 120 125 Pro Ala Leu Pro Glu Pro Pro Gly Ile
Leu Ala Pro Gln Pro Pro Asp 130 135 140 Val Gly Ser Ser Asp Pro Leu
Ser Met Val Gly Pro Ser Gln Gly Arg 145 150 155 160 Ser Pro Ser Tyr
Ala Ser 165 92177PRTArtificial sequenceSynthetic polypeptide 92Phe
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10
15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp
20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu
Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu
Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr
Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg
Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His
Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser
Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile
Leu Phe Leu Val Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu 130 135 140
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 145
150 155 160 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser
Tyr Ala 165 170 175 Ser 93168PRTArtificial sequenceSynthetic
polypeptide 93Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Gly
115 120 125 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro
Gln Pro 130 135 140 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val
Gly Pro Ser Gln 145 150 155 160 Gly Arg Ser Pro Ser Tyr Ala Ser 165
94166PRTArtificial sequenceSynthetic polypeptide 94Lys Leu Leu Tyr
Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly
Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30
Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35
40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr
Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg
Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His
Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly Ser
Val Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala Ile
Leu Trp Leu Pro Leu Pro Gly Leu Pro 115 120 125 Pro Ala Leu Pro Glu
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 130 135 140 Val Gly Ser
Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg 145 150 155 160
Ser Pro Ser Tyr Ala Ser 165 95177PRTArtificial sequenceSynthetic
polypeptide 95Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Thr
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Trp Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu
Pro Glu 130 135 140 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
Gly Ser Ser Asp 145 150 155 160 Pro Leu Ser Met Val Gly Pro Ser Gln
Gly Arg Ser Pro Ser Tyr Ala 165 170 175 Ser 96162PRTArtificial
sequenceSynthetic polypeptide 96Lys Pro Lys Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu
Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val 50 55 60 Tyr
Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70
75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Gly Trp
Phe 85 90 95 Leu Gly Ile Lys Lys Asn Gly Ser Val Lys Gly Thr His
Tyr Gly Gln 100 105 110 Lys Ala Ile Leu Phe Leu Pro Leu Pro Gly Leu
Pro Pro Ala Leu Pro 115 120 125 Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp Val Gly Ser Ser 130 135 140 Asp Pro Leu Ser Met Val Gly
Pro Ser Gln Gly Arg Ser Pro Ser Tyr 145 150 155 160 Ala Ser
97160PRTArtificial sequenceSynthetic polypeptide 97Lys Leu Leu Tyr
Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp Gly
Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25 30
Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr 35
40 45 Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr
Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg
Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His
Gly Trp Phe Leu Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys Gly
Thr His Tyr Gly Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu Pro
Gly Leu Pro Pro Ala Leu Pro Glu Pro 115 120 125 Pro Gly Ile Leu Ala
Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro 130 135 140 Leu Ser Met
Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 145 150 155 160
98171PRTArtificial sequenceSynthetic polypeptide 98Phe Asn Leu Pro
Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala 35
40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
Tyr 50 55 60 Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly Ser Gln
Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Gly Trp Phe
Leu Gly Ile Lys Lys Asn Gly 100 105 110 Ser Val Lys Gly Thr His Tyr
Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu Pro Gly Leu Pro
Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala 130 135 140 Pro Gln Pro
Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly 145 150 155 160
Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser 165 170
99216PRTArtificial sequenceSynthetic polypeptide 99Met 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
10048PRTArtificial sequenceSynthetic polypeptide 100Leu Pro Met Val
Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu 1 5 10 15 Ser Asp
Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 20 25 30
Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 35
40 45 101183PRTArtificial sequenceSynthetic polypeptide 101Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys
Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly
Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe
Leu Glu Arg Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser
Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120
125 Ala Ile Leu Phe Leu Pro Leu Leu Pro Met Val Pro Glu Glu Pro Glu
130 135 140 Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu Glu 145 150 155 160 Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr
Gly Leu Glu Ala Val 165 170 175 Arg Ser Pro Ser Phe Glu Lys 180
102174PRTArtificial sequenceSynthetic polypeptide 102Lys Pro Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu
Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25
30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys
Asn Gly Ser Cys Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Pro Leu Leu Pro 115 120 125 Met Val Pro Glu
Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp 130 135 140 Met Phe
Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 145 150 155
160 Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
103172PRTArtificial sequenceSynthetic polypeptide 103Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly
Ser Cys Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala
Ile Leu Phe Leu Pro Leu Leu Pro Met Val 115 120 125 Pro Glu Glu Pro
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe 130 135 140 Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr 145 150 155
160 Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
104183PRTArtificial sequenceSynthetic polypeptide 104Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Val Leu Leu Pro Met Val Pro Glu Glu Pro Glu 130 135 140 Asp Leu
Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu 145 150 155
160 Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val
165 170 175 Arg Ser Pro Ser Phe Glu Lys 180 105174PRTArtificial
sequenceSynthetic polypeptide 105Lys Pro Val Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val
Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr
Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65
70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala
Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val
Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
Phe Leu Val Leu Leu Pro 115 120 125 Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser Asp 130 135 140 Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 145 150 155 160 Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
106172PRTArtificial sequenceSynthetic polypeptide 106Val Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly
Ser Val Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala
Ile Leu Phe Leu Val Leu Leu Pro Met Val 115 120 125 Pro Glu Glu Pro
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe 130 135 140 Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr 145 150 155
160 Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
107183PRTArtificial sequenceSynthetic polypeptide 107Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Thr Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Trp
Leu Pro Leu Leu Pro Met Val Pro Glu Glu Pro Glu 130 135 140 Asp Leu
Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu 145 150 155
160 Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val
165 170 175 Arg Ser Pro Ser Phe Glu Lys 180 108174PRTArtificial
sequenceSynthetic polypeptide 108Lys Pro Lys Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val
Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr
Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65
70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala
Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val
Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
Trp Leu Pro Leu Leu Pro 115 120 125 Met Val Pro Glu Glu Pro Glu Asp
Leu Arg Gly His Leu Glu Ser Asp 130 135 140 Met Phe Ser Ser Pro Leu
Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 145 150 155 160 Val Thr Gly
Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
109172PRTArtificial sequenceSynthetic polypeptide 109Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Lys Lys Asn Gly
Ser Val Lys Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala
Ile Leu Trp Leu Pro Leu Leu Pro Met Val 115 120 125 Pro Glu Glu Pro
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe 130 135 140 Ser Ser
Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr 145 150 155
160 Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 165 170
110177PRTArtificial sequenceSynthetic polypeptide 110Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Ile Asp Thr Asp Gly Leu Val Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Gly Trp
Phe Leu Gly Ile Lys Lys Asn Gly 100 105 110 Ser Val Lys Gly Thr His
Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro 115 120 125 Leu Leu Pro Met
Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu 130 135 140 Glu Ser
Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro 145 150 155
160 Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu
165 170 175 Lys 111168PRTArtificial sequenceSynthetic polypeptide
111Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile
1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp
Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu
Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Ile
Asp Thr Asp Gly Leu Val 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu
Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn
Thr Tyr Ile Ser Lys Lys His Gly Trp Phe 85 90 95 Leu Gly Ile Lys
Lys Asn Gly Ser Val Lys Gly Thr His Tyr Gly Gln 100 105 110 Lys Ala
Ile Leu Phe Leu Pro Leu Leu Pro Met Val Pro Glu Glu Pro 115 120 125
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu 130
135 140 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu
Ala 145 150 155 160 Val Arg Ser Pro Ser Phe Glu Lys 165
112166PRTArtificial sequenceSynthetic polypeptide 112Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Ile Asp Thr Asp Gly Leu Val
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Gly Trp Phe Leu Gly 85 90 95 Ile Lys Lys Asn Gly Ser Val Lys
Gly Thr His Tyr Gly Gln Lys Ala 100 105 110 Ile Leu Phe Leu Pro Leu
Leu Pro Met Val Pro Glu Glu Pro Glu Asp 115 120 125 Leu Arg Gly His
Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr 130 135 140 Asp Ser
Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg 145 150 155
160 Ser Pro Ser Phe Glu Lys 165 113140PRTArtificial
sequenceSynthetic polypeptide 113Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 114140PRTArtificial sequenceSynthetic
polypeptide 114Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val
Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln
Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly
Ser Val Val Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140
115131PRTArtificial sequenceSynthetic polypeptide 115Lys Pro Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu
Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25
30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Asp Gln
Asn Gly Ser Cys Val Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
116129PRTArtificial sequenceSynthetic polypeptide 116Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Asp Gln Asn Gly
Ser Cys Val Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser 115 120 125 Asp
117131PRTArtificial sequenceSynthetic polypeptide 117Lys Pro Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu
Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25
30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Asp Gln
Asn Gly Ser Val Val Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
118129PRTArtificial sequenceSynthetic polypeptide 118Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro 1 5 10 15 Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln 20 25
30 Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr
35 40 45 Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu
Tyr Gly 50 55 60 Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu
Arg Leu Glu Glu 65 70 75 80 Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys
His Ala Glu Lys Asn Trp 85 90 95 Phe Val Gly Leu Asp Gln Asn Gly
Ser Val Val Arg Gly Pro Arg Thr 100 105 110 His Tyr Gly Gln Lys Ala
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser 115 120 125 Asp
119140PRTArtificial sequenceSynthetic polypeptide 119Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys Val
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 120140PRTArtificial
sequenceSynthetic polypeptide 120Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 12190PRTArtificial sequenceSynthetic
polypeptide 121Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys
Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly
Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile Cys Glu Thr Pro Thr
Cys Gln Ser Asn Glu Phe Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile
Pro Gln His Trp Leu Cys Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp
Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu
Pro Pro Gly Ser Leu Ser Leu 85 90 12237PRTArtificial
sequenceSynthetic polypeptide 122Cys Gly Ala Asp Gln Phe Arg Cys
Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly
Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile
Cys 35 12337PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(33)..(33)Xaa = Pro or
SerMISC_FEATURE(34)..(34)Xaa = Pro or GlnMISC_FEATURE(35)..(35)Xaa
= Ala or GlnMISC_FEATURE(36)..(36)Xaa = His or no amino acid 123Cys
Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln 1 5 10
15 His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu
20 25 30 Xaa Xaa Xaa Xaa Cys 35 12438PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(2)..(2)Xaa = Glu, Leu,
Pro, Gln or ArgMISC_FEATURE(3)..(3)Xaa = Ala, Pro or
SerMISC_FEATURE(4)..(4)Xaa = Asp, Gly, Ile or
AsnMISC_FEATURE(5)..(5)Xaa = Glu, Gly or GlnMISC_FEATURE(6)..(6)Xaa
= Phe or GlnMISC_FEATURE(7)..(7)Xaa = Phe, Lys, Pro, Gln, Arg or
ThrMISC_FEATURE(8)..(8)Xaa = Glu or no amino
acidMISC_FEATURE(10)..(10)Xaa = Gly, Asn, Arg or
SerMISC_FEATURE(11)..(11)Xaa = Asn or SerMISC_FEATURE(13)..(13)Xaa
= His, Asn, Gln or ArgMISC_FEATURE(15)..(15)Xaa = Ile or
ValMISC_FEATURE(17)..(17)Xaa = Ala, Glu, Leu, Pro, Gln, Arg or
ValMISC_FEATURE(18)..(18)Xaa = Ala, His, Pro, Gln, Arg or
ThrMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Glu or
ValMISC_FEATURE(25)..(25)Xaa = Asp, Asn or
ProMISC_FEATURE(28)..(28)Xaa = Gly, Leu or
GlnMISC_FEATURE(30)..(30)Xaa = Asp, Gly, Asn or
SerMISC_FEATURE(34)..(34)Xaa = Ala, Glu, Lys or
ThrMISC_FEATURE(35)..(35)Xaa = Asp, Gly, Leu, Asn or
SerMISC_FEATURE(36)..(36)Xaa = Ala or no amino
acidMISC_FEATURE(37)..(37)Xaa = His or no amino acid 124Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Xaa Cys Xaa Pro 1 5 10 15 Xaa
Xaa Trp Xaa Cys Asp Gly Xaa Xaa Asp Cys Xaa Asp Xaa Ser Asp 20 25
30 Glu Xaa Xaa Xaa Xaa Cys 35 12538PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(2)..(2)Xaa = Ala, Gly or
ProMISC_FEATURE(3)..(3)Xaa = Ala, Pro or SerMISC_FEATURE(4)..(4)Xaa
= Asp, Gly or AsnMISC_FEATURE(5)..(5)Xaa = Glu or
GlnMISC_FEATURE(7)..(7)Xaa = Gln, Arg or ThrMISC_FEATURE(9)..(9)Xaa
= Gly, Asn, Arg or SerMISC_FEATURE(10)..(10)Xaa = Gly, Asn or
SerMISC_FEATURE(11)..(11)Xaa = Gly or ThrMISC_FEATURE(12)..(12)Xaa
= Gly, Lys, Gln or SerMISC_FEATURE(13)..(13)Xaa = Ile, Lys or no
amino acidMISC_FEATURE(15)..(15)Xaa = Ile, Leu or
ValMISC_FEATURE(17)..(17)Xaa = Leu, Gln, Arg or
ValMISC_FEATURE(18)..(18)Xaa = Ala, Glu, His or
ProMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Leu or
ValMISC_FEATURE(25)..(25)Xaa = Asp or AsnMISC_FEATURE(30)..(30)Xaa
= Gly or AsnMISC_FEATURE(34)..(34)Xaa = Ala, Glu, Lys, Pro or
SerMISC_FEATURE(35)..(35)Xaa = Gly, Leu, Pro or
SerMISC_FEATURE(36)..(36)Xaa = Ala, Glu, Val or no amino
acidMISC_FEATURE(37)..(37)Xaa = Ile, Thr or no amino acid 125Cys
Xaa Xaa Xaa Xaa Phe Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 1 5 10
15 Xaa Xaa Trp Xaa Cys Asp Gly Xaa Xaa Asp Cys Gly Asp Xaa Ser Asp
20 25 30 Glu Xaa Xaa Xaa Xaa Cys 35 12647PRTArtificial
sequenceSynthetic polypeptide 126Cys Gln Ser Asn Glu Phe Arg Cys
Arg Ser Gly Arg Cys Ile Pro Gln 1 5 10 15 His Trp Leu Cys Asp Gly
Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ser Gln Gln Cys
Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35 40 45
12736PRTArtificial sequenceSynthetic polypeptide 127Cys Gln Ser Asn
Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln 1 5 10 15 His Trp
Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30
Ser Gln Gln Cys 35 12835PRTArtificial sequenceSynthetic polypeptide
128Cys Arg Ala Gly Glu Phe Arg Cys Ser Asn Gly Arg Cys Val Pro Leu
1 5 10 15 Thr Trp Leu Cys Asp Gly Glu Asp Asp Cys Gln Asp Asn Ser
Asp Glu 20 25 30 Lys Asn Cys 35 12938PRTArtificial
sequenceSynthetic polypeptide 129Cys Pro Ser Asn Gln Phe Pro Cys
Arg Ser Thr Gly Ile Cys Ile Pro 1 5 10 15 Leu Ala Trp Val Cys Asp
Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp 20 25 30 Glu Ser Pro Ala
His Cys 35 13037PRTArtificial sequenceSynthetic polypeptide 130Cys
Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln 1 5 10
15 His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu
20 25 30 Pro Pro Ala His Cys 35 13137PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(9)..(9)Xaa = Ser or
GlyMISC_FEATURE(17)..(17)Xaa = His or ThrMISC_FEATURE(19)..(19)Xaa
= Val or LeuMISC_FEATURE(33)..(33)Xaa = Pro or
SerMISC_FEATURE(36)..(36)Xaa = His or Thr 131Cys Leu Pro Asp Glu
Phe Gln Cys Xaa Ser Gly Arg Cys Ile Pro Gln 1 5 10 15 Xaa Trp Xaa
Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Xaa
Pro Ala Xaa Cys 35 13238PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(2)..(2)Xaa = Ala, Gly, Pro or
ArgMISC_FEATURE(3)..(3)Xaa = Ala or ProMISC_FEATURE(4)..(4)Xaa =
Asp, Gly, Asn or SerMISC_FEATURE(5)..(5)Xaa = Glu or
GlnMISC_FEATURE(7)..(7)Xaa = Gln, Arg or ThrMISC_FEATURE(9)..(9)Xaa
= Gly, Ser, LysMISC_FEATURE(11)..(11)Xaa = Gly or
ThrMISC_FEATURE(12)..(12)Xaa = Gly, His or
ArgMISC_FEATURE(13)..(13)Xaa = Arg or no amino
acidMISC_FEATURE(15)..(15)Xaa = Ile, Leu or
ValMISC_FEATURE(16)..(16)Xaa = Pro or SerMISC_FEATURE(17)..(17)Xaa
= Ala, Leu or GlnMISC_FEATURE(18)..(18)Xaa = Asn, Ser or
ThrMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Glu or
ValMISC_FEATURE(28)..(28)Xaa = Gly or LeuMISC_FEATURE(34)..(34)Xaa
= Ala, Glu or ThrMISC_FEATURE(35)..(35)Xaa = Asp, Asn or
SerMISC_FEATURE(36)..(36)Xaa = Ala or no amino
acidMISC_FEATURE(37)..(37)Xaa = Thr or no amino acid 132Cys Xaa Xaa
Xaa Xaa Phe Xaa Cys Xaa Asn Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa
Xaa Trp Xaa Cys Asp Gly Xaa Asp Asp Cys Xaa Asp Gly Ser Asp 20 25
30 Glu Xaa Xaa Xaa Xaa Cys 35 13337PRTArtificial sequenceSynthetic
polypeptide 133Cys Gly Pro Asp Gln Phe Arg Cys Ser Ser Gly Lys Cys
Ile Pro Gln 1 5 10 15 His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly
Asp Gly Ser Asp Glu 20 25 30 Ser Pro Ala Thr Cys 35
13437PRTArtificial sequenceSynthetic polypeptide 134Cys Gly Ala Asn
Glu Phe Gln Cys Arg Ser Thr Gly Ile Cys Val Pro 1 5 10 15 Val Glu
Trp Val Cys Asp Gly Asp Asn Asp Cys Gly Asp Gly Ser Asp 20 25 30
Glu Pro Pro Val Cys 35 13537PRTArtificial sequenceSynthetic
polypeptide 135Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys
Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly
Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile Cys 35
13637PRTArtificial sequenceSynthetic polypeptide 136Cys Gly Pro Asp
Glu Phe Arg Cys Asn Asn Gly Gln Cys Ile Pro Leu 1 5 10 15 Pro Trp
Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Asn Ser Asp Glu 20 25 30
Pro Leu Glu Ile Cys 35 13735PRTArtificial sequenceSynthetic
polypeptide 137Cys Ala Ser Gly Glu Phe Thr Cys Asn Asn Gly Gln Cys
Val Pro Leu 1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asn Asp Cys Gln
Asp Gly Ser Asp Glu 20 25 30 Lys Gly Cys 35 13836PRTArtificial
sequenceSynthetic polypeptide 138Cys Pro Pro Asp Glu Phe Gln Cys
Arg Gly Thr Lys Lys Cys Leu Pro 1 5 10 15 Leu Ala Trp Val Cys Asp
Gly Asp Asn Asp Cys Glu Asp Asp Ser Asp 20 25 30 Glu Glu Ser Cys 35
13937PRTArtificial sequenceSynthetic polypeptide 139Cys Leu Pro Asp
Glu Phe Gln Cys Gly Ser Gly Arg Cys Ile Pro Gln 1 5 10 15 His Trp
Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu
20 25 30 Pro Pro Ala His Cys 35 14036PRTArtificial
sequenceSynthetic polypeptide 140Cys Ala Pro Gly Glu Phe Thr Cys
Lys Asn Thr Gly Arg Cys Ile Pro 1 5 10 15 Leu Asn Trp Arg Cys Asp
Gly Asp Asp Asp Cys Gly Asp Gly Ser Asp 20 25 30 Glu Thr Asp Cys 35
14129PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(2)..(2)Xaa = Ala, Gly or
ProMISC_FEATURE(3)..(3)Xaa = Ala, Pro or SerMISC_FEATURE(4)..(4)Xaa
= Asp, Gly or AsnMISC_FEATURE(5)..(5)Xaa = Glu or
GlnMISC_FEATURE(7)..(7)Xaa = Gln, Arg or ThrMISC_FEATURE(9)..(9)Xaa
= Gly, Asn, Arg or SerMISC_FEATURE(10)..(10)Xaa = Gly, Asn or
SerMISC_FEATURE(11)..(11)Xaa = Gly or ThrMISC_FEATURE(12)..(12)Xaa
= Gly, Lys, Gln or SerMISC_FEATURE(13)..(13)Xaa = Ile, Lys or no
amino acidMISC_FEATURE(15)..(15)Xaa = Ile, Leu or
ValMISC_FEATURE(17)..(17)Xaa = Leu, Gln, Arg or
ValMISC_FEATURE(18)..(18)Xaa = Ala, Glu, His or
ProMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Leu or
ValMISC_FEATURE(25)..(25)Xaa = Asp or Asn 141Cys Xaa Xaa Xaa Xaa
Phe Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 1 5 10 15 Xaa Xaa Trp
Xaa Cys Asp Gly Xaa Xaa Asp Cys Gly Asp 20 25 14248PRTArtificial
sequenceSynthetic polypeptide 142Cys Gly Ala Asp Gln Phe Arg Cys
Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly
Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile
Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg 35 40 45
14340PRTArtificial sequenceSynthetic polypeptide 143Cys Arg Ser Gly
Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu 1 5 10 15 Asn Asp
Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro 20 25 30
Ala Ser Glu Pro Pro Gly Ser Leu 35 40 14438PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(2)..(2)Xaa = Ala, Gly,
Pro or ArgMISC_FEATURE(3)..(3)Xaa = Ala or
ProMISC_FEATURE(4)..(4)Xaa = Asp, Gly, Asn or
SerMISC_FEATURE(5)..(5)Xaa = Glu or GlnMISC_FEATURE(7)..(7)Xaa =
Gln, Arg or ThrMISC_FEATURE(9)..(9)Xaa = Gly, Ser or
LysMISC_FEATURE(11)..(11)Xaa = Gly or ThrMISC_FEATURE(12)..(12)Xaa
= Gly, His or ArgMISC_FEATURE(13)..(13)Xaa = Arg or no amino
acidMISC_FEATURE(15)..(15)Xaa = Ile, Leu or
ValMISC_FEATURE(16)..(16)Xaa = Pro or SerMISC_FEATURE(17)..(17)Xaa
= Ala, Leu or GlnMISC_FEATURE(18)..(18)Xaa = Asn, Ser or
ThrMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Glu or
ValMISC_FEATURE(28)..(28)Xaa = Gly or LeuMISC_FEATURE(34)..(34)Xaa
= Ala, Glu or ThrMISC_FEATURE(35)..(35)Xaa = Asp, Asn or
SerMISC_FEATURE(36)..(36)Xaa = Ala or no amino
acidMISC_FEATURE(37)..(37)Xaa = Thr or no amino acid 144Cys Xaa Xaa
Xaa Xaa Phe Xaa Cys Xaa Asn Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa
Xaa Trp Xaa Cys Asp Gly Xaa Asp Asp Cys Xaa Asp Gly Ser Asp 20 25
30 Glu Xaa Xaa Xaa Xaa Cys 35 14538PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(2)..(2)Xaa = Glu, Leu, Pro, Gln or
ArgMISC_FEATURE(3)..(3)Xaa = Ala, Pro or SerMISC_FEATURE(4)..(4)Xaa
= Asp, Gly, Ile or AsnMISC_FEATURE(5)..(5)Xaa = Glu, Gly or
GlnMISC_FEATURE(6)..(6)Xaa = Phe or GlnMISC_FEATURE(7)..(7)Xaa =
Phe, Lys, Pro, Gln, Arg or ThrMISC_FEATURE(8)..(8)Xaa = Glu or no
amino acidMISC_FEATURE(10)..(10)Xaa = Gly, Asn, Arg or
SerMISC_FEATURE(11)..(11)Xaa = Asn or SerMISC_FEATURE(13)..(13)Xaa
= His, Asn, Gln or ArgMISC_FEATURE(15)..(15)Xaa = Ile or
ValMISC_FEATURE(17)..(17)Xaa = Ala, Glu, Leu, Pro, Gln, Arg or
ValMISC_FEATURE(18)..(18)Xaa = Ala, His, Pro, Gln, Arg or
ThrMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Glu or
ValMISC_FEATURE(25)..(25)Xaa = Asp, Asn or
ProMISC_FEATURE(28)..(28)Xaa = Gly, Leu or
GlnMISC_FEATURE(30)..(30)Xaa = Asp, Gly, Asn or
SerMISC_FEATURE(34)..(34)Xaa = Ala, Glu, Lys or
ThrMISC_FEATURE(35)..(35)Xaa = Asp, Gly, Leu, Asn or
SerMISC_FEATURE(36)..(36)Xaa = Ala or no amino
acidMISC_FEATURE(37)..(37)Xaa = His or no amino acid 145Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Xaa Cys Xaa Pro 1 5 10 15 Xaa
Xaa Trp Xaa Cys Asp Gly Xaa Xaa Asp Cys Xaa Asp Xaa Ser Asp 20 25
30 Glu Xaa Xaa Xaa Xaa Cys 35 14638PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(2)..(2)Xaa = Ala, Gly or
ProMISC_FEATURE(3)..(3)Xaa = Ala, Pro or SerMISC_FEATURE(4)..(4)Xaa
= Asp, Gly or AsnMISC_FEATURE(5)..(5)Xaa = Glu or
GlnMISC_FEATURE(7)..(7)Xaa = Gln, Arg or ThrMISC_FEATURE(9)..(9)Xaa
= Gly, Asn, Arg or SerMISC_FEATURE(10)..(10)Xaa = Gly, Asn or
SerMISC_FEATURE(11)..(11)Xaa = Gly or ThrMISC_FEATURE(12)..(12)Xaa
= Gly, Lys, Gln or SerMISC_FEATURE(13)..(13)Xaa = Ile, Lys or no
amino acidMISC_FEATURE(15)..(15)Xaa = Ile, Leu or
ValMISC_FEATURE(17)..(17)Xaa = Leu, Gln, Arg or
ValMISC_FEATURE(18)..(18)Xaa = Ala, Glu, His or
ProMISC_FEATURE(20)..(20)Xaa = Leu, Arg or
ValMISC_FEATURE(24)..(24)Xaa = Asp, Leu or
ValMISC_FEATURE(25)..(25)Xaa = Asp or AsnMISC_FEATURE(30)..(30)Xaa
= Gly or AsnMISC_FEATURE(34)..(34)Xaa = Ala, Glu, Lys, Pro or
SerMISC_FEATURE(35)..(35)Xaa = Gly, Leu, Pro or
SerMISC_FEATURE(36)..(36)Xaa = Ala, Glu, Val or no amino
acidMISC_FEATURE(37)..(37)Xaa = Ile, Thr or no amino acid 146Cys
Xaa Xaa Xaa Xaa Phe Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 1 5 10
15 Xaa Xaa Trp Xaa Cys Asp Gly Xaa Xaa Asp Cys Gly Asp Xaa Ser Asp
20 25 30 Glu Xaa Xaa Xaa Xaa Cys 35 14736PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(3)..(3)Xaa = Ala or
GluMISC_FEATURE(4)..(4)Xaa = Gly or SerMISC_FEATURE(9)..(9)Xaa =
Gly or ArgMISC_FEATURE(10)..(10)Xaa = Asn, Arg or
SerMISC_FEATURE(11)..(11)Xaa = Ala, Ser or
ThrMISC_FEATURE(12)..(12)Xaa = Lys or AsnMISC_FEATURE(17)..(17)Xaa
= Glu, His, Gln or SerMISC_FEATURE(18)..(18)Xaa = Ala or
ValMISC_FEATURE(20)..(20)Xaa = Ile or ValMISC_FEATURE(24)..(24)Xaa
= Ile or ValMISC_FEATURE(28)..(28)Xaa = Asp or
GluMISC_FEATURE(34)..(34)Xaa = Asp, Lys, Met, Asn or
ThrMISC_FEATURE(35)..(35)Xaa = Asn, Ser or Tyr 147Cys Gly Xaa Xaa
Leu Phe Thr Cys Xaa Xaa Xaa Xaa Ile Cys Ile Ser 1 5 10 15 Xaa Xaa
Trp Xaa Cys Asp Gly Xaa Asp Asp Cys Xaa Asp Asn Ser Asp 20 25 30
Glu Xaa Xaa Cys 35 14847PRTArtificial sequenceSynthetic polypeptide
148Cys Gly Glu Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser
1 5 10 15 His Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn
Ser Asp 20 25 30 Glu Asn Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro
Gly Ser Leu 35 40 45 14936PRTArtificial sequenceSynthetic
polypeptide 149Cys Gly Ala Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile
Cys Ile Ser 1 5 10 15 Gln Val Trp Val Cys Asp Gly Val Asp Asp Cys
Glu Asp Asn Ser Asp 20 25 30 Glu Asp Ser Cys 35 15036PRTArtificial
sequenceSynthetic polypeptide 150Cys Gly Ala Gly Leu Phe Thr Cys
Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Gln Ala Trp Val Cys Asp
Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30 Glu Asn Tyr Cys 35
15136PRTArtificial sequenceSynthetic polypeptide 151Cys Gly Ala Gly
Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Gln Ala
Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30
Glu Thr Asn Cys 35 15236PRTArtificial sequenceSynthetic polypeptide
152Cys Gly Glu Gly Leu Phe Thr Cys Gly Ser Thr Asn Ile Cys Ile Ser
1 5 10 15 Ser Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn
Ser Asp 20 25 30 Glu Asn Asn Cys 35 15336PRTArtificial
sequenceSynthetic polypeptide 153Cys Gly Glu Gly Leu Phe Thr Cys
Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 His Ala Trp Val Cys Asp
Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30 Glu Asn Asn Cys 35
15436PRTArtificial sequenceSynthetic polypeptide 154Cys Gly Glu Gly
Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Glu Ala
Trp Ile Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30
Glu Lys Asn Cys 35 15536PRTArtificial sequenceSynthetic polypeptide
155Cys Gly Ala Gly Leu Phe Thr Cys Arg Ser Ala Lys Ile Cys Ile Ser
1 5 10 15 His Ala Trp Val Cys Asp Gly Ile Asp Asp Cys Glu Asp Asn
Ser Asp 20 25 30 Glu Asn Asn Cys 35 15636PRTArtificial
sequenceSynthetic polypeptide 156Cys Gly Ala Gly Leu Phe Thr Cys
Arg Asn Ser Lys Ile Cys Ile Ser 1 5 10 15 Gln Ala Trp Val Cys Asp
Gly Val Asp Asp Cys Asp Asp Asn Ser Asp 20 25 30 Glu Lys Tyr Cys 35
15736PRTArtificial sequenceSynthetic polypeptide 157Cys Gly Ala Ser
Leu Phe Thr Cys Arg Arg Ser Asn Ile Cys Ile Ser 1 5 10 15 Gln Ala
Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30
Glu Met Asn Cys 35 15836PRTArtificial sequenceSynthetic polypeptide
158Cys Gly Ala Gly Leu Phe Thr Cys Arg Ser Thr Lys Ile Cys Ile Ser
1 5 10 15 Gln Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn
Ser Asp 20 25 30 Glu Lys Asn Cys 35 15947PRTArtificial
sequenceSynthetic polypeptide 159Cys Gly Ala Gly Leu Phe Thr Cys
Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Gln Val Trp Val Cys Asp
Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30 Glu Asp Ser Cys
Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35 40 45
16047PRTArtificial sequenceSynthetic polypeptide 160Cys Gly Ala Gly
Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Gln Ala
Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30
Glu Asn Tyr Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35 40
45 16147PRTArtificial sequenceSynthetic polypeptide 161Cys Gly Ala
Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15 Gln
Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25
30 Glu Thr Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35
40 45 16247PRTArtificial sequenceSynthetic polypeptide 162Cys Gly
Glu Gly Leu Phe Thr Cys Gly Ser Thr Asn Ile Cys Ile Ser 1 5 10 15
Ser Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20
25 30 Glu Asn Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu
35 40 45 16347PRTArtificial sequenceSynthetic polypeptide 163Cys
Gly Glu Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10
15 Glu Ala Trp Ile Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp
20 25 30 Glu Lys Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser
Leu 35 40 45 16447PRTArtificial sequenceSynthetic polypeptide
164Cys Gly Ala Gly Leu Phe Thr Cys Arg Ser Ala Lys Ile Cys Ile Ser
1 5 10 15 His Ala Trp Val Cys Asp Gly Ile Asp Asp Cys Glu Asp Asn
Ser Asp 20 25 30 Glu Asn Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro
Gly Ser Leu 35 40 45 16547PRTArtificial sequenceSynthetic
polypeptide 165Cys Gly Ala Gly Leu Phe Thr Cys Arg Asn Ser Lys Ile
Cys Ile Ser 1 5 10 15 Gln Ala Trp Val Cys Asp Gly Val Asp Asp Cys
Asp Asp Asn Ser Asp 20 25 30 Glu Lys Tyr Cys Ser Ala Pro Ala Ser
Glu Pro Pro Gly Ser Leu 35 40 45 16647PRTArtificial
sequenceSynthetic polypeptide 166Cys Gly Ala Ser Leu Phe Thr Cys
Arg Arg Ser Asn Ile Cys Ile Ser 1 5 10 15 Gln Ala Trp Val Cys Asp
Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30 Glu Met Asn Cys
Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35 40 45
16747PRTArtificial sequenceSynthetic polypeptide 167Cys Gly Ala Gly
Leu Phe Thr Cys Arg Ser Thr Lys Ile Cys Ile Ser 1 5 10 15 Gln Ala
Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20 25 30
Glu Lys Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu 35 40
45 168135PRTArtificial sequenceSynthetic polypeptide 168Cys Gly Ala
Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala
Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25
30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg
35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp
Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln
Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu Cys Gly
Glu Gly Leu Phe Thr Cys 85 90 95 Arg Ser Thr Asn Ile Cys Ile Ser
His Ala Trp Val Cys Asp Gly Val 100 105 110 Asp Asp Cys Glu Asp Asn
Ser Asp Glu Asn Asn Cys Ser Ala Pro Ala 115 120 125 Ser Glu Pro Pro
Gly Ser Leu 130 135 169134PRTArtificial sequenceSynthetic
polypeptide 169Cys Leu Pro Asp Glu Phe Gln Cys Gly Ser Gly Arg Cys
Ile Pro Gln 1 5 10 15 His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly
Asp Gly Ser Asp Glu 20 25 30 Pro Pro Ala His Cys Ser Ala Pro Ala
Ser Glu Pro Pro Gly Ser Leu 35 40 45 Cys Arg Ala Gly Glu Phe Arg
Cys Ser Asn Gly Arg Cys Val Pro Leu 50 55 60 Thr Trp Leu Cys Asp
Gly Glu Asp Asp Cys Gln Asp Asn Ser Asp Glu 65 70 75 80 Lys Asn Cys
Ala Gln Pro Thr Cys Gly Glu Gly Leu Phe Thr Cys Arg 85 90 95 Ser
Thr Asn Ile Cys Ile Ser His Ala Trp Val Cys Asp Gly Val Asp 100 105
110 Asp Cys Glu Asp Asn Ser Asp Glu Asn Asn Cys Ser Ala Pro Ala Ser
115 120 125 Glu Pro Pro Gly Ser Leu 130 170136PRTArtificial
sequenceSynthetic polypeptide 170Cys Ala Pro Gly Glu Phe Thr Cys
Lys Asn Thr Gly Arg Cys Ile Pro 1 5 10 15 Leu Asn Trp Arg Cys Asp
Gly Asp Asp Asp Cys Gly Asp Gly Ser Asp 20 25 30 Glu Thr Asp Cys
Pro Ala Pro Thr Cys Pro Ser Asn Gln Phe Pro Cys 35 40 45 Arg Ser
Thr Gly Ile Cys Ile Pro Leu Ala Trp Val Cys Asp Gly Leu 50 55 60
Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Pro Ala His Cys Ser Ala 65
70
75 80 Pro Ala Ser Glu Pro Pro Gly Ser Leu Cys Gly Glu Gly Leu Phe
Thr 85 90 95 Cys Arg Ser Thr Asn Ile Cys Ile Ser His Ala Trp Val
Cys Asp Gly 100 105 110 Val Asp Asp Cys Glu Asp Asn Ser Asp Glu Asn
Asn Cys Ser Ala Pro 115 120 125 Ala Ser Glu Pro Pro Gly Ser Leu 130
135 171136PRTArtificial sequenceSynthetic polypeptide 171Cys Gly
Pro Asp Glu Phe Arg Cys Asn Asn Gly Gln Cys Ile Pro Leu 1 5 10 15
Pro Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Asn Ser Asp Glu 20
25 30 Pro Leu Glu Ile Cys Gln Ala Pro Thr Cys Gln Ser Asn Glu Phe
Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys
Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp Glu Pro Pro
Ala His Cys Ser Ala 65 70 75 80 Pro Ala Ser Glu Pro Pro Gly Ser Leu
Cys Gly Glu Gly Leu Phe Thr 85 90 95 Cys Arg Ser Thr Asn Ile Cys
Ile Ser His Ala Trp Val Cys Asp Gly 100 105 110 Val Asp Asp Cys Glu
Asp Asn Ser Asp Glu Asn Asn Cys Ser Ala Pro 115 120 125 Ala Ser Glu
Pro Pro Gly Ser Leu 130 135 172230PRTArtificial sequenceSynthetic
polypeptide 172Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Cys Gly
Ala Asp 130 135 140 Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg
Ala Trp Arg Cys 145 150 155 160 Asp Gly Val Asp Asp Cys Gly Asp Gly
Ser Asp Glu Ala Pro Glu Ile 165 170 175 Cys Glu Thr Pro Thr Cys Gln
Ser Asn Glu Phe Arg Cys Arg Ser Gly 180 185 190 Arg Cys Ile Pro Gln
His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly 195 200 205 Asp Gly Ser
Asp Glu Ser Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro 210 215 220 Pro
Gly Ser Leu Ser Leu 225 230 173221PRTArtificial sequenceSynthetic
polypeptide 173Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly
Ser Cys 130 135 140 Val Pro Arg Ala Trp Arg Cys Asp Gly Val Asp Asp
Cys Gly Asp Gly 145 150 155 160 Ser Asp Glu Ala Pro Glu Ile Cys Glu
Thr Pro Thr Cys Gln Ser Asn 165 170 175 Glu Phe Arg Cys Arg Ser Gly
Arg Cys Ile Pro Gln His Trp Leu Cys 180 185 190 Asp Gly Leu Asn Asp
Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln Cys 195 200 205 Ser Ala Pro
Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu 210 215 220
174230PRTArtificial sequenceSynthetic polypeptide 174Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp Cys Gly Ala Asp 130 135 140 Gln Phe
Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg Cys 145 150 155
160 Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile
165 170 175 Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg
Ser Gly 180 185 190 Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu
Asn Asp Cys Gly 195 200 205 Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser
Ala Pro Ala Ser Glu Pro 210 215 220 Pro Gly Ser Leu Ser Leu 225 230
175230PRTArtificial sequenceSynthetic polypeptide 175Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys Val
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp Cys Gly Ala Asp 130 135 140 Gln Phe
Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg Cys 145 150 155
160 Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile
165 170 175 Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg
Ser Gly 180 185 190 Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu
Asn Asp Cys Gly 195 200 205 Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser
Ala Pro Ala Ser Glu Pro 210 215 220 Pro Gly Ser Leu Ser Leu 225 230
176320PRTArtificial sequenceSynthetic polypeptide 176Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp Cys Gly Ala Asp 130 135 140 Gln Phe
Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg Cys 145 150 155
160 Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile
165 170 175 Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg
Ser Gly 180 185 190 Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu
Asn Asp Cys Gly 195 200 205 Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser
Ala Pro Ala Ser Glu Pro 210 215 220 Pro Gly Ser Leu Ser Leu Cys Gly
Ala Asp Gln Phe Arg Cys Gly Asn 225 230 235 240 Gly Ser Cys Val Pro
Arg Ala Trp Arg Cys Asp Gly Val Asp Asp Cys 245 250 255 Gly Asp Gly
Ser Asp Glu Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys 260 265 270 Gln
Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln His 275 280
285 Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser
290 295 300 Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu
Ser Leu 305 310 315 320 177311PRTArtificial sequenceSynthetic
polypeptide 177Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly
Ser Cys 130 135 140 Val Pro Arg Ala Trp Arg Cys Asp Gly Val Asp Asp
Cys Gly Asp Gly 145 150 155 160 Ser Asp Glu Ala Pro Glu Ile Cys Glu
Thr Pro Thr Cys Gln Ser Asn 165 170 175 Glu Phe Arg Cys Arg Ser Gly
Arg Cys Ile Pro Gln His Trp Leu Cys 180 185 190 Asp Gly Leu Asn Asp
Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln Cys 195 200 205 Ser Ala Pro
Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu Cys Gly Ala 210 215 220 Asp
Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg 225 230
235 240 Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro
Glu 245 250 255 Ile Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg
Cys Arg Ser 260 265 270 Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp
Gly Leu Asn Asp Cys 275 280 285 Gly Asp Gly Ser Asp Glu Ser Gln Gln
Cys Ser Ala Pro Ala Ser Glu 290 295 300 Pro Pro Gly Ser Leu Ser Leu
305 310 178320PRTArtificial sequenceSynthetic polypeptide 178Phe
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10
15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp
20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu
Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu
Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr
Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg
Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His
Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser
Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile
Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Cys Gly Ala Asp 130 135 140
Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg Cys 145
150 155 160 Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro
Glu Ile 165 170 175 Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg
Cys Arg Ser Gly 180 185 190 Arg Cys Ile Pro Gln His Trp Leu Cys Asp
Gly Leu Asn Asp Cys Gly 195 200 205 Asp Gly Ser Asp Glu Ser Gln Gln
Cys Ser Ala Pro Ala Ser Glu Pro 210 215 220 Pro Gly Ser Leu Ser Leu
Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn 225 230 235 240 Gly Ser Cys
Val Pro Arg Ala Trp Arg Cys Asp Gly Val Asp Asp Cys 245 250 255 Gly
Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys 260 265
270 Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln His
275 280 285 Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp
Glu Ser 290 295 300 Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly
Ser Leu Ser Leu 305 310 315 320 179320PRTArtificial
sequenceSynthetic polypeptide 179Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp Cys Gly Ala Asp 130 135 140 Gln Phe Arg Cys Gly Asn
Gly Ser Cys Val Pro Arg Ala Trp Arg Cys 145 150 155 160 Asp Gly Val
Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile 165 170 175 Cys
Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly
180 185 190 Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu Asn Asp
Cys Gly 195 200 205 Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro
Ala Ser Glu Pro 210 215 220 Pro Gly Ser Leu Ser Leu Cys Gly Ala Asp
Gln Phe Arg Cys Gly Asn 225 230 235 240 Gly Ser Cys Val Pro Arg Ala
Trp Arg Cys Asp Gly Val Asp Asp Cys 245 250 255 Gly Asp Gly Ser Asp
Glu Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys 260 265 270 Gln Ser Asn
Glu Phe Arg Cys Arg Ser Gly Arg Cys Ile Pro Gln His 275 280 285 Trp
Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser 290 295
300 Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu
305 310 315 320 180230PRTArtificial sequenceSynthetic polypeptide
180Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg
1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser
Asp Glu 20 25 30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser
Asn Glu Phe Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His
Trp Leu Cys Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp
Glu Ser Gln Gln Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly
Ser Leu Ser Leu Phe Asn Leu Pro Pro Gly 85 90 95 Asn Tyr Lys Lys
Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe 100 105 110 Leu Arg
Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser 115 120 125
Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val 130
135 140 Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr
Asp 145 150 155 160 Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu
Cys Leu Phe Leu 165 170 175 Glu Arg Leu Glu Glu Asn His Tyr Asn Thr
Tyr Ile Ser Lys Lys His 180 185 190 Ala Glu Lys Asn Trp Phe Val Gly
Leu Lys Lys Asn Gly Ser Cys Lys 195 200 205 Arg Gly Pro Arg Thr His
Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro 210 215 220 Leu Pro Val Ser
Ser Asp 225 230 181221PRTArtificial sequenceSynthetic polypeptide
181Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg
1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser
Asp Glu 20 25 30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser
Asn Glu Phe Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His
Trp Leu Cys Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp
Glu Ser Gln Gln Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly
Ser Leu Ser Leu Lys Pro Lys Leu Leu Tyr 85 90 95 Cys Ser Asn Gly
Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val 100 105 110 Asp Gly
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser 115 120 125
Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln 130
135 140 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr
Pro 145 150 155 160 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Asn 165 170 175 Thr Tyr Ile Ser Lys Lys His Ala Glu Lys
Asn Trp Phe Val Gly Leu 180 185 190 Lys Lys Asn Gly Ser Cys Lys Arg
Gly Pro Arg Thr His Tyr Gly Gln 195 200 205 Lys Ala Ile Leu Phe Leu
Pro Leu Pro Val Ser Ser Asp 210 215 220 182230PRTArtificial
sequenceSynthetic polypeptide 182Cys Gly Ala Asp Gln Phe Arg Cys
Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly
Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile
Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg 35 40 45 Cys Arg
Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu 50 55 60
Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro 65
70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu Phe Asn Leu Pro
Pro Gly 85 90 95 Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys Ser Asn
Gly Gly His Phe 100 105 110 Leu Arg Ile Leu Pro Asp Gly Thr Val Asp
Gly Thr Arg Asp Arg Ser 115 120 125 Asp Gln His Ile Gln Leu Gln Leu
Ser Ala Glu Ser Val Gly Glu Val 130 135 140 Tyr Ile Lys Ser Thr Glu
Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp 145 150 155 160 Gly Leu Leu
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu 165 170 175 Glu
Arg Leu Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His 180 185
190 Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys
195 200 205 Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
Leu Pro 210 215 220 Leu Pro Val Ser Ser Asp 225 230
183230PRTArtificial sequenceSynthetic polypeptide 183Cys Gly Ala
Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala
Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25
30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg
35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp
Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln
Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu
Phe Asn Leu Pro Pro Gly 85 90 95 Asn Tyr Lys Lys Pro Lys Leu Leu
Tyr Cys Ser Asn Gly Gly His Phe 100 105 110 Leu Arg Ile Leu Pro Asp
Gly Thr Val Asp Gly Thr Arg Asp Arg Ser 115 120 125 Asp Gln His Ile
Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val 130 135 140 Tyr Ile
Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp 145 150 155
160 Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu
165 170 175 Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys
Lys His 180 185 190 Ala Glu Lys Asn Trp Phe Val Gly Leu Asp Gln Asn
Gly Ser Cys Val 195 200 205 Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
Ala Ile Leu Phe Leu Pro 210 215 220 Leu Pro Val Ser Ser Asp 225 230
184320PRTArtificial sequenceSynthetic polypeptide 184Cys Gly Ala
Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala
Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25
30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg
35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp
Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln
Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu
Cys Gly Ala Asp Gln Phe 85 90 95 Arg Cys Gly Asn Gly Ser Cys Val
Pro Arg Ala Trp Arg Cys Asp Gly 100 105 110 Val Asp Asp Cys Gly Asp
Gly Ser Asp Glu Ala Pro Glu Ile Cys Glu 115 120 125 Thr Pro Thr Cys
Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys 130 135 140 Ile Pro
Gln His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly 145 150 155
160 Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly
165 170 175 Ser Leu Ser Leu Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys
Pro Lys 180 185 190 Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro Asp 195 200 205 Gly Thr Val Asp Gly Thr Arg Asp Arg Ser
Asp Gln His Ile Gln Leu 210 215 220 Gln Leu Ser Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser Thr Glu 225 230 235 240 Thr Gly Gln Tyr Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser 245 250 255 Gln Thr Pro
Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn 260 265 270 His
Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe 275 280
285 Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His
290 295 300 Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser
Ser Asp 305 310 315 320 185311PRTArtificial sequenceSynthetic
polypeptide 185Cys Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys
Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly
Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile Cys Glu Thr Pro Thr
Cys Gln Ser Asn Glu Phe Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile
Pro Gln His Trp Leu Cys Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp
Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu
Pro Pro Gly Ser Leu Ser Leu Cys Gly Ala Asp Gln Phe 85 90 95 Arg
Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp Arg Cys Asp Gly 100 105
110 Val Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys Glu
115 120 125 Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly
Arg Cys 130 135 140 Ile Pro Gln His Trp Leu Cys Asp Gly Leu Asn Asp
Cys Gly Asp Gly 145 150 155 160 Ser Asp Glu Ser Gln Gln Cys Ser Ala
Pro Ala Ser Glu Pro Pro Gly 165 170 175 Ser Leu Ser Leu Lys Pro Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His 180 185 190 Phe Leu Arg Ile Leu
Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg 195 200 205 Ser Asp Gln
His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu 210 215 220 Val
Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr 225 230
235 240 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe 245 250 255 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile
Ser Lys Lys 260 265 270 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys
Lys Asn Gly Ser Cys 275 280 285 Lys Arg Gly Pro Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu 290 295 300 Pro Leu Pro Val Ser Ser Asp
305 310 186320PRTArtificial sequenceSynthetic polypeptide 186Cys
Gly Ala Asp Gln Phe Arg Cys Gly Asn Gly Ser Cys Val Pro Arg 1 5 10
15 Ala Trp Arg Cys Asp Gly Val Asp Asp Cys Gly Asp Gly Ser Asp Glu
20 25 30 Ala Pro Glu Ile Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu
Phe Arg 35 40 45 Cys Arg Ser Gly Arg Cys Ile Pro Gln His Trp Leu
Cys Asp Gly Leu 50 55 60 Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser
Gln Gln Cys Ser Ala Pro 65 70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu
Ser Leu Cys Gly Ala Asp Gln Phe 85 90 95 Arg Cys Gly Asn Gly Ser
Cys Val Pro Arg Ala Trp Arg Cys Asp Gly 100 105 110 Val Asp Asp Cys
Gly Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys Glu 115 120 125 Thr Pro
Thr Cys Gln Ser Asn Glu Phe Arg Cys Arg Ser Gly Arg Cys 130 135 140
Ile Pro Gln His Trp Leu Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly 145
150 155 160 Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro
Pro Gly 165 170 175 Ser Leu Ser Leu Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val 180 185 190 Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp 195 200 205 Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu 210 215 220 Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu 225 230 235 240 Thr Gly Gln
Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser 245 250 255 Gln
Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn 260 265
270 His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
275 280 285 Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg
Thr His 290 295 300 Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 305 310 315 320 187320PRTArtificial
sequenceSynthetic polypeptide 187Cys Gly Ala Asp Gln Phe Arg Cys
Gly Asn Gly Ser Cys Val Pro Arg 1 5 10 15 Ala Trp Arg Cys Asp Gly
Val Asp Asp Cys Gly Asp Gly Ser Asp Glu 20 25 30 Ala Pro Glu Ile
Cys Glu Thr Pro Thr Cys Gln Ser Asn Glu Phe Arg 35 40 45 Cys Arg
Ser Gly Arg Cys Ile Pro Gln His Trp Leu Cys Asp Gly Leu 50 55 60
Asn Asp Cys Gly Asp Gly Ser Asp Glu Ser Gln Gln Cys Ser Ala Pro 65
70 75 80 Ala Ser Glu Pro Pro Gly Ser Leu Ser Leu Cys Gly Ala Asp
Gln Phe 85 90 95 Arg Cys Gly Asn Gly Ser Cys Val Pro Arg Ala Trp
Arg Cys Asp Gly 100 105 110 Val Asp Asp Cys Gly Asp Gly Ser Asp Glu
Ala Pro Glu Ile Cys Glu 115 120 125 Thr Pro Thr Cys Gln Ser Asn Glu
Phe Arg Cys Arg Ser Gly Arg Cys 130 135 140 Ile Pro Gln His Trp Leu
Cys Asp Gly Leu Asn Asp Cys Gly Asp Gly 145 150 155 160 Ser Asp Glu
Ser Gln Gln Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly 165 170 175 Ser
Leu Ser Leu Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys 180 185
190 Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp
195 200 205 Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile
Gln Leu 210 215 220 Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys Ser Thr Glu 225 230 235 240 Thr Gly Gln Tyr Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser 245 250
255 Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn
260 265 270 His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn
Trp Phe 275 280 285 Val Gly Leu Asp Gln Asn Gly Ser Cys Val Arg Gly
Pro Arg Thr His 290 295 300 Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro
Leu Pro Val Ser Ser Asp 305 310 315 320 188178PRTArtificial
sequenceSynthetic polypeptide 188Lys Pro Lys Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val
Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr
Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65
70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala
Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys
Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp Cys Gly Glu Gly Leu
Phe Thr Cys Arg Ser Thr Asn Ile 130 135 140 Cys Ile Ser His Ala Trp
Val Cys Asp Gly Val Asp Asp Cys Glu Asp 145 150 155 160 Asn Ser Asp
Glu Asn Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly 165 170 175 Ser
Leu 189178PRTArtificial sequenceSynthetic polypeptide 189Cys Gly
Glu Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile Cys Ile Ser 1 5 10 15
His Ala Trp Val Cys Asp Gly Val Asp Asp Cys Glu Asp Asn Ser Asp 20
25 30 Glu Asn Asn Cys Ser Ala Pro Ala Ser Glu Pro Pro Gly Ser Leu
Lys 35 40 45 Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu
Arg Ile Leu 50 55 60 Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg
Ser Asp Gln His Ile 65 70 75 80 Gln Leu Gln Leu Ser Ala Glu Ser Val
Gly Glu Val Tyr Ile Lys Ser 85 90 95 Thr Glu Thr Gly Gln Tyr Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr 100 105 110 Gly Ser Gln Thr Pro
Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu 115 120 125 Glu Asn His
Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn 130 135 140 Trp
Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg 145 150
155 160 Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser 165 170 175 Ser Asp 19047PRTArtificial sequenceSynthetic
polypeptide 190Cys Gly Glu Gly Leu Phe Thr Cys Arg Ser Thr Asn Ile
Cys Ile Ser 1 5 10 15 His Ala Trp Val Cys Asp Gly Val Asp Asp Cys
Glu Asp Asn Ser Asp 20 25 30 Glu Asn Asn Cys Ser Ala Pro Ala Ser
Glu Pro Pro Gly Ser Leu 35 40 45 191140PRTArtificial
sequenceSynthetic polypeptide 191Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Glu Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 192140PRTArtificial sequenceSynthetic
polypeptide 192Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Glu Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Asp 100 105
110 Gln Asn Gly Ser Val Val Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 193140PRTArtificial sequenceSynthetic polypeptide 193Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Glu Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 194131PRTArtificial
sequenceSynthetic polypeptide 194Lys Pro Lys Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val
Asp Gly Thr Glu Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr
Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65
70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala
Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val
Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
195131PRTArtificial sequenceSynthetic polypeptide 195Lys Pro Val
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu
Pro Asp Gly Thr Val Asp Gly Thr Glu Asp Arg Ser Asp Gln His 20 25
30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Asp Gln
Asn Gly Ser Val Val Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
196140PRTArtificial sequenceSynthetic polypeptide 196Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Glu Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val Val
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 197131PRTArtificial
sequenceSynthetic polypeptide 197Lys Pro Val Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val
Asp Gly Thr Glu Asp Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr
Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60
Tyr Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65
70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala
Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val
Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu
Trp Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
198131PRTArtificial sequenceSynthetic polypeptide 198Lys Pro Val
Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15 Leu
Pro Asp Gly Thr Val Asp Gly Thr Glu Asp Arg Ser Asp Gln His 20 25
30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys
35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly
Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu Phe
Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile Ser
Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Asp Gln
Asn Gly Ser Val Val Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Trp Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp 130
199140PRTArtificial sequenceSynthetic polypeptide 199Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25
30 Gly Thr Val Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 200140PRTArtificial
sequenceSynthetic polypeptide 200Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Val Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 201140PRTArtificial sequenceSynthetic
polypeptide 201Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Val Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 202131PRTArtificial sequenceSynthetic polypeptide 202Lys Pro
Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Val Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys
Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 203131PRTArtificial sequenceSynthetic polypeptide 203Lys Pro
Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Val Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys 85 90
95 Asn Trp Phe Val Gly Leu Asp Gln Asn Gly Ser Val Val Arg Gly Pro
100 105 110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu
Pro Val 115 120 125 Ser Ser Asp 130 204140PRTArtificial
sequenceSynthetic polypeptide 204Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Val Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 205131PRTArtificial sequenceSynthetic
polypeptide 205Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Val Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 206131PRTArtificial sequenceSynthetic
polypeptide 206Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Val Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Asp Gln Asn Gly Ser Val Val Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 207140PRTArtificial sequenceSynthetic
polypeptide 207Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Asp 100 105
110 Gln Asn Gly Ser Val Val Val Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 208140PRTArtificial sequenceSynthetic polypeptide 208Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Val Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 209140PRTArtificial
sequenceSynthetic polypeptide 209Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Val Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 210140PRTArtificial sequenceSynthetic
polypeptide 210Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Val Arg Leu Glu Glu Asn His Val Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 211140PRTArtificial sequenceSynthetic polypeptide 211Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Val Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 212140PRTArtificial
sequenceSynthetic polypeptide 212Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Val Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Val Arg Leu Glu Glu Asn His Tyr
Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Val Leu Pro
Val Ser Ser Asp 130 135 140 213140PRTArtificial sequenceSynthetic
polypeptide 213Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Val Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 214131PRTArtificial sequenceSynthetic polypeptide 214Lys Pro
Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Thr Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys
Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 215131PRTArtificial sequenceSynthetic polypeptide 215Lys Pro
Val Leu Leu Tyr Thr Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Ser Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys
Lys Asn Gly Ser Ala Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 216131PRTArtificial sequenceSynthetic polypeptide 216Lys Pro
Lys Leu Leu Tyr Cys Ser Asn Gly Gly Tyr Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Asn Thr Tyr Ile
Ser Lys Lys Tyr Ala Glu Lys 85 90 95 Asn Trp Tyr Val Gly Leu Lys
Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 217131PRTArtificial sequenceSynthetic polypeptide 217Lys Pro
Val Leu Leu Tyr Cys Ser Asn Gly Gly Tyr Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys Tyr Ala Glu Lys 85 90 95 Asn Trp Tyr Val Gly Leu Lys
Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 218140PRTArtificial sequenceSynthetic polypeptide 218Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly Tyr Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Phe Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr Ile Ser Lys Lys Tyr Ala
Glu Lys Asn Trp Tyr Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 219177PRTArtificial
sequenceSynthetic polypeptide 219Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110
Gln Asn Gly Ser Cys Val Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115
120 125 Ala Ile Leu Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro
Glu 130 135 140 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly
Ser Ser Asp 145 150 155 160 Pro Leu Ser Met Val Gly Pro Ser Gln Gly
Arg Ser Pro Ser Tyr Ala 165 170 175 Ser 220183PRTArtificial
sequenceSynthetic polypeptide 220Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Leu
Pro Met Val Pro Glu Glu Pro Glu 130 135 140 Asp Leu Arg Gly His Leu
Glu Ser Asp Met Phe Ser Ser Pro Leu Glu 145 150 155 160 Thr Asp Ser
Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val 165 170 175 Arg
Ser Pro Ser Phe Glu Lys 180 221147PRTArtificial sequenceSynthetic
polypeptide 221His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln Val 1 5 10 15 Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly
His Phe Leu Arg Ile 20 25 30 Leu Pro Asp Gly Thr Val Asp Gly Thr
Arg Asp Arg Ser Asp Gln His 35 40 45 Ile Gln Leu Gln Leu Ser Ala
Glu Ser Val Gly Glu Val Tyr Ile Lys 50 55 60 Ser Thr Glu Thr Gly
Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 65 70 75 80 Tyr Gly Ser
Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 85 90 95 Glu
Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 100 105
110 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro
115 120 125 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu
Pro Val 130 135 140 Ser Ser Asp 145 222135PRTArtificial
sequenceSynthetic polypeptide 222Gly Gly Gln Val Lys Pro Lys Leu
Leu Tyr Cys Ser Asn Gly Gly His 1 5 10 15 Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp Gly Thr Arg Asp Arg 20 25 30 Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu 35 40 45 Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr 50 55 60
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe 65
70 75 80 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys 85 90 95 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
Asn Gly Ser Val 100 105 110 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu 115 120 125 Pro Leu Pro Val Ser Ser Asp 130
135 223177PRTArtificial sequenceSynthetic polypeptide 223Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu 130 135 140 Pro
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 145 150
155 160 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr
Ala 165 170 175 Ser 224183PRTArtificial sequenceSynthetic
polypeptide 224Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Leu Pro Met Val Pro Glu Glu
Pro Glu 130 135 140 Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser
Ser Pro Leu Glu 145 150 155 160 Thr Asp Ser Met Asp Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala Val 165 170 175 Arg Ser Pro Ser Phe Glu Lys
180 225131PRTArtificial sequenceSynthetic polypeptide 225Lys Pro
Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile 1 5 10 15
Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His 20
25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn His Tyr Val Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys 85 90 95 Asn Trp Phe Val Gly Leu Lys
Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105 110 Arg Thr His Tyr Gly
Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 115 120 125 Ser Ser Asp
130 226140PRTArtificial sequenceSynthetic polypeptide 226Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Cys
Val Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 227140PRTArtificial
sequenceSynthetic polypeptide 227Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Pro His Ile Gln Leu Gln Leu Ile Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn Gly Tyr
Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 228131PRTArtificial sequenceSynthetic
polypeptide 228Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Pro His 20 25 30 Ile Gln Leu Gln Leu Ile Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
Gly Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 229140PRTArtificial sequenceSynthetic
polypeptide 229Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Phe Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Thr
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Val Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Trp Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 230140PRTArtificial sequenceSynthetic polypeptide 230Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val
Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 231140PRTArtificial
sequenceSynthetic polypeptide 231Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65
70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr
Val Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe
Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val Val Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro
Val Ser Ser Asp 130 135 140 232131PRTArtificial sequenceSynthetic
polypeptide 232Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Phe Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Thr Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Trp Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130 233140PRTArtificial sequenceSynthetic
polypeptide 233Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Glu Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 234140PRTArtificial sequenceSynthetic polypeptide 234Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70
75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Val Asn
Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val
Gly Leu Lys 100 105 110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr
His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val
Ser Ser Asp 130 135 140 235140PRTArtificial sequenceSynthetic
polypeptide 235Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Val Leu
Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys
Leu Phe Leu Val Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105
110 Lys Asn Gly Ser Val Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
140 236140PRTArtificial sequenceSynthetic polypeptide 236Phe Asn
Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15
Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20
25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr
Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly
Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu
Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala
Glu Lys Asn Trp Phe Val Gly Leu Asp 100 105 110 Gln Asn Gly Ser Val
Val Val Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu
Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 237140PRTArtificial
sequenceSynthetic polypeptide 237Phe Asn Leu Pro Pro Gly Asn Tyr
Lys Lys Pro Val Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe
Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Glu Asp
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser
Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60
Leu Ala Met Asp Thr Asp Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys 65
70 75 80 Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg
Ile Leu 85 90 95 Pro Asp Gly Thr Val Asp Gly Thr Glu Asp Arg Ser
Asp Gln His Ile 100 105 110 Gln Leu Gln Leu Ser Ala Glu Ser Val Gly
Glu Val Tyr Ile Lys Ser 115 120 125 Thr Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr Asp 130 135 140 238131PRTArtificial sequenceSynthetic
polypeptide 238Lys Pro Val Leu Leu Tyr Cys Ser Asn Gly Gly His Phe
Leu Arg Ile 1 5 10 15 Leu Pro Asp Gly Thr Val Asp Gly Thr Glu Asp
Arg Ser Asp Gln His 20 25 30 Ile Gln Leu Gln Leu Ser Ala Glu Ser
Val Gly Glu Val Tyr Ile Lys 35 40 45 Ser Thr Glu Thr Gly Gln Tyr
Leu Ala Met Asp Thr Asp Gly Leu Leu 50 55 60 Tyr Gly Ser Gln Thr
Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 65 70 75 80 Glu Glu Asn
His Tyr Val Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 85 90 95 Asn
Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Val Lys Arg Gly Pro 100 105
110 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val
115 120 125 Ser Ser Asp 130
* * * * *