U.S. patent application number 17/494001 was filed with the patent office on 2022-01-20 for growth differentiation factor 15 fusion proteins.
This patent application is currently assigned to Amgen Inc.. The applicant listed for this patent is Amgen Inc.. Invention is credited to Murielle Marie VENIANT ELLISON, Kenneth William WALKER, YuMei XIONG.
Application Number | 20220017584 17/494001 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220017584 |
Kind Code |
A1 |
XIONG; YuMei ; et
al. |
January 20, 2022 |
GROWTH DIFFERENTIATION FACTOR 15 FUSION PROTEINS
Abstract
GDF15 molecules are provided herein. In some embodiments, the
GDF15 molecule is a GDF15-Fc fusion, in which a GDF15 region is
fused to an Fc region. In some embodiments, the GDF15 region is
fused to the Fc region via a linker. Also, provided herein are
methods for making and using GDF15 molecules.
Inventors: |
XIONG; YuMei; (Palo Alto,
CA) ; WALKER; Kenneth William; (Newbury Park, CA)
; VENIANT ELLISON; Murielle Marie; (Thousand Oaks,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amgen Inc. |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
Amgen Inc.
Thousand Oaks
CA
|
Appl. No.: |
17/494001 |
Filed: |
October 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17148761 |
Jan 14, 2021 |
11161889 |
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17494001 |
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16620029 |
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PCT/US19/26369 |
Apr 8, 2019 |
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17148761 |
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62655108 |
Apr 9, 2018 |
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International
Class: |
C07K 14/495 20060101
C07K014/495 |
Claims
1. A fusion protein comprising a GDF15 region joined to an Fc
region via a linker, wherein the GDF15 region comprises the amino
acid sequence of SEQ ID NO: 6 and at least one mutation, wherein at
least one of the mutations is of the aspartate at position 5.
2. The fusion protein of claim 1, wherein the aspartate at position
5 is mutated to glutamate.
3. The fusion protein of claim 2, wherein the GDF15 region
comprises the amino acid sequence of SEQ ID NO: 16.
4. The fusion protein of claim 1 or 2, wherein the GDF15 region
further comprises a mutation of the asparagine at position 3.
5. The fusion protein of claim 3, wherein the asparagine at
position 3 mutated to glutamine.
6. The fusion protein of claim 5, wherein the GDF15 region
comprises the amino acid sequence of SEQ ID NO: 18.
7. The fusion protein of any one of claims 1-6, wherein the linker
is a (G4S)n or (G4Q)n linker, wherein n is greater than 0.
8. The fusion protein of claim 7, wherein n is 1 or 2.
9. A fusion protein comprising a GDF15 region joined to an Fc
region via a linker, wherein the GDF15 region comprises the amino
acid sequence of SEQ ID NO 6 and at least one mutation, wherein at
least one of the mutations is of the asparagine at position 3 or
the aspartate at position 5, and wherein the linker is a
(G.sub.4Q).sub.n linker and n is greater than 2.
10. The fusion protein of claim 7 or 9, wherein the n is 3 or
4.
11. The fusion protein of claim 9 or 10, wherein the GDF15 region
comprises a mutation of aspartate at position 5 to glutamate.
12. The fusion protein of claim 11, wherein the GDF15 region
comprises the amino acid sequence of SEQ ID NO: 16.
13. The fusion protein of any one of claims 9-11, wherein the GDF15
region comprises a mutation of the asparagine at position 3 to
glutamine.
14. The fusion protein of claim 13, wherein the GDF15 region
comprises the amino acid sequence of SEQ ID NO: 18.
15. The fusion protein of any one of claims 1-14, wherein the Fc
region comprises a charged pair mutation.
16. The fusion protein of any one of claims 1-14, wherein the Fc
region comprises a truncated hinge region.
17. The fusion protein of any one of claims 1-10, wherein the Fc
region is selected from Table 3.
18. A method for treating a metabolic condition or disorder
comprising administering a a fusion protein of any one of claims
1-17.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 17/148,761, filed on Jan. 14, 2021, which is a continuation of
U.S. application Ser. No. 16/620,029, filed on Dec. 6, 2019, which
is a U.S. national stage filing under 35 U.S.C. .sctn. 371 of PCT
Application No. PCT/US2019/026369, filed on Apr. 8, 2019, which
claims the benefit of U.S. Provisional Application No. 62/655,108,
filed on Apr. 9, 2018, which are all hereby incorporated by
reference in its entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled A-2239-US-CNT2_SeqList_ST25.txt, created Sep. 20,
2021, which is 109 kb in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] The instant disclosure relates to GDF15 molecules, such as
GDF15 fusion proteins, compositions thereof, and methods for making
and using such proteins.
BACKGROUND
[0004] Growth differentiation factor 15 (GDF15), also referred to
as macrophage inhibitory cytokine 1 (MIC1) (Bootcov M R, 1997, Proc
Natl Acad Sci 94:11514-9), placental bone morphogenetic factor
(PLAB) (Hromas R 1997, Biochim Biophys Acta. 1354:40-4), placental
transforming growth factor beta (PTGFB) (Lawton L N 1997, Gene.
203:17-26), prostate derived factor (PDF) (Paralkar V M 1998, J
Biol Chem. 273:13760-7), and nonsteroidal anti-inflammatory
drug-activated gene (NAG-1) (Baek S J 2001, J Biol Chem. 276:
33384-92), is a secreted protein that circulates in plasma as an
.about.25 kDa homodimer. GDF15 binds to GDNF family receptor
.alpha.-like (GFRAL) with high affinity. GDF15-induced cell
signaling is believed to require the interaction of GFRAL with the
coreceptor RET.
[0005] GDF15 has been linked to multiple biological activities.
Elevated GDF15 has been shown to be correlated with weight loss and
administration of GDF15 has been shown to reduce food intake and
body weight. Accordingly, there is a need for efficacious GDF15
molecules that can be administered as a therapeutic. The present
disclosure provides GDF15 molecules that meets this need and
provide related advantages.
SUMMARY
[0006] Provided herein are GDF15 molecules, methods of making the
molecules and methods of using the molecules. In some embodiments,
the GDF15 molecule is a GDF15-Fc fusion protein. The fusion protein
can comprise a GDF15 region joined to an Fc region. In some
embodiments, the GDF15 region is joined to the Fc via a linker.
[0007] In some embodiments, the GDF15 region comprises the amino
acid sequence of SEQ ID NO: 6 and at least one mutation, such as a
mutation of the asparagine at position 3 (N3), as a mutation of the
aspartate at position 5 (D5), or mutations of the asparagine at
position 3 and the aspartate at position 5. In some embodiments,
the GDF15 region comprises a mutation of the aspartate at position
5 to glutamate (D5E). In some embodiments, the GDF15 region
comprises the amino acid sequence of SEQ ID NO: 16. In some
embodiments, the GDF15 region comprises a mutation of the
asparagine at position 3 to glutamine (N3Q), for example, having an
amino acid sequence SEQ ID NO: 14. In yet other embodiments, the
GDF15 region comprises both N3Q and D5E mutations. In some
embodiments, the GDF15 region comprises the amino acid sequence of
SEQ ID NO: 18.
[0008] In some embodiments, the fusion protein has a linker that is
a G4S (SEQ ID NO: 19) or G4Q (SEQ ID NO: 24) linker, such as a
(G4S)n or (G4Q)n linker, wherein n is greater than 0. In some
embodiments, the fusion protein has a linker that is a G4A (SEQ ID
NO: 58) linker, such as a (G4A)n linker, wherein n is greater than
0. In some embodiments, n is 1 or 2. In some embodiments, n is
greater than 2, such as 3, 4, 5, 6, 7, or 8. In some embodiments,
the linker comprises the amino acid sequence of SEQ ID NO: 19, 20,
21, 22, 23, 24, 25, or 58.
[0009] In some embodiments, the fusion protein has an Fc region
comprises a charged pair mutation. In some embodiments, the Fc
region has a truncated hinge region. In some embodiments, the Fc
region is selected from Table 3.
[0010] Also provided herein are dimers and tetramers comprising the
fusion proteins disclosed herein. In one embodiment, the dimer
comprises a GDF15-Fc fusion comprising the amino acid sequence of
any one of SEQ ID NOs: 39-57. In some embodiments, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57
dimerizes with an Fc domain comprising the amino acid sequence of
SEQ ID NO: 32, 33, 34, 35, 36, or 37, such as shown in Table 6. In
some embodiments, the dimers form tetramers. Methods of producing
and using the GDF15 molecules disclosed herein are also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graph showing the effect on the body weight of
cynomologus monkeys dosed with vehicle, 3 mg/kg of the positive
control FGF21-Fc, 1.5 mg/kg of scFc-GDF15, or 1.5 mg/kg of the
dimer Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K) weekly for six
weeks, followed by a five-week washout.
[0012] FIG. 2 is a plot showing the effect on the triglyceride
levels of cynomologus monkeys dosed with vehicle, 3 mg/kg of the
positive control FGF21-Fc, 1.5 mg/kg of scFc-GDF15, or 1.5 mg/kg of
the dimer Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K) weekly for
six weeks, followed by a five-week washout.
[0013] FIG. 3 shows the profile of Fc.DELTA.10(-)-(G4S)4-GDF15
after cation exchange.
[0014] FIG. 4 is a peptide map of Fc.DELTA.10(+)-(G4)-GDF15.
[0015] FIG. 5 is a graph showing the effect on food intake in mice
as a function of dose of the dimers
Fc.DELTA.10(-)-GDF15(.DELTA.3):Fc.DELTA.10(+,K) (SEQ ID NOs: 41 and
32); Fc.DELTA.10(-)-GDF15(N3D):Fc.DELTA.10(+,K) (SEQ ID NOs: 42 and
32); Fc.DELTA.10(-,CC)-GDF15(.DELTA.3):Fc.DELTA.10(+,K,CC) (SEQ ID
NOs: 43 and 34); Fc.DELTA.10(-,CC)-GDF15(N3D):Fc.DELTA.10(+,K,CC)
(SEQ ID NOs: 44 and 34)) and
Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K) (SEQ ID NOs:39 and
32).
[0016] FIG. 6 is a graph of the serum concentration of
Fc.DELTA.10(-)-GDF15(.DELTA.3) (SEQ ID NO: 41);
Fc.DELTA.10(-)-GDF15(N3D) (SEQ ID NO: 42);
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3) (SEQ ID NO: 43); and
Fc.DELTA.10(-,CC)-GDF15(N3D) (SEQ ID NO: 44)) as a function of time
in mice.
[0017] FIG. 7 is a graph showing the effect on the body weight of
cynomologus monkeys dosed with vehicle, 3 mg/kg of the positive
control FGF21-Fc, 0.5 mg/kg or 3.0 mg/kg of
Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E):Fc.DELTA.16(+,K,CC) (SEQ ID
NOs: 45 and 35), 0.5 mg/kg or 3.0 mg/kg of
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E):Fc.DELTA.16(+,K,CC)) (SEQ ID NOs:
46 and 35) or 0.5 mg/kg or 3.0 mg/kg of
Fc.DELTA.16(-)-GDF15(N3Q/D5E):Fc.DELTA.16(+,K) (SEQ ID NOs: 47 and
36) weekly for four weeks, followed by a four-week washout.
[0018] FIG. 8 is a graph showing the effect on the body weight of
cynomologus monkeys dosed with vehicle, 1.5 mg/kg of
Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K) (SEQ ID NOs: 39 and
32), 1.5 mg/kg of Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q):Fc.DELTA.16(+,K)
(SEQ ID NOs: 49 and 36); 1.5 mg/kg of
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.16(+,K) (SEQ ID NOs:
50 and 36), 1.5 mg/kg of
Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E):Fc.DELTA.16(+,K) (SEQ ID NOs: 54
and 36), or 1.5 mg/kg of
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.10(+,K,L234A/L2-
35A) (SEQ ID NOs: 57 and 37) weekly for two weeks.
[0019] FIG. 9 is a graph of food intake as a function of dose in
ob/ob mice administered
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.16(+,K) (SEQ ID NOs:
50 and 36).
[0020] FIG. 10 is a graph of food intake as a function of dose in
ob/ob mice administered
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.10(+,K,L234A/L2-
35A) (SEQ ID NOs: 57 and 37).
DETAILED DESCRIPTION
[0021] Provided herein are GDF15 molecules, methods of making the
molecules and methods of using the molecules. In some embodiments,
the GDF15 molecule is a GDF15-Fc fusion protein. The fusion protein
can comprise a GDF15 region joined to an Fc region. In some
embodiments, the GDF15 region is joined to the Fc via a linker.
[0022] In some embodiments, the GDF15 region comprises wild type
GDF15. Both the human and murine GDF15 have a signal peptide and
prodomain. The nucleotide sequence for full-length human GDF15
is:
TABLE-US-00001 (SEQ ID NO: 1) atgcccgggc aagaactcag gacggtgaat
ggctctcaga tgctcctggt gttgctggtg ctctcgtggc tgccgcatgg gggcgccctg
tctctggccg aggcgagccg cgcaagtttc ccgggaccct cagagttgca ctccgaagac
tccagattcc gagagttgcg gaaacgctac gaggacctgc taaccaggct gcgggccaac
cagagctggg aagattcgaa caccgacctc gtcccggccc ctgcagtccg gatactcacg
ccagaagtgc ggctgggatc cggcggccac ctgcacctgc gtatctctcg ggccgccctt
cccgaggggc tccccgaggc ctcccgcctt caccgggctc tgttccggct gtccccgacg
gcgtcaaggt cgtgggacgt gacacgaccg ctgcggcgtc agctcagcct tgcaagaccc
caggcgcccg cgctgcacct gcgactgtcg ccgccgccgt cgcagtcgga ccaactgctg
gcagaatctt cgtccgcacg gccccagctg gagttgcact tgcggccgca agccgccagg
gggcgccgca gagcgcgtgc gcgcaacggg gaccactgtc cgctcgggcc cgggcgttgc
tgccgtctgc acacggtccg cgcgtcgctg gaagacctgg gctgggccga ttgggtgctg
tcgccacggg aggtgcaagt gaccatgtgc atcggcgcgt gcccgagcca gttccgggcg
gcaaacatgc acgcgcagat caagacgagc ctgcaccgcc tgaagcccga cacggtgcca
gcgccctgct gcgtgcccgc cagctacaat cccatggtgc tcattcaaaa gaccgacacc
ggggtgtcgc tccagaccta tgatgacttg ttagccaaag actgccactg catatga
[0023] The amino acid sequence for full-length human GDF15 (308
amino acids) is:
TABLE-US-00002 (SEQ ID NO: 2)
MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDS
RFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLH
LRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAP
ALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCP
LGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHA
QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCH CI
[0024] The nucleotide sequence for human GDF15 without its signal
sequence is:
TABLE-US-00003 (SEQ ID NO: 3) ctgtctctgg ccgaggcgag ccgcgcaagt
ttcccgggac cctcagagtt gcactccgaa gactccagat tccgagagtt gcggaaacgc
tacgaggacc tgctaaccag gctgcgggcc aaccagagct gggaagattc gaacaccgac
ctcgtcccgg cccctgcagt ccggatactc acgccagaag tgcggctggg atccggcggc
cacctgcacc tgcgtatctc tcgggccgcc cttcccgagg ggctccccga ggcctcccgc
cttcaccggg ctctgttccg gctgtccccg acggcgtcaa ggtcgtggga cgtgacacga
ccgctgcggc gtcagctcag ccttgcaaga ccccaggcgc ccgcgctgca cctgcgactg
tcgccgccgc cgtcgcagtc ggaccaactg ctggcagaat cttcgtccgc acggccccag
ctggagttgc acttgcggcc gcaagccgcc agggggcgcc gcagagcgcg tgcgcgcaac
ggggaccact gtccgctcgg gcccgggcgt tgctgccgtc tgcacacggt ccgcgcgtcg
ctggaagacc tgggctgggc cgattgggtg ctgtcgccac gggaggtgca agtgaccatg
tgcatcggcg cgtgcccgag ccagttccgg gcggcaaaca tgcacgcgca gatcaagacg
agcctgcacc gcctgaagcc cgacacggtg ccagcgccct gctgcgtgcc cgccagctac
aatcccatgg tgctcattca aaagaccgac accggggtgt cgctccagac ctatgatgac
ttgttagcca aagactgcca ctgcatatga
[0025] The amino acid sequence for human GDF15 without its 29 amino
acid signal sequence (279 amino acids) is:
TABLE-US-00004 (SEQ ID NO: 4)
LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDL
VPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTA
SRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLEL
HLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPR
EVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLAKDCHCI
[0026] The nucleotide sequence for human GDF15 without its signal
peptide or prodomain is:
TABLE-US-00005 (SEQ ID NO: 5)
gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctg
cacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctg
tcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccag
ttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctg
aagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatccc
atggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgat
gacttgttagccaaagactgccactgcatatga
[0027] The amino acid sequence for human GDF15 without its signal
peptide or pro-domain (the active domain of GDF15 of 112 amino
acids) is:
TABLE-US-00006 (SEQ ID NO: 6)
ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQ
FRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DLLAKDCHCI
[0028] The nucleotide sequence for full-length murine GDF15 is:
TABLE-US-00007 (SEQ ID NO: 7) atggccccgc ccgcgctcca ggcccagcct
ccaggcggct ctcaactgag gttcctgctg ttcctgctgc tgttgctgct gctgctgtca
tggccatcgc agggggacgc cctggcaatg cctgaacagc gaccctccgg ccctgagtcc
caactcaacg ccgacgagct acggggtcgc ttccaggacc tgctgagccg gctgcatgcc
aaccagagcc gagaggactc gaactcagaa ccaagtcctg acccagctgt ccggatactc
agtccagagg tgagattggg gtcccacggc cagctgctac tccgcgtcaa ccgggcgtcg
ctgagtcagg gtctccccga agcctaccgc gtgcaccgag cgctgctcct gctgacgccg
acggcccgcc cctgggacat cactaggccc ctgaagcgtg cgctcagcct ccggggaccc
cgtgctcccg cattacgcct gcgcctgacg ccgcctccgg acctggctat gctgccctct
ggcggcacgc agctggaact gcgcttacgg gtagccgccg gcagggggcg ccgaagcgcg
catgcgcacc caagagactc gtgcccactg ggtccggggc gctgctgtca cttggagact
gtgcaggcaa ctcttgaaga cttgggctgg agcgactggg tgctgtcccc gcgccagctg
cagctgagca tgtgcgtggg cgagtgtccc cacctgtatc gctccgcgaa cacgcatgcg
cagatcaaag cacgcctgca tggcctgcag cctgacaagg tgcctgcccc gtgctgtgtc
ccctccagct acaccccggt ggttcttatg cacaggacag acagtggtgt gtcactgcag
acttatgatg acctggtggc ccggggctgc cactgcgctt ga
[0029] The amino acid sequence for full-length murine GDF15 (303
amino acids) is:
TABLE-US-00008 (SEQ ID NO: 8)
MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPESQ
LNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHGQL
LLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAP
ALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGR
CCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKAR
LHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGCHCA
[0030] The nucleotide sequence for murine GDF15 without its signal
sequence is:
TABLE-US-00009 (SEQ ID NO: 9)
tcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctga
gtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctga
gccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagt
cctgacccagctgtccggatactcagtccagaggtgagattggggtccca
cggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctcc
ccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcc
cgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccgggg
accccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctgg
ctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagcc
gccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgccc
actgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttg
aagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctg
agcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgca
tgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctg
ccccgtgctgtgtcccctccagctacaccccggtggacttatgcacagga
cagacagtggtgtgtcactgcagacttatgatgacctggtggcccggggc
tgccactgcgcttga
[0031] The amino acid sequence for murine GDF15 without its 32
amino acid signal sequence (271 amino acids) is:
TABLE-US-00010 (SEQ ID NO: 10)
SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSP
DPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARP
WDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGR
GRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCV
GECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGV
SLQTYDDLVARGCHCA
[0032] The nucleotide sequence for murine GDF15 without its signal
sequence or pro-domain is:
TABLE-US-00011 (SEQ ID NO: 11)
agcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgc
tgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgac
tgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgt
ccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctg
catggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagc
tacaccccggtggacttatgcacaggacagacagtggtgtgtcactgcaga
cttatgatgacctggtggcccggggctgccactgcgcttga
[0033] The amino acid sequence for murine GDF15 without its signal
peptide or prodomain (active domain of 115 amino acids) is:
TABLE-US-00012 (SEQ ID NO: 12)
SAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGEC
PHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQ
TYDDLVARGCHCA
[0034] In some embodiments, the GDF15 molecule comprises a GDF15
region comprising an active domain of GDF15, e.g., GDF15 without
its signal peptide or pro-domain. In some embodiments, the GDF15
region comprises the amino acid sequence of SEQ ID NO: 6 or 12. In
some embodiments, the GDF15 region comprises a GDF15 sequence with
one or more mutations, such as at least one mutation in the active
domain of GDF15. In particular embodiments, the mutation or
mutations do not reduce or eliminate the activity of GDF15. In some
embodiments, the GDF15 region comprises a mutation in the active
domain of human GDF15. In one embodiment, the mutation is a
deletion of the first three amino acids of the active domain, such
as "GDF15(.DELTA.3)" which is an active domain of human GDF15 in
which the first three amino acids removed (i.e., SEQ ID NO:
13).
[0035] In some embodiments, the GDF15 region comprises a mutation
of the asparagine at position 3 (N3) of the active domain of human
GDF15 (SEQ ID NO: 6). An N3 mutation can refer to the mutation of
the asparagine residue at position 3 of SEQ ID NO: 6 or the
mutation of an asparagine residue corresponding to the asparagine
at position 3 of SEQ ID NO: 6 in a GDF15 amino acid sequence. In
some embodiments, the asparagine at position 3 is mutated to
glutamine (N3Q) or aspartate (N3D). Accordingly, in some
embodiments, the GDF15 molecule comprises a GDF15 region of
GDF15(N3Q), which has the amino acid sequence of SEQ ID NO: 14. In
other embodiments, the GDF15 molecule comprises a GDF15 region of
GDF15(N3D), which has the amino acid sequence of SEQ ID NO: 15. In
some embodiments, the GDF15 region comprises a mutation of the
aspartate at position 5 (D5) of the active domain of human GDF15
(SEQ ID NO: 6). A D5 mutation can refer to the mutation of the
aspartate residue at position 5 of SEQ ID NO: 6 or the mutation of
an aspartate residue corresponding to the aspartate at position 5
of SEQ ID NO: 6 in a GDF15 amino acid sequence. In one embodiment,
the aspartate at position 5 is mutated to glutamate (D5E).
Accordingly, in some embodiments, the GDF15 molecule comprises a
GDF15 region of GDF15(D5E), which has the amino acid sequence of
SEQ ID NO: 16.
[0036] In yet other embodiments, the GDF15 region comprises a
combination of mutations, such as a combination of .DELTA.3 and D5
mutations, e.g., GDF15(.DELTA.3/D5E) (SEQ ID NO: 17) or a
combination of N3 and D5 mutations, e.g., GDF15(N3D/D5E) or
GDF15(N3Q/D5E). In, the GDF15 region comprises the amino acid
sequence of SEQ ID NO: 18.
[0037] Table 1 provides examples of GDF15 regions that can be used
in the GDF15 molecules.
TABLE-US-00013 TABLE 1 GDF15 Regions SEQ ID NO: Designation
Sequence 6 GDF15 ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD
TVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI 13 GDF15(.DELTA.3)
GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPRE
VQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC HCI First third amino acids at
N-terminus of GDF15 sequence (SEQ ID NO: 6) is deleted in this
GDF15 region. 14 GDF15(N3Q) ARQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD
TVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI Underlined and bolded
residue is N3Q mutation. 15 GDF15(N3D)
ARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD
TVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI Underlined and bolded
residue is N3D mutation. 16 GDF15(D5E)
ARNGEHCPLGPGRCCRLHTVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD
TVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI Underlined and bolded
residue is D5E mutation. 17 GDF15(.DELTA.3/D5E)
GEHCPLGPGRCCRLHTVRASLEDLGWADWVLSPRE
VQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP
APCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC HCI First third amino acids at
N-terminus of GDF15 sequence (SEQ ID NO: 6) is deleted in this
GDF15 region; underlined and bolded residue is D5E mutation
(position in reference to wild-type GDF15 sequence of SEQ ID NO:
6). 18 GDF15(N3Q/D5E) ARQGEHCPLGPGRCCRLHTVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD
TVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI Underlined and bolded
residues are N3Q and D5E mutations.
[0038] In some embodiments, the GDF15 molecule is fused to an Fc
directly. In other embodiments, the Fc is fused to the GDF15
molecule via a linker. In some embodiments, the linker comprises a
G4S (SEQ ID NO: 19) linker. In other embodiments, the linker
comprises a G4Q (SEQ ID NO: 24) linker. In other embodiments, the
linker comprises a G4A (SEQ ID NO: 58) linker. The linker can be a
(G4S)n or (G4Q)n linker, wherein n is greater than 0. The linker
can be a (G4A)n linker, wherein n is greater than 0. In some
embodiments, n is 1 or 2. In some embodiments, n is greater than or
equal to 2, such as 3, 4, 5, 6, 7, or 8. In some embodiments, the
linker comprises the amino acid sequence of SEQ ID NO: 19, 20, 21,
22, 23, 24, 25, or 58 as shown in Table 2.
TABLE-US-00014 TABLE 2 Linkers SEQ ID NO: Designation Sequence 19
G4S GGGGS 20 (G4S)2 GGGGSGGGGS 21 (G4S)4 GGGGSGGGGSGGGGSGGGGS 22
(G4S)8 GGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGS 23 G4 GGGG 24 G4Q
GGGGQ 25 (G4Q)4 GGGGQGGGGQGGGGQGGGGQ 58 G4A GGGGA
[0039] In some embodiments, the GDF15 molecule comprises an Fc
region. The Fc region can comprise or be derived from the Fc domain
of a heavy chain of an antibody. In some embodiments, the Fc region
may comprise an Fc domain with a mutation, such as a charged pair
mutation, a mutation in a glycosylation site or the inclusion of an
unnatural amino acid. The Fc region can be derived from a human IgG
constant domain of IgG1, IgG2, IgG3 or IgG4. In some embodiments,
the Fc region comprises the constant domain of an IgA, IgD, IgE,
and IgM heavy chain.
[0040] In some embodiments, the Fc region comprises an Fc domain
with a charged pair mutation. By introducing a mutation resulting
in a charged Fc region, the GDF15 molecule can dimerize with a
corresponding Fc molecule having the opposite charge. For example,
an aspartate-to-lysine mutation (E356K, wherein 356 is the position
using EU numbering, and corresponds to the positions as noted in
Tables 3-5) and a glutamate-to-lysine mutation (D399K wherein 399
is the position using EU numbering, and corresponds to positions as
noted in Tables 3-5) can be introduced into the Fc region that is
joined to a GDF15 region, optionally via a linker, resulting in a
positively charged Fc region for the GDF15 molecule.
Lysine-to-aspartate mutations (K392D, K409D; wherein 392 and 409
are the positions using EU numbering and corresponds to the
positions as noted in Tables 3-5) can be introduced into an Fc
domain of a separate molecule, resulting in a negatively charged Fc
molecule. The aspartate residues in the negatively charged Fc
molecule can associate with the lysine residues of the positively
charged Fc region of the GDF15 molecule through electrostatic
force, facilitating formation of Fc heterodimers between the Fc
region of the GDF15 molecule and the Fc molecule, while reducing or
preventing formation of Fc homodimers between the Fc regions of the
GDF15 molecules or between Fc molecules.
[0041] In some embodiments, one or more lysine-to-aspartate
mutations (K392D, K409D) are introduced into the Fc region that is
joined to a GDF15 region, optionally via a linker and an
aspartate-to-lysine mutation (E356K) and a glutamate-to-lysine
mutation (D399K) is introduced into the Fc domain of another
molecule. The aspartate residues in the Fc region of the GDF15
molecule can associate with the lysine residues of the Fc molecule
through electrostatic force, facilitating formation of Fc
heterodimers between the Fc region of the GDF15 molecule and the Fc
molecule, and reducing or preventing formation of Fc homodimers
between the Fc regions of the GDF15 molecules or between Fc
molecules.
[0042] In some embodiments, the GDF15 molecule comprises an Fc
region comprising an Fc domain with a mutated hinge region. In some
embodiments, the Fc domain comprises a deletion in the hinge. In
some embodiments, ten amino acids from the hinge are deleted, e.g.,
Fc.DELTA.10. In other embodiments, sixteen amino acids from the
hinge are deleted, e.g., Fc.DELTA.16. In some embodiments, the Fc
domain comprises a hinge deletion (e.g., Fc.DELTA.10 or
Fc.DELTA.16) and a charged pair mutation, such that the Fc domain
is positively or negatively charged. For example, the Fc domain can
comprise a ten-amino acid deletion in the hinge and
lysine-to-aspartate mutations (K392D, K409D), such as
Fc.DELTA.10(-). In another embodiment, the Fc domain can comprise a
ten-amino acid deletion in the hinge and an aspartate-to-lysine
mutation (E356K) and a glutamate-to-lysine mutation (D399K), such
as an Fc.DELTA.10(+). In another embodiment, the Fc domain can
comprise a sixteen-amino acid deletion in the hinge and
lysine-to-aspartate mutations (K392D, K409D), such as
Fc.DELTA.16(-). In another embodiment, the Fc domain can comprise a
sixteen-amino acid deletion in the hinge and an aspartate-to-lysine
mutation (E356K) and a glutamate-to-lysine mutation (D399K), such
as an Fc.DELTA.16(+).
[0043] In some embodiments, an Fc molecule comprising a hinge
deletion and a charged pair mutation heterodimerizes with such a
GDF15 molecule. For example, the Fc molecule can have a hinge
deletion and charged pair mutation that complements the hinge
deletion and charged pair mutation of the Fc region of a GDF15
molecule. For example, an Fc molecule can comprise an Fc domain
with a ten-amino acid deletion in the hinge and lysine-to-aspartate
mutations (K392D, K409D), such as Fc.DELTA.10(-), which can
optionally comprise a C-terminal lysine (e.g., Fc.DELTA.10(-, K)).
The Fc molecule can heterodimerize with a GDF15 molecule that
comprises an Fc.DELTA.10(+). In another embodiment, the Fc molecule
can comprise a ten-amino acid deletion in the hinge and an
aspartate-to-lysine mutation (E356K) and a glutamate-to-lysine
mutation (D399K), such as an Fc.DELTA.10(+), which can optionally
comprise a C-terminal lysine (e.g., Fc.DELTA.10(+, K)). The Fc
molecule can heterodimerize with a GDF15 molecule that comprises an
Fc.DELTA.10(-). In another embodiment, the Fc molecule can comprise
a sixteen-amino acid deletion in the hinge and lysine-to-aspartate
mutations (K392D, K409D), such as Fc.DELTA.16(-), which can
optionally comprise a C-terminal lysine (e.g., Fc.DELTA.16(-, K)).
The Fc molecule which can heterodimerize with a GDF15 molecule that
comprises an Fc.DELTA.16(+). In another embodiment, the Fc molecule
can comprise a sixteen-amino acid deletion in the hinge and an
aspartate-to-lysine mutation (E356K) and a glutamate-to-lysine
mutation (D399K), such as an Fc.DELTA.16(+), which can optionally
comprise a C-terminal lysine (e.g., Fc.DELTA.16(-, K)). The Fc
molecule can heterodimerize with a GDF15 molecule that comprises an
Fc.DELTA.16(-).
[0044] In some embodiments, the Fc region or Fc molecule comprises
an Fc domain with an L234A and/or L235A mutation, wherein 234 and
235 are the positions using EU numbering and corresponds to the
positions as noted in Tables 3-5. The Fc domain can comprise an
L234A mutation, an L235A mutation, a charged pair mutation, a hinge
deletion, or any combination thereof. In some embodiments, the Fc
domain comprises both an L234A mutation and an L235A mutation. In
some embodiments, the Fc domain comprises a hinge deletion, an
L234A mutation, an L235A mutation, and a charged pair mutation,
such as Fc.DELTA.10(+, L234A/L235A), Fc.DELTA.10(-, L234A/L235A),
Fc.DELTA.16(+, L234A/L235A), or Fc.DELTA.16(-, L234A/L235A). In
some embodiments, the Fc domain comprises an optional C-terminal
lysine, e.g., Fc.DELTA.10(+,K,L234A/L235A),
Fc.DELTA.10(-,K,L234A/L235A), Fc.DELTA.16(+,K,L234A/L235A), or
Fc.DELTA.16(-,K,L234A/L235A).
[0045] In some embodiments, the Fc region or Fc molecule comprises
an Fc domain with a "cysteine clamp." A cysteine clamp mutation
involves the introduction of a cysteine into the Fc domain at a
specific location through mutation so that when incubated with
another Fc domain that also has a cysteine introduced at a specific
location through mutation, a disulfide bond (cysteine clamp) may be
formed between the two Fc domains (e.g., between an Fc.DELTA.16(+)
domain having a "cysteine clamp" mutation and an Fc.DELTA.16(-)
domain having a "cysteine clamp" mutation). The cysteine can be
introduced into the CH3 domain of an Fc domain. In some
embodiments, an Fc domain may contain one or more such cysteine
clamp mutations. In one embodiment, a cysteine clamp is provided by
introducing a serine to cysteine mutation (S354C, wherein 354 is
the position using EU numbering, and corresponds to the position as
noted in Tables 3-5) into a first Fc domain and a tyrosine to
cysteine mutation (Y349C, wherein 349 is the position using EU
numbering, and corresponds to the position as noted in Tables 3-5)
into a second Fc domain. In one embodiment, a GDF15 molecule
comprises an Fc region comprising an Fc domain with a cysteine
clamp, a negatively charged pair mutation and a sixteen-amino acid
hinge deletion (e.g., GDF15-Fc.DELTA.16(-,CC)), and an Fc molecule
comprising an Fc domain comprising a cysteine clamp, a positively
charged pair mutation and a sixteen-amino acid hinge deletion, and
an optional C-terminal lysine (e.g., Fc.DELTA.16(+,K,CC)). The
cysteine clamp may augment the heterodimerization of the GDF-Fc
molecule with the Fc molecule.
[0046] Examples of Fc regions that can be used in a GDF15 molecule
are shown in Table 3.
TABLE-US-00015 TABLE 3 Fc Regions SEQ ID NO: Designation Sequence
26 Fc.DELTA.10(-) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYDT TPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG Underlined and bolded residues are K392D and
K409D mutations. 27 Fc.DELTA.10(+)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG Underlined and bolded residues are E356K and
D399K mutations. 28 Fc.DELTA.10(-, CC)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYDT TPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG Underlined and italicized residue is Y349C
mutation;underlined and bolded residues are K392D and K409D
mutations. 29 Fc.DELTA.16(-, CC) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVCTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYDTTPPVLDSD GSFFLYSDLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPG Underlined and italicized residue is Y349C
mutation;underlined and bolded residues are K392D and K409D
mutations. 30 FcA16(-) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYDTTPPVLDSD GSFFLYSDLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPG Underlined and bolded residues are K392D and K409D
mutations. 31 FcA10(-, APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
L234A/L235A) DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYDT TPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG Underlined and italicized residues are L234A
and L235A mutations; underlined and bolded residues are K392D and
K409D mutations.
[0047] Examples of Fc molecules are shown in Table 4, in which the
C-terminal lysine is optional.
TABLE-US-00016 TABLE 4 Fc Molecules SEQ ID NO: Designation Sequence
32 Fc.DELTA.10(+, K) APELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRKEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLKSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
Underlined and bolded residues are E356K and D399K mutations. 33
Fc.DELTA.10(-, K) APELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYDTTPPVLDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
Underlined and bolded residues are K392D and K409D mutations. 34
Fc.DELTA.10(+, K, CC) APELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPP CRKEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLKSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
Underlined and italicized residue is S354C mutation; underlined and
bolded residues are E356K and D399K mutations. 35 Fc.DELTA.16(+, K,
CC) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCRKEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLKSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK Underlined and italicized residue is
S354C mutation; underlined and bolded residues are E356K and D399K
mutations. 36 Fc.DELTA.16(+, K) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRKEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLKSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK
Underlined and bolded residues are E356K and D399K mutations. 37
Fc.DELTA.10(+, K, APEAAGGPSVFLFPPKPKDTLMISRTPEVTCV L234A/L235A)
VVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRKEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLKSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
Underlined and italicized residues are L234A and L235A mutations;
underlined and bolded residues are E356K and D399K mutations.
[0048] The Fc molecules can be used to dimerize with a molecule
comprising a complementary Fc domain. For example, an Fc molecule
of Fc.DELTA.10(+,K) can dimerize with a molecule comprising an Fc
region comprising a ten-amino acid hinge deletion and a negatively
charged pair mutation such as Fc.DELTA.10(-) (e.g., a GDF15
molecule comprising an Fc region of Fc.DELTA.10(-)). An Fc molecule
of Fc.DELTA.10(-,K) can dimerize with a molecule comprising an Fc
region comprising a ten-amino acid hinge deletion and a negatively
charged pair mutation such as Fc.DELTA.10(+) (e.g., a GDF15
molecule comprising an Fc region of Fc.DELTA.10(+)).
[0049] An Fc molecule of Fc.DELTA.10(+,K,CC) can dimerize with a
molecule comprising an Fc region comprising a ten-amino acid hinge
deletion and a negatively charged pair mutation such as
Fc.DELTA.10(-,CC) (e.g., a GDF15 molecule comprising an Fc region
of Fc.DELTA.10(-, CC)). An Fc molecule of Fc.DELTA.16(+,K,CC) can
dimerize with a molecule comprising an Fc region comprising a
ten-amino acid hinge deletion and a negatively charged pair
mutation such as Fc.DELTA.16(-, CC) (e.g., a GDF15 molecule
comprising an Fc region of Fc.DELTA.16(-, CC)). An Fc molecule of
Fc.DELTA.16(+,K) can dimerize with a molecule comprising an Fc
region comprising a ten-amino acid hinge deletion and a negatively
charged pair mutation such as Fc.DELTA.16(-) (e.g., a GDF15
molecule comprising an Fc region of Fc.DELTA.16(+)). An Fc molecule
of Fc.DELTA.10(+,K,L234A/L235A) can dimerize with a molecule
comprising an Fc region comprising a ten-amino acid hinge deletion
and a negatively charged pair mutation such as
Fc.DELTA.10(-,L234A/L235A) (e.g., a GDF15 molecule comprising an Fc
region of Fc.DELTA.10(-, L234A/L235A)).
[0050] Examples of GDF15 molecules that are GDF15-Fc fusion
proteins are shown in Table 5.
TABLE-US-00017 TABLE 5 GDF15 Molecules GDF15-Fc Fusion Protein
Components GDF15-Fc Fusion Protein SEQ ID NOs SEQ Fc GDF15 ID NO.
Designation Sequence Region Linker Region 38 scFc7-
GGGERKSSVECPPCPAPP -- -- -- GDF15 VAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNA KTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMT
KNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGG GGGSGGGGSGGGGSGGG
GSGGGGSGGGGSGGGGS GGGGSERKSSVECPPCPA PPVAGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGS GGGGSGGGGSGGGGSGG
GGSARNGDHCPLGPGRC CRLHTVRASLEDLGWAD WVLSPREVQVTMCIGACP
SQFRAANMHAQIKTSLHR LKPDTVPAPCCVPASYNP MVLIQKTDTGVSLQTYDD LLAKDCHCI
39 Fc.DELTA.10(-)- APELLGGPSVFLFPPKPKD 26 21 6 (G4S)4-
TLMISRTPEVTCVVVDVS GDF15 HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS
DLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGG
GSGGGGSARNGDHCPLG PGRCCRLHTVRASLEDLG WADWVLSPREVQVTMCI
GACPSQFRAANMHAQIKT SLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ
TYDDLLAKDCHCI Underlined and bolded residues are K392D and K409D
mutations. 40 Fc.DELTA.10(+)- APELLGGPSVFLFPPKPKD 27 23 6
(G4)-GDF15 TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR
KEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YKTTPPVLKSDGSFFLYS
KLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGGGGGARNGDHCPL
GPGRCCRLHTVRASLEDL GWADWVLSPREVQVTM CIGACPSQFRAANMHAQI
KTSLHRLKPDTVPAPCCV PASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCI Underlined
and and bolded residues are E356K and D399K mutations. 41 FcA10(-)-
APELLGGPSVFLFPPKPKD 26 -- 13 GDF15(.DELTA.3) TLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLS LSPGGDHCPLGPGRCCRL HTVRASLEDLGWADWVL
SPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLA KDCHCI Underlined and bolded residues are K392D
and K409D mutations. 42 FcA10(-)- APELLGGPSVFLFPPKPKD 26 -- 15
GDF15(N3D) TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS
DLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGARDGDHCPLGPGRC
CRLHTVRASLEDLGWAD WVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHR
LKPDTVPAPCCVPASYNP MVLIQKTDTGVSLQTYDD LLAKDCHCI Underlined and
bolded residues are K392D and K409D mutations. 43 FcA10(-,CC)-
APELLGGPSVFLFPPKPKD 28 -- 13 GDF15(.DELTA.3) TLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSR EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLS LSPGGDHCPLGPGRCCRL HTVRASLEDLGWADWVL
SPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLA KDCHCI Underlined and italicized residue is
Y349C mutation; underlined and bolded residues are K392D and K409D
mutations. 44 Fc.DELTA.10(-,CC)- APELLGGPSVFLFPPKPKD 28 -- 15
GDF15(N3D) TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSR
EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS
DLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGARDGDHCPLGPGRC
CRLHTVRASLEDLGWAD WVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHR
LKPDTVPAPCCVPASYNP MVLIQKTDTGVSLQTYDD LLAKDCHCI Underlined and
italicized residue is Y349C mutation; underlined and bolded
residues are K392D and K409D mutations. 45 Fc.DELTA.16(-,CC)-
GPSVFLFPPKPKDTLMISR 29 -- 17 GDF15(.DELTA.3/D5E) TPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP REPQVCTLPPSREEMTKN QVSLTCLVKGFYPSDIAV
EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGGEHC PLGPGRCCRLHTVRASLE DLGWADWVLSPREVQVT
MCIGACPSQFRAANMHA QIKTSLHRLKPDTVPAPCC VPASYNPMVLIQKTDTGV
SLQTYDDLLAKDCHCI Underlined and italicized residue is Y349C
mutation; underlined and bolded residues are K392D and K409D
mutations. 46 Fc.DELTA.16(-,CC)- GPSVFLFPPKPKDTLMISR 29 -- 18
GDF15(N3Q/ TPEVTCVVVDVSHEDPEV D5E) KFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP
REPQVCTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV
LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGARQG
EHCPLGPGRCCRLHTVRA SLEDLGWADWVLSPREV QVTMCIGACPSQFRAAN
MHAQIKTSLHRLKPDTVP APCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCI
Underlined and italicized residue is Y349C mutation; underlined and
bolded residues are K392D and K409D mutations. 47 Fc.DELTA.16(-)-
GPSVFLFPPKPKDTLMISR 30 -- 18 GDF15(N3Q/ TPEVTCVVVDVSHEDPEV D5E)
KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV
EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGARQG
EHCPLGPGRCCRLHTVRA SLEDLGWADWVLSPREV QVTMCIGACPSQFRAAN
MHAQIKTSLHRLKPDTVP APCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCI
Underlined and bolded residues are K392D and K409D mutations. 48
Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 25 6 (G4Q)4-
TPEVTCVVVDVSHEDPEV GDF15 KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS
RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG QGGGGQGGGGQGGGGQ
ARNGDHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQF
RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCI
Underlined and bolded residues are K392D and K409D mutations. 49
Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 25 14 (G4Q)4-
TPEVTCVVVDVSHEDPEV GDF15(N3Q) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS
RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG QGGGGQGGGGQGGGGQ
ARQGDHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQF
RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCI
Underlined and bolded residues are K392D and K409D mutations. 50
Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 25 18 (G4Q)4-
TPEVTCVVVDVSHEDPEV GDF15(N3Q/ KFNWYVDGVEVHNAKT D5E)
KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV
LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG
QGGGGQGGGGQGGGGQ ARQGEHCPLGPGRCCRLH TVRASLEDLGWADWVLS
PREVQVTMCIGACPSQFR AANMHAQIKTSLHRLKPD TVPAPCCVPASYNPMVLI
QKTDTGVSLQTYDDLLA KDCHCI Underlined and bolded residues are K392D
and K409D mutations. 51 Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 20
14 (G4S)2- TPEVTCVVVDVSHEDPEV GDF15(N3Q) KFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV
LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG
SGGGGSARQGDHCPLGP GRCCRLHTVRASLEDLG WADWVLSPREVQVTMCI
GACPSQFRAANMHAQIKT SLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ
TYDDLLAKDCHCI Underlined and bolded residues are K392D and K409D
mutations. 52 Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 20 18 (G4S)2-
TPEVTCVVVDVSHEDPEV GDF15(N3Q/ KFNWYVDGVEVHNAKT D5E)
KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV
LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG
SGGGGSARQGEHCPLGPG RCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIG
ACPSQFRAANMHAQIKTS LHRLKPDTVPAPCCVPAS YNPMVLIQKTDTGVSLQT
YDDLLAKDCHCI Underlined and bolded residues are K392D and K409D
mutations. 53 Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 19 14 G4S-
TPEVTCVVVDVSHEDPEV GDF15(N3Q) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS
RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG SARQGDHCPLGPGRCCRL
HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP
DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCI Underlined and bolded
residues are K392D and K409D mutations. 54 Fc.DELTA.16(-)-
GPSVFLFPPKPKDTLMISR 30 19 18 G4S- TPEVTCVVVDVSHEDPEV GDF15(N3Q/
KFNWYVDGVEVHNAKT D5E) KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV
EWESNGQPENNYDTTPPV LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGGGGG SARQGEHCPLGPGRCCRL HTVRASLEDLGWADWVL
SPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL
IQKTDTGVSLQTYDDLLA KDCHCI Underlined and bolded residues are K392D
and K409D mutations. 55 Fc.DELTA.16(-)- GPSVFLFPPKPKDTLMISR 30 --
14 GDF15(N3Q) TPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNYDTTPPV
LDSDGSFFLYSDLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGARQG
DHCPLGPGRCCRLHTVRA SLEDLGWADWVLSPREV QVTMCIGACPSQFRAAN
MHAQIKTSLHRLKPDTVP APCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCI
Underlined and bolded residues are K392D and K409D mutations. 56
Fc.DELTA.10(-, APEAAGGPSVFLFPPKPKD 31 25 14 L234A/L235A)-
TLMISRTPEVTCVVVDVS (G4Q)4- HEDPEVKFNWYVDGVEV GDF15(N3Q)
HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN
YDTTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS
LSPGGGGGQGGGGQGGG GQGGGGQARQGDHCPLG PGRCCRLHTVRASLEDLG
WADWVLSPREVQVTMCI GACPSQFRAANMHAQIKT SLHRLKPDTVPAPCCVPA
SYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI Underlined and italicized residues
are L234A and L235A mutations; underlined and bolded residues are
K392D and K409D mutations. 57 Fc.DELTA.10(-, APEAAGGPSVFLFPPKPKD 31
25 18 L234A/L235A)- TLMISRTPEVTCVVVDVS (G4Q)4- HEDPEVKFNWYVDGVEV
GDF15(N3Q/ HNAKTKPREEQYNSTYR D5E) VVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLS LSPGGGGGQGGGGQGGG GQGGGGQARQGEHCPLG
PGRCCRLHTVRASLEDLG WADWVLSPREVQVTMCI GACPSQFRAANMHAQIKT
SLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI Underlined and
italicized residues are L234A and L235A mutations; underlined and
bolded residues are K392D and K409D mutations.
[0051] In some embodiments, the fusion protein is an scFc-GDF15 in
which the GDF15 region is joined to two Fc regions. In some
embodiments, the fusion protein comprises an amino acid sequence
that has at least 85%, 90%, 95% or 99% sequence identity to SEQ ID
NO: 38. In some embodiments, the fusion protein comprises an amino
acid sequence of SEQ ID NO: 38. In calculating percent sequence
identity, the sequences being compared are aligned in a way that
gives the largest match between the sequences. A computer program
that can be used to determine percent identity is the GCG program
package, which includes GAP (Devereux et al., (1984) Nucl. Acid
Res. 12:387; Genetics Computer Group, University of Wisconsin,
Madison, Wis.). The computer algorithm GAP can be used to align the
two polypeptides or polynucleotides for which the percent sequence
identity is to be determined. The sequences are aligned for optimal
matching of their respective amino acid or nucleotide (the "matched
span", as determined by the algorithm). A gap opening penalty
(which is calculated as 3.times. the average diagonal, wherein the
"average diagonal" is the average of the diagonal of the comparison
matrix being used; the "diagonal" is the score or number assigned
to each perfect amino acid match by the particular comparison
matrix) and a gap extension penalty (which is usually 1/10 times
the gap opening penalty), as well as a comparison matrix such as
PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In
certain embodiments, a standard comparison matrix (see, Dayhoff et
al., (1978) Atlas of Protein Sequence and Structure 5:345-352 for
the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl.
Acad. Sci. U.S.A. 9:10915-10919 for the BLOSUM 62 comparison
matrix) is also used by the algorithm. Parameters that can be used
for determining percent identity using the GAP program are the
following: [0052] Algorithm: Needleman et al., 1970, J. Mol. Biol.
48:443-453; [0053] Comparison matrix: BLOSUM 62 from Henikoff et
al., 1992, supra; [0054] Gap Penalty: 12 (but with no penalty for
end gaps) [0055] Gap Length Penalty: 4 [0056] Threshold of
Similarity: 0 Certain alignment schemes for aligning two amino acid
sequences can result in matching of only a short region of the two
sequences, and this small aligned region can have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, the selected
alignment method (e.g., the GAP program) can be adjusted if so
desired to result in an alignment that spans at least 50 contiguous
amino acids of the target polypeptide.
[0057] In some embodiments, the GDF15 molecule is
Fc.DELTA.10(-)-(G4S)4-GDF15, Fc.DELTA.10(+)-(G4)-GDF15,
Fc.DELTA.10(-)-GDF15(.DELTA.3), Fc.DELTA.10(-)-GDF15(N3D),
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3), Fc.DELTA.10(-,CC)-GDF15(N3D),
Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E),
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E), Fc.DELTA.16(-)-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4Q)4-GDF15, Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q),
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-G4S-GDF15(N3Q), Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-GDF15(N3Q),
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q), or
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E).
[0058] In some embodiments, the GDF15 molecule comprises the amino
acid sequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, or 57. In some embodiments, the
GDF15 molecule comprises an amino acid sequence that has 80-99%,
85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO: 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or
57. In some embodiments, the GDF15 molecule comprises an amino acid
sequence that has at least 85% sequence identity to SEQ ID NO: 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
or 57. In some embodiments, the GDF15 molecule comprises an amino
acid sequence that has at least 90% sequence identity to SEQ ID NO:
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, or 57. In some embodiments, the GDF15 molecule comprises an
amino acid sequence that has at least 95% sequence identity to SEQ
ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, or 57. In some embodiments, the GDF15 molecule
comprises an amino acid sequence that has at least 99% sequence
identity to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, or 57.
[0059] In some embodiments, the GDF15 molecule is a
Fc.DELTA.10(-)-(G4S)4-GDF15, Fc.DELTA.10(+)-(G4)-GDF15,
Fc.DELTA.10(-)-GDF15(A3), Fc.DELTA.10(-)-GDF15(N3D),
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3), Fc.DELTA.10(-,CC)-GDF15(N3D),
Fc.DELTA.16(-,CC)-GDF15(A3/D5E), Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-GDF15(N3Q/D5E), Fc.DELTA.16(-)-(G4Q)4-GDF15,
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q),
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-G4S-GDF15(N3Q), Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-GDF15(N3Q),
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q), or
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule. In some
embodiments, the GDF15 molecule is a Fc.DELTA.10(-)-(G4S)4-GDF15,
Fc.DELTA.10(+)-(G4)-GDF15, Fc.DELTA.10(-)-GDF15(.DELTA.3),
Fc.DELTA.10(-)-GDF15(N3D), Fc.DELTA.10(-,CC)-GDF15(.DELTA.3),
Fc.DELTA.10(-,CC)-GDF15(N3D),
Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E),
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E), Fc.DELTA.16(-)-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4Q)4-GDF15, Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q),
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-G4S-GDF15(N3Q), Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-GDF15(N3Q),
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q), or Fc.DELTA.10(-,
L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule that has 80-99%,
85%-99%, 90-99%, or 95-99% sequence identity to its Fc region
and/or GDF15 region.
[0060] In some embodiments, the GDF15 molecule is a
Fc.DELTA.10(-)-(G4S)4-GDF15, Fc.DELTA.10(+)-(G4)-GDF15,
Fc.DELTA.10(-)-GDF15(.DELTA.3), Fc.DELTA.10(-)-GDF15(N3D),
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3), Fc.DELTA.10(-,CC)-GDF15(N3D),
Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E),
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E), Fc.DELTA.16(-)-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4Q)4-GDF15, Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q),
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q),
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-G4S-GDF15(N3Q), Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E),
Fc.DELTA.16(-)-GDF15(N3Q),
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q), or
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule that has
at least 85%, 90%, 95% or 99% sequence identity to its Fc region
and/or GDF15 region. For example, a Fc.DELTA.10(-)-(G4S)4-GDF15
molecule with 80-99%, 85%-99%, 90-99%, or 95-99% sequence identity
to its Fc region and/or GDF15 region, includes a GDF15 molecule
with an Fc region that has a ten-amino acid deletion of the hinge
region and a negatively charged pair mutation, and has 80-99%,
85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO: 26
and/or a GDF15 region that has 80-99%, 85%-99%, 90-99%, or 95-99%
sequence identity to SEQ ID NO: 6. For example, a
Fc.DELTA.10(-)-(G4S)4-GDF15 molecule with at least 85%, 90%, 95% or
99% sequence identity to its Fc region and/or GDF15 region,
includes a GDF15 molecule with an Fc region that has a ten-amino
acid deletion of the hinge region and a negatively charged pair
mutation, and has at least 85%, 90%, 95% or 99% sequence identity
to SEQ ID NO: 26 and/or a GDF15 region that has at least 85%, 90%,
95% or 99% sequence identity to SEQ ID NO: 6.
[0061] In another example, a Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E)
molecule with 80-99%, 85%-99%, 90-99%, or 95-99% sequence identity
to its Fc region and/or a GDF15 region, includes a GDF15 molecule
with an Fc region that has a sixteen-amino acid deletion of the
hinge region and a negatively charged pair mutation that has
80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO:
30 and/or a GDF15 region that has 80-99%, 85%-99%, 90-99%, or
95-99% sequence identity to SEQ ID NO: 18. In another example, a
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E) molecule with at least 85%,
90%, 95% or 99% sequence identity to its Fc region and/or a GDF15
region, includes a GDF15 molecule with an Fc region that has a
sixteen-amino acid deletion of the hinge region and a negatively
charged pair mutation that has at least 85%, 90%, 95% or 99%
sequence identity to SEQ ID NO: 30 and/or a GDF15 region that has
at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:
18.
[0062] In yet another example, a
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule with
80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to its Fc
region and/or a GDF15 region, includes a GDF15 molecule with an Fc
region that has a ten-amino acid deletion of the hinge region, a
negatively charged pair mutation and leucine to alanine mutations
at positions 234 and 235 and has 80-99%, 85%-99%, 90-99%, or 95-99%
sequence identity to SEQ ID NO: 31 and/or a GDF15 region that has
80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO:
18. In yet another example, a Fc.DELTA.10(-,
L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule with at least 85%, 90%,
95% or 99% sequence identity to its Fc region and/or a GDF15
region, includes a GDF15 molecule with an Fc region that has a
ten-amino acid deletion of the hinge region, a negatively charged
pair mutation and leucine to alanine mutations at positions 234 and
235 and has at least 85%, 90%, 95% or 99% sequence identity to SEQ
ID NO: 31 and/or a GDF15 region that has at least 85%, 90%, 95% or
99% sequence identity to SEQ ID NO: 18.
[0063] Also provided herein are dimers and tetramers comprising a
GDF15 molecule provided herein. In one embodiment, the dimer
comprises a GDF15-Fc fusion comprising the amino acid sequence of
any one of SEQ ID NOs: 39-57. In some embodiments, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57
dimerizes with an Fc molecule comprising the amino acid sequence of
SEQ ID NO: 32, 33, 34, 35, 36, or 37 (in which the C-terminal
lysine is optional), such as shown in Table 6. For example, in some
embodiments, the dimer is Fc.DELTA.10(-)-(G4S)4-GDF15:
Fc.DELTA.10(+,K). In another embodiment, the dimer is
Fc.DELTA.10(-, L234A/L235A)-(G4Q)4-GDF15(N3Q):
Fc.DELTA.10(+,K,L234A/L235A). In yet another embodiment, the dimer
is
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q):Fc.DELTA.10(+,K,L234A/L235A)-
.
TABLE-US-00018 TABLE 6 Dimers GDF15- Fc Fc Corresponding Fusion
Molecule Fc SEQ ID GDF15-Fc Fusion SEQ Molecule NO. Designation ID
NO. Designation 39 Fc.DELTA.10(-)-(G4S)4-GDF15 32 Fc.DELTA.10(+, K)
40 Fc.DELTA.10(+)-(G4)-GDF15 33 Fc.DELTA.10(-, K) 41
Fc.DELTA.10(-)-GDF15(.DELTA.3) 32 Fc.DELTA.10(+, K) 42
Fc.DELTA.10(-)-GDF15(N3D) 32 Fc.DELTA.10(+, K) 43 Fc.DELTA.10(-,
CC)-GDF15(.DELTA.3) 34 Fc.DELTA.10(+, K, CC) 44 Fc.DELTA.10(-,
CC)-GDF15(N3D) 34 Fc.DELTA.10(+, K, CC) 45 Fc.DELTA.16(-,
CC)-GDF15(.DELTA.3/D5E) 35 Fc.DELTA.16(+, K, CC) 46 Fc.DELTA.16(-,
CC)- 35 Fc.DELTA.16(+, K, CC) GDF15(N3Q/D5E) 47
Fc.DELTA.16(-)-GDF15(N3Q/D5E) 36 Fc.DELTA.16(+, K) 48
Fc.DELTA.16(-)-(G4Q)4-GDF15 36 Fc.DELTA.16(+, K) 49
Fc.DELTA.16(-)-(G4Q)4- 36 Fc.DELTA.16(+, K) GDF15(N3Q) 50
Fc.DELTA.16(-)-(G4Q)4- 36 Fc.DELTA.16(+, K) GDF15(N3Q/D5E) 51
Fc.DELTA.16(-)-(G4S)2- 36 Fc.DELTA.16(+, K) GDF15(N3Q) 52
Fc.DELTA.16(-)-(G4S)2- 36 Fc.DELTA.16(+, K) GDF15(N3Q/D5E) 53
Fc.DELTA.16(-)-G4S-GDF15(N3Q) 36 Fc.DELTA.16(+, K) 54
Fc.DELTA.16(-)-G4S- 36 Fc.DELTA.16(+, K) GDF15(N3Q/D5E) 55
Fc.DELTA.16(-)-GDF15(N3Q) 36 Fc.DELTA.16(+, K) 56 Fc.DELTA.10(-,
L234A/L235A)- 37 Fc.DELTA.10(+, K, (G4Q)4-GDF15(N3Q) L234A/L235A)
57 Fc.DELTA.10(-, L234A/L235A)- 37 Fc.DELTA.10(+, K,
(G4Q)4-GDF15(N3Q/D5E) L234A/L235A)
[0064] In one embodiment, a GDF15-Fc fusion comprising the amino
acid sequence of SEQ ID NO: 39 dimerizes with an Fc molecule
comprising SEQ ID NO: 32 (C-terminal lysine optional). In another
embodiment, a GDF15-Fc fusion comprising the amino acid sequence of
SEQ ID NO: 40 dimerizes with an Fc molecule comprising SEQ ID NO:
33 (C-terminal lysine optional). In another embodiment, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 41
dimerizes with an Fc molecule comprising SEQ ID NO: 32 (C-terminal
lysine optional). In another embodiment, a GDF15-Fc fusion
comprising the amino acid sequence of SEQ ID NO: 42 dimerizes with
an Fc molecule comprising SEQ ID NO: 32 (C-terminal lysine
optional). In another embodiment, a GDF15-Fc fusion comprising the
amino acid sequence of SEQ ID NO: 43 dimerizes with an Fc molecule
comprising SEQ ID NO: 34 (C-terminal lysine optional). In another
embodiment, a GDF15-Fc fusion comprising the amino acid sequence of
SEQ ID NO: 44 dimerizes with an Fc molecule comprising SEQ ID NO:
34 (C-terminal lysine optional). In another embodiment, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 44
dimerizes with an Fc molecule comprising SEQ ID NO: 34 (C-terminal
lysine optional). In another embodiment, a GDF15-Fc fusion
comprising the amino acid sequence of SEQ ID NO: 45 dimerizes with
an Fc molecule comprising SEQ ID NO: 35 (C-terminal lysine
optional). In another embodiment, a GDF15-Fc fusion comprising the
amino acid sequence of SEQ ID NO: 46 dimerizes with an Fc molecule
comprising SEQ ID NO: 35 (C-terminal lysine optional). In another
embodiment, a GDF15-Fc fusion comprising the amino acid sequence of
SEQ ID NO: 47 dimerizes with an Fc molecule comprising SEQ ID NO:
36 (C-terminal lysine optional). In another embodiment, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 48
dimerizes with an Fc molecule comprising SEQ ID NO: 36 (C-terminal
lysine optional). In another embodiment, a GDF15-Fc fusion
comprising the amino acid sequence of SEQ ID NO: 49 dimerizes with
an Fc molecule comprising SEQ ID NO: 36 (C-terminal lysine
optional). In another embodiment, a GDF15-Fc fusion comprising the
amino acid sequence of SEQ ID NO: 50 dimerizes with an Fc molecule
comprising SEQ ID NO: 36 (C-terminal lysine optional). In another
embodiment, a GDF15-Fc fusion comprising the amino acid sequence of
SEQ ID NO: 51 dimerizes with an Fc molecule comprising SEQ ID NO:
36 (C-terminal lysine optional). In another embodiment, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 52
dimerizes with an Fc molecule comprising SEQ ID NO: 36 (C-terminal
lysine optional). In another embodiment, a GDF15-Fc fusion
comprising the amino acid sequence of SEQ ID NO: 53 dimerizes with
an Fc molecule comprising SEQ ID NO: 36 (C-terminal lysine
optional). In another embodiment, a GDF15-Fc fusion comprising the
amino acid sequence of SEQ ID NO: 54 dimerizes with an Fc molecule
comprising SEQ ID NO: 36 (C-terminal lysine optional). In another
embodiment, a GDF15-Fc fusion comprising the amino acid sequence of
SEQ ID NO: 55 dimerizes with an Fc molecule comprising SEQ ID NO:
36 (C-terminal lysine optional). In another embodiment, a GDF15-Fc
fusion comprising the amino acid sequence of SEQ ID NO: 56
dimerizes with an Fc molecule comprising SEQ ID NO: 37 (C-terminal
lysine optional). In another embodiment, a GDF15-Fc fusion
comprising the amino acid sequence of SEQ ID NO: 57 dimerizes with
an Fc molecule comprising SEQ ID NO: 37 (C-terminal lysine
optional).
[0065] In some embodiments, the dimers form tetramers. For example,
the dimers in Table 6 can form tetramers. In some embodiments, the
tetramers are formed from the same dimers. In some embodiments, two
dimers of Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K);
Fc.DELTA.10(+)-(G4)-GDF15:Fc.DELTA.10(-,K);
Fc.DELTA.10(-)-GDF15(.DELTA.3):Fc.DELTA.10(+,K);
Fc.DELTA.10(-)-GDF15(N3D):Fc.DELTA.10(+,K);
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3):Fc.DELTA.10(+,K,CC);
Fc.DELTA.10(-,CC)-GDF15(N3D):Fc.DELTA.10(+,K,CC);
Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E):Fc.DELTA.16(+,K,CC);
Fc.DELTA.16(-, CC)-GDF15(N3Q/D5E):Fc.DELTA.16(+,K,CC);
Fc.DELTA.16(-)-GDF15(N3Q/D5E):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-(G4Q)4-GDF15:Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-(G4S)2-GDF15(N3Q/D5E):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-G4S-GDF15(N3Q):Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E): Fc.DELTA.16(+,K);
Fc.DELTA.16(-)-GDF15(N3Q): Fc.DELTA.16(+,K);
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q):Fc.DELTA.10(+,K,L234A/L235A)-
; or
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.10(+,K,L234-
A/L235A) form a tetramer, such as through the dimerization of the
two GDF15 regions.
[0066] Also provided herein are host cells comprising the nucleic
acids and vectors for producing the GDF15 and Fc molecules
disclosed herein. In various embodiments, the vector or nucleic
acid is integrated into the host cell genome, which in other
embodiments the vector or nucleic acid is extra-chromosomal.
[0067] Recombinant cells, such as yeast, bacterial (e.g., E. coli),
and mammalian cells (e.g., immortalized mammalian cells) comprising
such a nucleic acid, vector, or combinations of either or both
thereof are provided. In various embodiments, cells comprising a
non-integrated nucleic acid, such as a plasmid, cosmid, phagemid,
or linear expression element, which comprises a sequence coding for
expression of a GDF15 molecule and/or an Fc molecule. In some
embodiments, the cell comprises a nucleic acid for producing a
GDF15 molecule and another cell comprises a nucleic acid for
producing an Fc molecule for dimerization with the GDF15 molecule
(e.g., a vector for encoding a GDF15 molecule in one cell and a
second vector for encoding an Fc molecule in a second cell). In
other embodiments, a host cell comprises a nucleic acid for
producing a GDF15 molecule and an Fc molecule (e.g., a vector that
encodes both molecules). In another embodiment, a host cell
comprises a nucleic acid for producing a GDF15 molecule and another
nucleic acid for producing an Fc molecule (e.g., two separate
vectors, one that encodes a GDF15 molecule and one that encodes an
Fc molecule, in a single host cell).
[0068] A vector comprising a nucleic acid sequence encoding a GDF15
molecule and/or an Fc molecule can be introduced into a host cell
by transformation or by transfection, such as by methods known in
the art.
[0069] A nucleic acid encoding a GDF15 molecule can be positioned
in and/or delivered to a host cell or host animal via a viral
vector. A viral vector can comprise any number of viral
polynucleotides, alone or in combination with one or more viral
proteins, which facilitate delivery, replication, and/or expression
of the nucleic acid of the invention in a desired host cell. The
viral vector can be a polynucleotide comprising all or part of a
viral genome, a viral protein/nucleic acid conjugate, a virus-like
particle (VLP), or an intact virus particle comprising viral
nucleic acids and a nucleic acid encoding a polypeptide comprising
a GDF15 region. A viral particle viral vector can comprise a
wild-type viral particle or a modified viral particle. The viral
vector can be a vector which requires the presence of another
vector or wild-type virus for replication and/or expression (e.g.,
a viral vector can be a helper-dependent virus), such as an
adenoviral vector amplicon. Suitable viral vector particles in this
respect, include, for example, adenoviral vector particles
(including any virus of or derived from a virus of the
adenoviridae), adeno-associated viral vector particles (AAV vector
particles) or other parvoviruses and parvoviral vector particles,
papillomaviral vector particles, flaviviral vectors, alphaviral
vectors, herpes viral vectors, pox virus vectors, retroviral
vectors, including lentiviral vectors.
[0070] A GDF15 molecule can be isolated using standard protein
purification methods. A polypeptide comprising a GDF15 region can
be isolated from a cell that has been engineered to express a
polypeptide comprising a GDF15 region, for example a cell that does
not naturally express native GDF15. Protein purification methods
known in the art can be employed to isolate GDF15 molecules, as
well as associated materials and reagents. Methods of purifying a
GDF15 molecule are also provided in the Examples herein. Additional
purification methods that may be useful for isolating GDF15
molecules can be found in references such as Bootcov M R, 1997,
Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie W D, 2000, Gene 254:
67-76.
[0071] Pharmaceutical compositions comprising a GDF15 molecule (and
optionally, an Fc molecule, such as a dimer or tetramer disclosed
herein) are also provided. Such polypeptide pharmaceutical
compositions can comprise a therapeutically effective amount of a
GDF15 molecule in admixture with a pharmaceutically or
physiologically acceptable formulation agent or carrier selected
for suitability with the mode of administration. The
pharmaceutically or physiologically acceptable formulation agent
can be one or more formulation agents suitable for accomplishing or
enhancing the delivery of a GDF15 molecule into the body of a human
or non-human subject. Pharmaceutically acceptable substances such
as wetting or emulsifying agents, preservatives or buffers, which
enhance the shelf life or effectiveness of the GDF15 molecule can
also act as, or form a component of, a formulation carrier.
Acceptable pharmaceutically acceptable carriers are preferably
nontoxic to recipients at the dosages and concentrations employed.
The pharmaceutical composition can contain formulation agent(s) for
modifying, maintaining, or preserving, for example, the pH,
osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition.
[0072] The effective amount of pharmaceutical composition
comprising a GDF15 molecule which is to be employed therapeutically
will depend, for example, upon the therapeutic context and
objectives. One skilled in the art will appreciate that the
appropriate dosage levels for treatment will thus vary depending,
in part, upon the molecule delivered, the indication for which a
GDF15 molecule is being used, the route of administration, and the
size (body weight, body surface, or organ size) and condition (the
age and general health) of the subject. The frequency of dosing
will depend upon the pharmacokinetic parameters of the GDF15
molecule in the formulation being used.
[0073] The route of administration of the pharmaceutical
composition can be orally; through injection by intravenous,
intraperitoneal, intracerebral (intraparenchymal),
intracerebroventricular, intramuscular, intraocular, intraarterial,
intraportal, or intralesional routes; by sustained release systems
(which may also be injected); or by implantation devices. Where
desired, the compositions can be administered by bolus injection or
continuously by infusion, or by an implantation device. The
composition can also be administered locally via implantation of a
membrane, sponge, or other appropriate material onto which the
desired molecule has been absorbed or encapsulated. Where an
implantation device is used, the device can be implanted into any
suitable tissue or organ, and delivery of the desired molecule can
be via diffusion, timed-release bolus, or continuous
administration.
[0074] A GDF15 molecule can be used to treat, diagnose or
ameliorate, a metabolic condition or disorder. In one embodiment,
the metabolic disorder is diabetes, e.g., type 2 diabetes. In
another embodiment, the metabolic condition or disorder is obesity.
In other embodiments, the metabolic condition or disorder is
dyslipidemia, elevated glucose levels, elevated insulin levels or
diabetic nephropathy. For example, a metabolic condition or
disorder that can be treated or ameliorated using a GDF15 molecule
includes a state in which a human subject has a fasting blood
glucose level of 125 mg/dL or greater, for example 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or
greater than 200 mg/dL. Blood glucose levels can be determined in
the fed or fasted state, or at random. The metabolic condition or
disorder can also comprise a condition in which a subject is at
increased risk of developing a metabolic condition. For a human
subject, such conditions include a fasting blood glucose level of
100 mg/dL. Conditions that can be treated using a pharmaceutical
composition comprising a GDF15 molecule can also be found in the
American Diabetes Association Standards of Medical Care in Diabetes
Care 2011, American Diabetes Association, Diabetes Care Vol. 34,
No. Supplement 1, S11-S61, 2010.
[0075] The administration can be performed such as by intravenous
(IV) injection, intraperitoneal (IP) injection, subcutaneous
injection, intramuscular injection, or orally in the form of a
tablet or liquid formation. A therapeutically effective dose of a
GDF15 molecule will depend upon the administration schedule, the
unit dose of agent administered, whether the GDF15 molecule is
administered in combination with other therapeutic agents, the
immune status and the health of the recipient. A therapeutically
effective dose is an amount of a GDF15 molecule that elicits a
biological or medicinal response in a tissue system, animal, or
human being sought by a researcher, medical doctor, or other
clinician, which includes alleviation or amelioration of the
symptoms of the disease or disorder being treated, i.e., an amount
of a GDF15 molecule that supports an observable level of one or
more desired biological or medicinal response, for example,
lowering blood glucose, insulin, triglyceride, or cholesterol
levels; reducing body weight; improving glucose tolerance, energy
expenditure, or insulin sensitivity; or reducing food intake. A
therapeutically effective dose of a GDF15 molecule can also vary
with the desired result.
[0076] Also provided herein is a method comprising measuring a
baseline level of one or more metabolically-relevant compounds such
as glucose, insulin, cholesterol, lipid in a subject, administering
a pharmaceutical composition comprising a GDF15 molecule to the
subject, and after a desired period of time, measure the level of
the one or more metabolically-relevant compounds (e.g., blood
glucose, insulin, cholesterol, lipid) in the subject. The two
levels can then be compared to determine the relative change in the
metabolically-relevant compound in the subject. Depending on the
outcome of that comparison another dose of the pharmaceutical
composition can be administered to achieve a desired level of one
or more metabolically-relevant compound.
[0077] A pharmaceutical composition comprising a GDF15 molecule can
be co-administered with another compound or therapeutic agent. A
GDF15 molecule (and optionally, its corresponding Fc molecule) can
be administered in combination with another therapeutic agent, such
as an agent that lowers blood glucose, insulin, triglyceride, or
cholesterol levels; lowers body weight; reduces food intake;
improves glucose tolerance, energy expenditure, or insulin
sensitivity; or any combination thereof (e.g., antidiabetic agent,
hypolipidemic agent, anti-obesity agent, anti-hypertensive agent,
or agonist of peroxisome proliferator-activator receptor). The
identity and properties of a compound co-administered with the
GDF15 molecule will depend on the nature of the condition to be
treated or ameliorated. The agent administered with a GDF15
molecule disclosed herein can be a GLP-1R agonist, such as GLP-1 or
an analog thereof; or an exendin, exendin analog, or exendin
agonist. A non-limiting list of examples of compounds that can be
administered in combination with the pharmaceutical composition
include liraglutide, rosiglitizone, pioglitizone, repaglinide,
nateglitinide, metformin, exenatide, stiagliptin, pramlintide,
glipizide, glimeprirideacarbose, orlistat, lorcaserin,
phenterminetopiramate, naltrexonebupropion, setmelanotide,
semaglutide, efpeglenatide, canagliflozin, LIK-066, SAR-425899,
Tt-401, FGFR4Rx, HDV-biotin and miglitol.
[0078] In one embodiment, a GDF15 molecule comprising the amino
acid sequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56 or 57 is administered with another
compound or therapeutic agent, such as liraglutide.
[0079] In another embodiment, a GDF15 molecule and corresponding Fc
molecule comprising the amino acid sequences of SEQ ID NOs: 39 and
32 (C-terminal lysine optional), respectively; SEQ ID NOs: 40 and
33 (C-terminal lysine optional), SEQ ID NOs: 41 and 32 (C-terminal
lysine optional), respectively; SEQ ID NOs: 42 and 32 (C-terminal
lysine optional), respectively; SEQ ID NOs: 43 and 34 (C-terminal
lysine optional), respectively; SEQ ID NOs: 44 and 34 (C-terminal
lysine optional), respectively; SEQ ID NOs: 45 and 35 (C-terminal
lysine optional), respectively; SEQ ID NOs: 46 and 35 (C-terminal
lysine optional), respectively; SEQ ID NOs: 47 and 36 (C-terminal
lysine optional) respectively; SEQ ID NOs: 48 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 49 and 36 (C-terminal
lysine optional) respectively; SEQ ID NOs: 50 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 51 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 52 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 53 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 54 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 55 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 56 and 37 (C-terminal
lysine optional), respectively; or SEQ ID NOs: 57 and 37
(C-terminal lysine optional), respectively; is administered with
another compound or therapeutic agent, such as liraglutide.
[0080] In another embodiment, a GDF15 molecule and corresponding Fc
molecule comprising the amino acid sequences of SEQ ID NOs: 50 and
36 (C-terminal lysine optional), respectively, is administered with
another compound or therapeutic agent, such as liraglutide. In
another embodiment, a GDF15 molecule and corresponding Fc molecule
comprising the amino acid sequences of SEQ ID NOs: 57 and 37
(C-terminal lysine optional), respectively, is administered with
another compound or therapeutic agent, such as liraglutide.
[0081] A GDF15 molecule administered with another therapeutic agent
can include concurrent administration of a therapeutically
effective amount of the GDF15 molecule (and optionally, its
corresponding Fc molecule) and a therapeutically effective amount
of the other therapeutic agent. A GDF15 molecule administered with
another therapeutic agent can include subsequent administration of
a therapeutically effective amount of the GDF15 molecule (and
optionally, its corresponding Fc molecule) and a therapeutically
effective amount of the other therapeutic agent, e.g.,
administration of a therapeutically effective amount of the GDF15
molecule (and optionally, its corresponding Fc molecule) followed
by a therapeutically effective amount of the other therapeutic
agent or administration of a therapeutically effective amount of
the other therapeutic agent followed by administration of a
therapeutically effective amount of the GDF15 molecule (and
optionally, its corresponding Fc molecule). Administration of a
therapeutically effective amount of the GDF15 molecule (and
optionally, its corresponding Fc molecule) can be at least 1, 2, 3,
4, 5, 6, or 7 days after administration of a therapeutically
effective amount of the other therapeutic agent. In another
embodiment, administration of a therapeutically effective amount of
a therapeutically effective amount of the other therapeutic agent
can be at least 1, 2, 3, 4, 5, 6, or 7 days after at least 1, 2, 3,
4, 5, 6, or 7 days after administration of a therapeutically
effective amount of the GDF15 molecule (and optionally, its
corresponding Fc molecule).
[0082] A GDF15 molecule administered concurrently with another
therapeutic agent can comprise administration of a composition
comprising both the GDF15 molecule (and optionally its
corresponding Fc molecule) and the other therapeutic agent, e.g., a
therapeutically effective amount of the GDF15 molecule (and
optionally its corresponding Fc molecule) is combined with a
therapeutically effective amount of the other agent prior to
administration. In another embodiment, concurrent administration of
GDF15 molecule (and optionally its corresponding Fc molecule) and
another therapeutic agent can comprise concurrent administration of
a first composition comprising the GDF15 molecule and a second
composition comprising the other therapeutic agent.
[0083] In some embodiments, administration of a GDF15 molecule with
another therapeutic agent has a synergistic effect. In one
embodiment, the effect is greater than the GDF15 molecule (and
optionally its corresponding Fc molecule) alone or the other agent.
In another embodiment, the effect is greater than an additive
effect of both agents (the GDF15 molecule, and optionally its
corresponding Fc molecule, plus the other agent). In one
embodiment, combination therapy (i.e., administration of a GDF15
molecule, optionally with its corresponding Fc molecule, with
another therapeutic agent) has a greater than 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 fold
effect than GDF15 monotherapy (administration of the GDF15
molecule, and optionally its corresponding Fc molecule). In another
embodiment, combination therapy (i.e., administration of a GDF15
molecule, optionally with its corresponding Fc molecule, with
another therapeutic agent) has a greater than 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 fold
effect than monotherapy with the other agent. The effect can be the
amount of body weight lost (e.g., the decrease in total mass or
percent body change); the decrease in blood glucose, insulin,
triglyceride, or cholesterol levels; the improvement in glucose
tolerance, energy expenditure, or insulin sensitivity; or the
reduction food intake. The synergistic effect can be about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, 63,
or 70 days after administration.
[0084] In one embodiment, a GDF15 molecule and corresponding Fc
molecule comprising the amino acid sequences of SEQ ID NOs: 39 and
32 (C-terminal lysine optional), respectively; SEQ ID NOs: 40 and
33 (C-terminal lysine optional), SEQ ID NOs: 41 and 32 (C-terminal
lysine optional), respectively; SEQ ID NOs: 42 and 32 (C-terminal
lysine optional), respectively; SEQ ID NOs: 43 and 34 (C-terminal
lysine optional), respectively; SEQ ID NOs: 44 and 34 (C-terminal
lysine optional), respectively; SEQ ID NOs: 45 and 35 (C-terminal
lysine optional), respectively; SEQ ID NOs: 46 and 35 (C-terminal
lysine optional), respectively; SEQ ID NOs: 47 and 36 (C-terminal
lysine optional) respectively; SEQ ID NOs: 48 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 49 and 36 (C-terminal
lysine optional) respectively; SEQ ID NOs: 50 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 51 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 52 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 53 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 54 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 55 and 36 (C-terminal
lysine optional), respectively; SEQ ID NOs: 56 and 37 (C-terminal
lysine optional), respectively; or SEQ ID NOs: 57 and 37
(C-terminal lysine optional), respectively; administered with a
GLP-1R agonist (e.g., liraglutide or exendin, or an analog or
agonist thereof) has a greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 fold effect than
GDF15 monotherapy; a greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 fold effect than
GLP-1R agonist monotherapy (i.e., administration of GLP-1R agonist
alone); or both, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 21, 28, 35, 42, 49, 56, 63, or 70 days after administration of
the agent(s).
[0085] The detailed description and following examples illustrate
the present invention and are not to be construed as limiting the
present invention thereto. Various changes and modifications can be
made by those skilled in the art on the basis of the description of
the invention, and such changes and modifications are also included
in the present invention.
EXAMPLES
[0086] The following examples, including the experiments conducted
and results achieved, are provided for illustrative purposes only
and are not to be construed as limiting the present invention.
Example 1
GDF15(WT)-Linker-Fc Molecules
[0087] The GDF15 molecules of scFc-GDF15 (SEQ ID NO: 38) and
Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39) were produced and the
activity of the molecules tested.
[0088] Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39) was stably
expressed in a serum free, suspension adapted CHO-K1 cell line. It
was cloned into a stable expression vector containing puromycin
resistance while the Fc chain for forming a heterodimer with
Fc.DELTA.10(-)-(G4S)4-GDF15, Fc.DELTA.10(+,K) (SEQ ID NO: 32), was
cloned into a hygromycin containing expression vector (Selexis,
Inc.). The plasmids were transfected at a 1:1 ratio using
lipofectamine LTX and cells were selected 2 days post transfection
in a proprietary growth media containing 10 ug/mL puromycin and 600
ug/mL hygromycin. Media was exchanged 2 times per week during
selection. When cells reached about 90% viability, they were scaled
up for a batch production run. Cells were seeded at
2.times.10.sup.6/mL in production media. The conditioned medium
(CM) produced by the cells was harvested on day 7 and clarified.
Endpoint viabilities typically were above 90%.
[0089] Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39) (and any paired
Fc) were clarified. Conditioned media was purified using a two-step
chromatography procedure. Approximately 5 L of the CM was applied
directly to a GE Mab Select SuRe column that had previously been
equilibrated with Dulbecco's Phosphate Buffered Saline (PBS). The
bound protein underwent three wash steps: first, 3 column volumes
(CV) of PBS; next, 1 CV of 20 mM Tris, 100 mM sodium chloride, pH
7.4; and finally, 3 CV of 500 mM L-arginine, pH 7.5. These wash
steps remove unbound or lightly bound media components and host
cell impurities. The column was then re-equilibrated with 5 CV of
20 mM Tris, 100 mM sodium chloride at pH 7.4 which brought the UV
absorbance back to baseline. The desired protein was eluted with
100 mM acetic acid at pH 3.6 and collected in bulk. The protein
pool was quickly titrated to within a pH range of 5.0 to 5.5 with 1
M Tris-HCl, pH 9.2. The pH adjusted protein pool was next loaded
onto a GE SP Sepharose.RTM. HP column that had been previously
equilibrated with 20 mM 2-ethanesulfonic acid (MES) at pH 6.0. The
bound protein was then washed with 5 CV of equilibration buffer,
and finally eluted over a 20 CV, 0 to 50% linear gradient from 0 to
400 mM sodium chloride in 20 mM MES at pH 6.0. Fractions were
collected during the elution and analyzed by analytical
size-exclusion chromatography (Superdex.RTM. 200) to determine the
appropriate fractions to pool for a homogeneous product. The SP HP
chromatography removes product-related impurities such as free Fc,
clipped species, and Fc-GDF15 multimers. The SP HP pool was then
buffer exchanged into 10 mM sodium acetate, 5% proline, pH 5.2 by
dialysis. It was concentrated to approximately 15 mg/ml using the
Sartorius Vivaspin.RTM. 20 ten kilo-dalton molecular weight cut-off
centrifugal device. Finally, it was sterile filtered and the
resulting solution containing the purified Fc-GDF15 molecules was
stored at 5.degree. C. Final products were assessed for identity
and purity using mass spectral analysis, sodium dodecyl sulfate
polyacrylamide electrophoresis and size exclusion high performance
liquid chromatography.
[0090] ScFc-GDF15 (SEQ ID NO: 38) was produced in a similar manner.
This GDF15 molecule was stably expressed in a CHO/CS9 cell line.
The molecules were cloned into a stable expression vector. The
plasmids (linearized) were transfected at a 1:1 ratio using
electroporation and cells were selected 2 days post transfection.
Media was exchanged 3 times per week during selection. When cells
reached about 90% viability, they were scaled up for a fed batch
production run. Cells were seeded at 1.times.10.sup.6/mL in
production media and fed once when the cell number reached to
4-5.times.10.sup.6/ml. The conditioned medium (CM) produced by the
cells was harvested on day 10 and clarified. Endpoint viabilities
typically were above 90%. ScFc-GDF15 was clarified and conditioned
media was purified using a two-step coupled chromatography
procedure. Conditioned media from multiple harvests were pooled and
concentrated nearly 5 fold by ultrafiltration using a 1 sq ft
Pellicon.RTM. 2 10 kD regenerated cellulose membrane (Millipore) by
tangential flow filtration. Approximately 5 L of the concentrated
CM was applied directly to a GE MabSelect SuRe column that had
previously been equilibrated with Dulbecco's Phosphate Buffered
Saline (PBS). The non-specifically bound protein was removed by a
12CV PBS wash step. The desired protein was eluted with 0.5% acetic
acid at pH 3.5, 150 mM NaCl in 3 CV and collected in a storage
loop. The collected protein pool was directly loaded onto a GE
HiLoad 26/60 Superdex 200 Prep Grade sizing column that had been
previously equilibrated with 30 mM acetate at pH 5.0, 150 mM NaCl.
Peak fractions collected during the sizing run were analyzed by
sodium dodecyl sulfate polyacrylamide gel electrophoresis to
determine the appropriate fractions to pool for a homogeneous
product. The pH of the final sizing-pool was adjusted to pH 4.5,
with the addition of 10% glacial acetic acid and then buffer
exchanged into 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5 by
dialysis. It was concentrated to above 15 mg/ml using a ten
kilo-dalton molecular weight cut-off centrifugal device. Protein
stability to freezing was tested by 3 cycles of freezing and
thawing. Finally, the final lot was sterile filtered and the
resulting solution containing the purified GDF15 molecules was
stored at -80.degree. C. Final products were assessed for identity
and purity using mass spectral analysis, n-terminal sequencing,
sodium dodecyl sulfate polyacrylamide electrophoresis and size
exclusion high performance liquid chromatography.
[0091] Activity of scFc-GDF15 and Fc.DELTA.10(-)-(G4S)4-GDF15 was
then analyzed for in vivo activity. Cynomologus monkeys (n=10 per
group) were administered vehicle, 3 mg/kg of the positive control
FGF21-Fc, 1.5 mg/kg of scFc-GDF15, or 1.5 mg/kg of
Fc.DELTA.10(-)-(G4S)4-GDF15:Fc.DELTA.10(+,K) weekly for six weeks,
followed by a five-week washout. Body weight and triglyceride
levels were determined. Naive male spontaneously obese cynomolgus
monkeys were prescreened for health and had a body mass index
>41. Monkeys were acclimated to single housing, experimental
procedures and handling for 6 weeks prior to treatment. Monkeys
were sorted into 4 groups receiving once weekly SC injection for 6
weeks (days 0, 7, 14, 21, 28, and 35) for each group to have
similar baseline. Overnight fasting blood samples were collected at
pre-dose days -24, -17 and -10, and on days 6, 13, 20, 27, 34, and
41 (6 days after each weekly dose) during the treatment phase.
During the washout phase, blood samples were collected on days 48,
55, 62, 69 and 76. Body weight was measured once a week and food
intake was monitored daily for each monkey throughout the study.
Each monkey received unlimited feed for a limited amount of time (1
hour) at the morning and evening feeding, approximately 8 hours
apart. A 150 g apple snack, for a limited amount of time (1 hour),
was provided between meals. The remaining food or apple was removed
and weighed after each meal or snack to calculate food intake.
[0092] GDF15-Fc fusion proteins reduced body weight (FIG. 1) and
triglyceride levels (FIG. 2), similar to FGF21-Fc.
Example 2
GDF15(WT)-Linker-Fc Molecule Attributes
[0093] The Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39) molecule as
described in Example 1 and Fc.DELTA.10(+)-(G4)-GDF15 (SEQ ID NO:
40) were analyzed for attributes that may affect its stability and
manufacturability (e.g., for commercial manufacturing). The GDF15
molecules (e.g., Fc.DELTA.10(-)-(G4S)4-GDF15 and
Fc.DELTA.10(+)-(G4)-GDF15) were determined to be highly
heterogeneous (e.g., analysis of an ion exchange column fraction of
Fc.DELTA.10(-)-(G4S)4-GDF15 shows the molecule is highly
heterogeneous, FIG. 3), an undesirable feature for
manufacturability of a molecule. To determine the attributes of the
GDF15 molecules that result in a highly heterogenous population,
analysis of the molecules by size exclusion chromatography, SDS
PAGE gel, and mass spectrometry was performed. A lack of difference
in retention time by size exclusion chromatography indicated that
aggregation or gross degradation are unlikely to be responsible for
the heterogeneity. There was also a lack of difference on an SDS
PAGE gel, which indicated that disulfide mispairing or gross
degradation are also unlikely to be responsible for the
heterogeneity.
[0094] MS analysis was also performed to evaluate the heterogeneity
of Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39). The GDF15 molecule
was purified using mono S, 1 ml column and fraction number 25 (P1),
fraction number 28 (P2), and fraction number 31 (P3) (FIG. 3) were
collected and submitted for MS analysis. About 50 .mu.g of the
fractions were dried down, resuspended in 25 .mu.L of 150 mM Tris,
pH 7.5/8M urea/40 mM hydroxylamine/10 mM DTT, and then incubated
for 1 hour at 37.degree. C. The samples were alkylated with 20 mM
iodoacetamide (IAM) for 30 minutes at room temperature in the dark.
The samples were then diluted to 100 .mu.L with water and 2 .mu.g
of trypsin (1:25) and digested overnight at 37.degree. C. The
digests were acidified, followed by injection onto a Waters
(Milford, Mass.) NanoAcquity UPLC system. Samples were first loaded
onto a 180 .mu.m.times.20 mm Symmetry C18 trapping column at 15
.mu.L/min, followed by peptide separation on an Agilent (Santa
Clara, Calif.) Zorbax 0.5 mm.times.250 mm 300SB-C18column. Buffer A
was 0.1% formic acid/water, while buffer B was 0.1% formic
acid/99.9% acetonitrile. The gradient consisted of initial
conditions at 1% B, followed by an increase to 45% B over 85
minutes, to 97% B over 1 minute, isocratic at 97% B for 6 minutes,
to 1% B over 3 minutes, and then isocratic at 1% B for 20 minutes.
The UPLC column effluent was sprayed into a Thermo Fisher
Scientific (San Jose, Calif.) Orbitrap Velos Pro mass spectrometer
using the standard heated electrospray ionization II (HESI II)
ionization source. The mass spectrometer method consisted of a full
MS scan of m/z [300-2000] at 30K resolution, followed by MS/MS (CID
activation) of the top 10 most abundant precursor ions. The
following instrument parameters were used for the analysis: source
voltage=3.5 kV; capillary temperature=275.degree. C.; S-lens RF
level=50%; activation time=10 msec; normalized collision energy=35;
isolation width=2.0 Da; and threshold=1.0E4. The Xtract component
of the Thermo Xcalibur 2.1 software was used for deconvolution of
high-resolution MS data. Averaged data from [300-2000] were
deconvoluted using a S/N threshold of 1.2 and a resolution of
100,000 at m/z 400. Deconvoluted peptide masses (glycosylated and
non-glycosylated) were displayed as monoisotopic [M+H].sup.+. The
various glycosylated species were confirmed by the stepwise loss of
glycan subunits and the presence of the unglycosylated precursor
ion as the most intense fragment following CID activation.
[0095] The MS results showed that the varying degrees of deamidated
species (e.g., 70% of P1, 47% of P2, and 24% of P3) and
glycosylation distribution (mostly monosaccharide and
trisaccharide) on the linker contributed to highly heterogeneous
nature of the GDF15 molecule as shown in its CEX profile (FIG. 3).
It was determined that the (G4S)4 linker (e.g., present in
Fc.DELTA.10(-)-(G4S)4-GDF15) was highly glycosylated and
phosphorylated, with varying degrees and types of glycosylation
and/or phosphorylation, and the N-terminus of the active fragment
of wildtype human GDF15 was highly susceptible to deamidation and
isomerization (see e.g., FIG. 4, which shows certain masses
extracted from the full mass spec data that correspond to the
unmodified, deamidated, and isomerized species of the peptide that
contains asparagine at position 3. The extracted masses were m/z
[590.25-590.75] from the doubly charged versions of the peptide).
The asparagine at position 3 (in reference to SEQ ID NO: 6, the
amino acid sequence encoding the active fragment of hGDF15) was
highly susceptible to deamidation and isomerization and the
aspartate at position 5 (in reference to SEQ ID NO: 6, the amino
acid sequence encoding the active fragment of hGDF15) was highly
susceptible to isomerization.
[0096] Based on these attributes, manufacturing a generally
homogenous population of a GDF15-Fc fusion protein having the
active fragment of wild type human GDF15 with a linker to the Fc
region (e.g., for commercial manufacturing) would be
challenging.
Example 3
Activity of GDF15-Fc Fusion Proteins without a Linker
[0097] To address the heterogeneity issues described in Example 2,
new GDF15-Fc fusion proteins that 1) eliminated the linker between
the GDF15 region and the Fc region and 2) eliminated or substituted
the N-terminal residues of the active fragment of wild-type human
GDF15 (e.g., GDF15(.DELTA.3) (SEQ ID NO: 13), where the first three
amino acids of the active fragment of wild type human GDF15 is
deleted, or GDF15(N3D) (SEQ ID NO: 16), in which the asparagine at
position 3 of the active fragment of wild type human GDF15 is
mutated to aspartate).
[0098] In addition to the charged pair mutation in the Fc region of
the GDF15-Fc fusion protein and the Fc molecule for the
non-covalent association of the Fc molecule to the Fc region of the
GDF15-Fc fusion protein to form a heterodimer, some of the new
molecules were designed to also include an interchain disulfide
bond in the CH3 region, or "cysteine clamp" (molecules that include
"CC" in their designation) to augment the heterodimerization of the
GDF-Fc molecule with an Fc molecule.
[0099] Four new GDF15-Fc fusion proteins in which 1) the linker
between the GDF15 region and the Fc region was deleted and 2) the
N-terminal residues of GDF15 were eliminated or substituted were
generated. In two of the four molecules, an interchain disulfide
bond was also introduced into the CH3 domain of the Fc region of
the GDF15-Fc fusion protein (as well as its corresponding Fc
molecule for heterodimerization). The potency and pharmacokinetic
(PK) properties of these molecules (Fc.DELTA.10(-)-GDF15(.DELTA.3)
(SEQ ID NO: 41); Fc.DELTA.10(-)-GDF15(N3D) (SEQ ID NO: 42);
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3) (SEQ ID NO: 43);
Fc.DELTA.10(-,CC)-GDF15(N3D) (SEQ ID NO: 44)) were compared to the
earlier generation Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39), in
mice.
[0100] To determine the potency of the molecules, food intake was
determined. Seven to eight-week old single-housed male ob/ob mice
were sorted into different treatment groups with each group having
comparable pretreatment body weight and food intake levels. Animals
were treated with 0.32 ug/kg, 1.6 ug/kg, 8 ug/kg, 40 ug/kg, 0.2
mg/kg, 1 mg/kg, or 5 mg/kg of a GDF15-Fc fusion protein (a dimer of
Fc.DELTA.10(-)-GDF15(.DELTA.3):Fc.DELTA.10(+,K) (SEQ ID NOs: 41 and
32); Fc.DELTA.10(-)-GDF15(N3D):Fc.DELTA.10(+,K) (SEQ ID NOs: 42 and
32); or Fc.DELTA.10(-,CC)-GDF15(.DELTA.3):Fc.DELTA.10(+,K,CC) (SEQ
ID NOs: 39 and 32)) through subcutaneous injection, and overnight
food intake was measured. Data presented is an average of 2-4
independent studies (FIG. 5). The four new molecules,
Fc.DELTA.10(-)-GDF15(.DELTA.3) (SEQ ID NO: 41);
Fc.DELTA.10(-)-GDF15(N3D) (SEQ ID NO: 42);
Fc.DELTA.10(-,CC)-GDF15(.DELTA.3) (SEQ ID NO: 43); and
Fc.DELTA.10(-,CC)-GDF15(N3D) (SEQ ID NO: 44), had comparable
potency as the earlier generation GDF15-Fc fusion protein,
Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39).
[0101] To determine the pharmacokinetics of the molecules, 18-wk
old male diet-induced obese C57Bl/6 mice were dosed with 1 mg/kg
protein subcutaneously, and serial sampling was performed at 1, 4,
8, 24, 72, 168, 240, and 336 hr post-dose. The four new molecules,
Fc.DELTA.10(-)-GDF15(.DELTA.3) (SEQ ID NO: 41);
Fc.DELTA.10(-)-GDF15(N3D) (SEQ ID NO: 42); Fc.DELTA.10(-,
CC)-GDF15(.DELTA.3) (SEQ ID NO: 43); and
Fc.DELTA.10(-,CC)-GDF15(N3D) (SEQ ID NO: 44), had comparable
pharmacokinetic properties as the earlier generation GDF15-Fc
fusion protein, Fc.DELTA.10(-)-(G4S)4-GDF15 (SEQ ID NO: 39) (FIG.
6).
Example 4
Further Engineering of GDF15-Fc Fusion Proteins without a
Linker
[0102] As the newly designed molecules with improved
manufacturability and stability attributes had similar potency and
PK properties as the earlier generation molecule, the molecules
were further engineered to reduce possible heterogeneity and reduce
Fc effector function and increase potency.
[0103] To further reduce heterogeneity of the GDF15 region, instead
of substituting the asparagine at position 3 with aspartate, the
asparagine was substituted with glutamine. In addition, the
molecules were engineered to have two changes introduced in the
N-terminus of GDF15, e.g., GDF15(.DELTA.3/D5E) (SEQ ID NO: 17),
GDF15(N3Q/D5E) (SEQ ID NO: 18) to eliminate the high rate of
deamidation and isomerization of the native GDF15 protein. To
reduce Fc effector function by and improve potency, the molecules
were also engineered to have the hinge region of the Fc region
deleted further by having an additional six amino acids deleted
from the Fc hinge region (e.g., Fc.DELTA.16 instead of Fc.DELTA.10)
to decrease binding to Fc.gamma.R. The same engineering of the
hinge region was performed for the corresponding Fc molecules to
which the GDF15-Fc fusion proteins heterodimerize with.
[0104] The activity of the further engineered GDF15-Fc fusion
proteins, Fc.DELTA.16(-,CC)-GDF15(A3/D5E) (SEQ ID NO: 45),
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E) (SEQ ID NO: 46), and
Fc.DELTA.16(-)-GDF15(N3Q/D5E) (SEQ ID NO: 47), were tested in
cynomologus monkeys. Naive male spontaneously obese cynomolgus
monkeys were acclimated/trained to procedural manipulations (e.g.,
blood collection, subcutaneous injection, body weight measurement,
feeding schedule) for 10 weeks prior to treatment initiation.
Eighty (80) monkeys were sorted into 8 treatment groups of n=10
monkeys each based on data collected during acclimation/training
phase (blood chemistries and body weight). Each treatment group was
administered vehicle, 3 mg/kg of the positive control FGF21-Fc, 0.5
mg/kg of Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E) (along with its
heterodimerization partner, Fc.DELTA.16(+,K,CC) (SEQ ID NO: 35)),
3.0 mg/kg of Fc.DELTA.16(-,CC)-GDF15(.DELTA.3/D5E) (along with its
heterodimerization partner, Fc.DELTA.16(+,K,CC) (SEQ ID NO: 35)),
0.5 mg/kg of Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E) (along with its
heterodimerization partner, Fc.DELTA.16(+,K,CC)), 3.0 mg/kg of
Fc.DELTA.16(-,CC)-GDF15(N3Q/D5E) (along with its heterodimerization
partner, Fc.DELTA.16(+,K,CC)), 0.5 mg/kg of
Fc.DELTA.16(-)-GDF15(N3Q/D5E) (along with its heterodimerization
partner, Fc.DELTA.16(+,K) (SEQ ID NO: 36)), or 3.0 mg/kg of
Fc.DELTA.16(-)-GDF15(N3Q/D5E) (along with its heterodimerization
partner, Fc.DELTA.16(+,K) (SEQ ID NO: 36)). Subcutaneous injections
of each were given once a week for 4 weeks during the treatment
phase followed by a 4-week washout phase; blood collection and body
weight monitoring occurred weekly and food intake occurred daily
during treatment and washout phases. The graph represents
n=5-6/group and data are represented as group means +SEM.
Statistical analysis was performed by ANCOVA and statistical
significance is denoted as *p<0.05, **p<0.01 and
***p<0.001 versus vehicle. Monkeys with rapid drug clearance
were suspect of anti-drug antibodies (ADAs) and were excluded from
analysis.
[0105] Unexpectedly, the newly engineered GDF15-Fc fusion proteins
lost almost all potency (FIG. 7). None of the newly engineered
GDF15-Fc fusion proteins reduced body weight to a similar degree as
to FGF21-Fc, in contrast to the previously generated GDF15-Fc
fusion proteins (see Example 1, FIG. 1)
Example 5
Restoration of GDF15-Fc Fusion Protein Activity in Cynomologus
Monkeys
[0106] The GDF15-Fc fusion proteins in Example 4 as compared to the
GDF15-Fc fusion protein in Example 1 had the following differences
as shown in Table 7:
TABLE-US-00019 TABLE 7 Differences between GDF15-Fc Fusion Proteins
in Examples 1 and 4 GDF15-Fc Molecules GDF15-Fc Molecules in
Example 1: in Example 5: Efficacious in Not Efficacious in
Cynomologus Monkeys Cynomologus Monkeys .DELTA.10 in Fc region
.DELTA.16 in Fc region No cysteine clamp Cysteine clamp Has linker
No linker Wild type GDF15 Two mutations in N- terminus of GDF15
[0107] To restore potency, different aspects of the molecules that
were efficacious in the monkeys were re-introduced into new
GDF15-Fc fusion proteins. The cysteine clamp (CH3 interchain
disulfide bond) was eliminated and a linker reintroduced for
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q) (SEQ ID NO: 49);
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E) (SEQ ID NO: 50) and
Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E) (SEQ ID NO: 54). However, the
linker used in this Example cannot be glycosylated (e.g., G4Q) or
was shorter (G45 instead of (G4S)4), to reduce glycosylation. Also,
for Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q), the mutation at position 5
was eliminated. Lastly, for the new molecule
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) (SEQ ID NO: 57),
the smaller deletion of the hinge region of the Fc region was
reintroduced, however with L234A/L235A mutations in the Fc region,
which should eliminate Fc.gamma.R binding.
[0108] These new molecules were compared to
Fc.DELTA.10(-)-(G4S)4-GDF15, which was shown to be efficacious in
cynomologus monkeys in Example 1. Naive male spontaneously obese
cynomolgus monkeys were acclimated/trained to procedural
manipulations (e.g., blood collection, subcutaneous injection, body
weight measurement, feeding schedule) for 2 weeks prior to
treatment initiation. Forty-two (42) monkeys were sorted into 6
treatment groups of n=7 monkeys each based on data collected during
acclimation/training phase (blood chemistries and body weight).
Each treatment group was administered vehicle, 1.5 mg/kg of
Fc.DELTA.10(-)-(G4S)4-GDF15 (along with its heterodimerization
partner, Fc.DELTA.10(+,K)), 1.5 mg/kg of
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q) (along with its heterodimerization
partner, Fc.DELTA.16(+,K)), 1.5 mg/kg of
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E) (along with its
heterodimerization partner, Fc.DELTA.16(+,K)), 1.5 mg/kg of
Fc.DELTA.16(-)-G4S-GDF15(N3Q/D5E) (along with its
heterodimerization partner, Fc.DELTA.16(+,K)), or 1.5 mg/kg of
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) (along with its
heterodimerization partner, Fc.DELTA.10(+,K,L234A/L235A) (SEQ ID
NO: 37). Subcutaneous injections were given once a week for 2 weeks
during the treatment phase; blood collection and body weight
monitoring occurred weekly and food intake was monitored daily
during the treatment phase. The graph represents n=7/group and data
is represented as group means.+-.SEM. Statistical analysis was
performed by ANCOVA and statistical significance is denoted as
*p<0.05, **p<0.01 and ***p<0.001 versus vehicle. The new
molecules restored potency (FIG. 8).
[0109] Based on these results, the N3Q mutation was determined to
not impact the GDF15 activity in the monkeys, and that the double
mutation in GDF15 (N3Q/D5E) also did not impact GDF15 activity in
the monkeys. The 16-amino acid Fc hinge deletion (.DELTA.16) was
also shown to have a similar effect as the 10-amino acid Fc hinge
deletion (.DELTA.10) in the monkeys. Lastly, the linker was shown
to be a critical component for activity in the monkeys. Though
whether the linker is a G4S or G4Q does not affect activity, the
length of the linker is important for activity. The longer linkers
(e.g., (G4S)4 and (G4Q)4 in FIG. 8) are more potent as compared to
a shorter linker (e.g., G4S).
Example 6
Food Intake Assay in ob/ob Mice for
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E) and
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E)
[0110] A food intake assay was used to evaluate efficacy of two
different GDF15-Fc fusion proteins. Seven to eight weeks-old
single-housed male ob/ob mice were sorted into different treatment
groups (n=5 per group) with each group having comparable
pretreatment body weight and food intake levels. Animals were
treated with 0.32 ug/kg, 1.6 ug/kg, 8 ug/kg, 40 ug/kg, 0.2 mg/kg, 1
mg/kg, or 5 mg/kg of the heterodimer
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.16(+,K) or
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):Fc.DELTA.10(+,K,L234A/L2-
35A) through subcutaneous injection, and overnight food intake was
measured. The results of a representative experiment for each
GDF15-Fc fusion protein is shown in a dose response curve for
Fc.DELTA.16(-)-(G4Q)4-GDF15(N3Q/D5E) (FIG. 9) and
Fc.DELTA.10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) (FIG. 10). The
results show both GDF15-Fc fusion proteins reduce food intake in
acute ob/ob mice. The ED50 in this assay is shown in Table 8.
TABLE-US-00020 TABLE 8 ED50 in Food Intake Assay ED50 (mg/kg)
Molecule n = 3-5 Fc.DELTA.16(-)-(G4Q)4- 7.4 .+-. 4.2 GDF15(N3Q/D5E)
Fc.DELTA.10(-, L234A/L235A)- 5.8 .+-. 0.7 (G4Q)4-GDF15(N3Q/D5E)
[0111] While the present invention has been described in terms of
various embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations that come within the scope of the invention as claimed.
In addition, the section headings used herein are for
organizational purposes only and are not to be construed as
limiting the subject matter described.
[0112] All references cited in this application are expressly
incorporated by reference herein for any purpose.
Sequence CWU 1
1
581927DNAHomo sapiens 1atgcccgggc aagaactcag gacggtgaat ggctctcaga
tgctcctggt gttgctggtg 60ctctcgtggc tgccgcatgg gggcgccctg tctctggccg
aggcgagccg cgcaagtttc 120ccgggaccct cagagttgca ctccgaagac
tccagattcc gagagttgcg gaaacgctac 180gaggacctgc taaccaggct
gcgggccaac cagagctggg aagattcgaa caccgacctc 240gtcccggccc
ctgcagtccg gatactcacg ccagaagtgc ggctgggatc cggcggccac
300ctgcacctgc gtatctctcg ggccgccctt cccgaggggc tccccgaggc
ctcccgcctt 360caccgggctc tgttccggct gtccccgacg gcgtcaaggt
cgtgggacgt gacacgaccg 420ctgcggcgtc agctcagcct tgcaagaccc
caggcgcccg cgctgcacct gcgactgtcg 480ccgccgccgt cgcagtcgga
ccaactgctg gcagaatctt cgtccgcacg gccccagctg 540gagttgcact
tgcggccgca agccgccagg gggcgccgca gagcgcgtgc gcgcaacggg
600gaccactgtc cgctcgggcc cgggcgttgc tgccgtctgc acacggtccg
cgcgtcgctg 660gaagacctgg gctgggccga ttgggtgctg tcgccacggg
aggtgcaagt gaccatgtgc 720atcggcgcgt gcccgagcca gttccgggcg
gcaaacatgc acgcgcagat caagacgagc 780ctgcaccgcc tgaagcccga
cacggtgcca gcgccctgct gcgtgcccgc cagctacaat 840cccatggtgc
tcattcaaaa gaccgacacc ggggtgtcgc tccagaccta tgatgacttg
900ttagccaaag actgccactg catatga 9272308PRTHomo sapiens 2Met Pro
Gly Gln Glu Leu Arg Thr Val Asn Gly Ser Gln Met Leu Leu1 5 10 15Val
Leu Leu Val Leu Ser Trp Leu Pro His Gly Gly Ala Leu Ser Leu 20 25
30Ala Glu Ala Ser Arg Ala Ser Phe Pro Gly Pro Ser Glu Leu His Ser
35 40 45Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu
Leu 50 55 60Thr Arg Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr
Asp Leu65 70 75 80Val Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu
Val Arg Leu Gly 85 90 95Ser Gly Gly His Leu His Leu Arg Ile Ser Arg
Ala Ala Leu Pro Glu 100 105 110Gly Leu Pro Glu Ala Ser Arg Leu His
Arg Ala Leu Phe Arg Leu Ser 115 120 125Pro Thr Ala Ser Arg Ser Trp
Asp Val Thr Arg Pro Leu Arg Arg Gln 130 135 140Leu Ser Leu Ala Arg
Pro Gln Ala Pro Ala Leu His Leu Arg Leu Ser145 150 155 160Pro Pro
Pro Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser Ser Ser Ala 165 170
175Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg
180 185 190Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly
Pro Gly 195 200 205Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu
Glu Asp Leu Gly 210 215 220Trp Ala Asp Trp Val Leu Ser Pro Arg Glu
Val Gln Val Thr Met Cys225 230 235 240Ile Gly Ala Cys Pro Ser Gln
Phe Arg Ala Ala Asn Met His Ala Gln 245 250 255Ile Lys Thr Ser Leu
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro 260 265 270Cys Cys Val
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr 275 280 285Asp
Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp 290 295
300Cys His Cys Ile3053840DNAHomo sapiens 3ctgtctctgg ccgaggcgag
ccgcgcaagt ttcccgggac cctcagagtt gcactccgaa 60gactccagat tccgagagtt
gcggaaacgc tacgaggacc tgctaaccag gctgcgggcc 120aaccagagct
gggaagattc gaacaccgac ctcgtcccgg cccctgcagt ccggatactc
180acgccagaag tgcggctggg atccggcggc cacctgcacc tgcgtatctc
tcgggccgcc 240cttcccgagg ggctccccga ggcctcccgc cttcaccggg
ctctgttccg gctgtccccg 300acggcgtcaa ggtcgtggga cgtgacacga
ccgctgcggc gtcagctcag ccttgcaaga 360ccccaggcgc ccgcgctgca
cctgcgactg tcgccgccgc cgtcgcagtc ggaccaactg 420ctggcagaat
cttcgtccgc acggccccag ctggagttgc acttgcggcc gcaagccgcc
480agggggcgcc gcagagcgcg tgcgcgcaac ggggaccact gtccgctcgg
gcccgggcgt 540tgctgccgtc tgcacacggt ccgcgcgtcg ctggaagacc
tgggctgggc cgattgggtg 600ctgtcgccac gggaggtgca agtgaccatg
tgcatcggcg cgtgcccgag ccagttccgg 660gcggcaaaca tgcacgcgca
gatcaagacg agcctgcacc gcctgaagcc cgacacggtg 720ccagcgccct
gctgcgtgcc cgccagctac aatcccatgg tgctcattca aaagaccgac
780accggggtgt cgctccagac ctatgatgac ttgttagcca aagactgcca
ctgcatatga 8404279PRTHomo sapiens 4Leu Ser Leu Ala Glu Ala Ser Arg
Ala Ser Phe Pro Gly Pro Ser Glu1 5 10 15Leu His Ser Glu Asp Ser Arg
Phe Arg Glu Leu Arg Lys Arg Tyr Glu 20 25 30Asp Leu Leu Thr Arg Leu
Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn 35 40 45Thr Asp Leu Val Pro
Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val 50 55 60Arg Leu Gly Ser
Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala65 70 75 80Leu Pro
Glu Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe 85 90 95Arg
Leu Ser Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu 100 105
110Arg Arg Gln Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu
115 120 125Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala
Glu Ser 130 135 140Ser Ser Ala Arg Pro Gln Leu Glu Leu His Leu Arg
Pro Gln Ala Ala145 150 155 160Arg Gly Arg Arg Arg Ala Arg Ala Arg
Asn Gly Asp His Cys Pro Leu 165 170 175Gly Pro Gly Arg Cys Cys Arg
Leu His Thr Val Arg Ala Ser Leu Glu 180 185 190Asp Leu Gly Trp Ala
Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val 195 200 205Thr Met Cys
Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met 210 215 220His
Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val225 230
235 240Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu
Ile 245 250 255Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu Leu 260 265 270Ala Lys Asp Cys His Cys Ile 2755339DNAHomo
sapiens 5gcgcgcaacg gggaccactg tccgctcggg cccgggcgtt gctgccgtct
gcacacggtc 60cgcgcgtcgc tggaagacct gggctgggcc gattgggtgc tgtcgccacg
ggaggtgcaa 120gtgaccatgt gcatcggcgc gtgcccgagc cagttccggg
cggcaaacat gcacgcgcag 180atcaagacga gcctgcaccg cctgaagccc
gacacggtgc cagcgccctg ctgcgtgccc 240gccagctaca atcccatggt
gctcattcaa aagaccgaca ccggggtgtc gctccagacc 300tatgatgact
tgttagccaa agactgccac tgcatatga 3396112PRTHomo sapiens 6Ala Arg Asn
Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg1 5 10 15Leu His
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30Val
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40
45Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
50 55 60Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
Pro65 70 75 80Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp
Thr Gly Val 85 90 95Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 1107912DNAMus musculus 7atggccccgc
ccgcgctcca ggcccagcct ccaggcggct ctcaactgag gttcctgctg 60ttcctgctgc
tgttgctgct gctgctgtca tggccatcgc agggggacgc cctggcaatg
120cctgaacagc gaccctccgg ccctgagtcc caactcaacg ccgacgagct
acggggtcgc 180ttccaggacc tgctgagccg gctgcatgcc aaccagagcc
gagaggactc gaactcagaa 240ccaagtcctg acccagctgt ccggatactc
agtccagagg tgagattggg gtcccacggc 300cagctgctac tccgcgtcaa
ccgggcgtcg ctgagtcagg gtctccccga agcctaccgc 360gtgcaccgag
cgctgctcct gctgacgccg acggcccgcc cctgggacat cactaggccc
420ctgaagcgtg cgctcagcct ccggggaccc cgtgctcccg cattacgcct
gcgcctgacg 480ccgcctccgg acctggctat gctgccctct ggcggcacgc
agctggaact gcgcttacgg 540gtagccgccg gcagggggcg ccgaagcgcg
catgcgcacc caagagactc gtgcccactg 600ggtccggggc gctgctgtca
cttggagact gtgcaggcaa ctcttgaaga cttgggctgg 660agcgactggg
tgctgtcccc gcgccagctg cagctgagca tgtgcgtggg cgagtgtccc
720cacctgtatc gctccgcgaa cacgcatgcg cagatcaaag cacgcctgca
tggcctgcag 780cctgacaagg tgcctgcccc gtgctgtgtc ccctccagct
acaccccggt ggttcttatg 840cacaggacag acagtggtgt gtcactgcag
acttatgatg acctggtggc ccggggctgc 900cactgcgctt ga 9128303PRTMus
musculus 8Met Ala Pro Pro Ala Leu Gln Ala Gln Pro Pro Gly Gly Ser
Gln Leu1 5 10 15Arg Phe Leu Leu Phe Leu Leu Leu Leu Leu Leu Leu Leu
Ser Trp Pro 20 25 30Ser Gln Gly Asp Ala Leu Ala Met Pro Glu Gln Arg
Pro Ser Gly Pro 35 40 45Glu Ser Gln Leu Asn Ala Asp Glu Leu Arg Gly
Arg Phe Gln Asp Leu 50 55 60Leu Ser Arg Leu His Ala Asn Gln Ser Arg
Glu Asp Ser Asn Ser Glu65 70 75 80Pro Ser Pro Asp Pro Ala Val Arg
Ile Leu Ser Pro Glu Val Arg Leu 85 90 95Gly Ser His Gly Gln Leu Leu
Leu Arg Val Asn Arg Ala Ser Leu Ser 100 105 110Gln Gly Leu Pro Glu
Ala Tyr Arg Val His Arg Ala Leu Leu Leu Leu 115 120 125Thr Pro Thr
Ala Arg Pro Trp Asp Ile Thr Arg Pro Leu Lys Arg Ala 130 135 140Leu
Ser Leu Arg Gly Pro Arg Ala Pro Ala Leu Arg Leu Arg Leu Thr145 150
155 160Pro Pro Pro Asp Leu Ala Met Leu Pro Ser Gly Gly Thr Gln Leu
Glu 165 170 175Leu Arg Leu Arg Val Ala Ala Gly Arg Gly Arg Arg Ser
Ala His Ala 180 185 190His Pro Arg Asp Ser Cys Pro Leu Gly Pro Gly
Arg Cys Cys His Leu 195 200 205Glu Thr Val Gln Ala Thr Leu Glu Asp
Leu Gly Trp Ser Asp Trp Val 210 215 220Leu Ser Pro Arg Gln Leu Gln
Leu Ser Met Cys Val Gly Glu Cys Pro225 230 235 240His Leu Tyr Arg
Ser Ala Asn Thr His Ala Gln Ile Lys Ala Arg Leu 245 250 255His Gly
Leu Gln Pro Asp Lys Val Pro Ala Pro Cys Cys Val Pro Ser 260 265
270Ser Tyr Thr Pro Val Val Leu Met His Arg Thr Asp Ser Gly Val Ser
275 280 285Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg Gly Cys His Cys
Ala 290 295 3009816DNAMus musculus 9tcgcaggggg acgccctggc
aatgcctgaa cagcgaccct ccggccctga gtcccaactc 60aacgccgacg agctacgggg
tcgcttccag gacctgctga gccggctgca tgccaaccag 120agccgagagg
actcgaactc agaaccaagt cctgacccag ctgtccggat actcagtcca
180gaggtgagat tggggtccca cggccagctg ctactccgcg tcaaccgggc
gtcgctgagt 240cagggtctcc ccgaagccta ccgcgtgcac cgagcgctgc
tcctgctgac gccgacggcc 300cgcccctggg acatcactag gcccctgaag
cgtgcgctca gcctccgggg accccgtgct 360cccgcattac gcctgcgcct
gacgccgcct ccggacctgg ctatgctgcc ctctggcggc 420acgcagctgg
aactgcgctt acgggtagcc gccggcaggg ggcgccgaag cgcgcatgcg
480cacccaagag actcgtgccc actgggtccg gggcgctgct gtcacttgga
gactgtgcag 540gcaactcttg aagacttggg ctggagcgac tgggtgctgt
ccccgcgcca gctgcagctg 600agcatgtgcg tgggcgagtg tccccacctg
tatcgctccg cgaacacgca tgcgcagatc 660aaagcacgcc tgcatggcct
gcagcctgac aaggtgcctg ccccgtgctg tgtcccctcc 720agctacaccc
cggtggttct tatgcacagg acagacagtg gtgtgtcact gcagacttat
780gatgacctgg tggcccgggg ctgccactgc gcttga 81610271PRTMus musculus
10Ser Gln Gly Asp Ala Leu Ala Met Pro Glu Gln Arg Pro Ser Gly Pro1
5 10 15Glu Ser Gln Leu Asn Ala Asp Glu Leu Arg Gly Arg Phe Gln Asp
Leu 20 25 30Leu Ser Arg Leu His Ala Asn Gln Ser Arg Glu Asp Ser Asn
Ser Glu 35 40 45Pro Ser Pro Asp Pro Ala Val Arg Ile Leu Ser Pro Glu
Val Arg Leu 50 55 60Gly Ser His Gly Gln Leu Leu Leu Arg Val Asn Arg
Ala Ser Leu Ser65 70 75 80Gln Gly Leu Pro Glu Ala Tyr Arg Val His
Arg Ala Leu Leu Leu Leu 85 90 95Thr Pro Thr Ala Arg Pro Trp Asp Ile
Thr Arg Pro Leu Lys Arg Ala 100 105 110Leu Ser Leu Arg Gly Pro Arg
Ala Pro Ala Leu Arg Leu Arg Leu Thr 115 120 125Pro Pro Pro Asp Leu
Ala Met Leu Pro Ser Gly Gly Thr Gln Leu Glu 130 135 140Leu Arg Leu
Arg Val Ala Ala Gly Arg Gly Arg Arg Ser Ala His Ala145 150 155
160His Pro Arg Asp Ser Cys Pro Leu Gly Pro Gly Arg Cys Cys His Leu
165 170 175Glu Thr Val Gln Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp
Trp Val 180 185 190Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val
Gly Glu Cys Pro 195 200 205His Leu Tyr Arg Ser Ala Asn Thr His Ala
Gln Ile Lys Ala Arg Leu 210 215 220His Gly Leu Gln Pro Asp Lys Val
Pro Ala Pro Cys Cys Val Pro Ser225 230 235 240Ser Tyr Thr Pro Val
Val Leu Met His Arg Thr Asp Ser Gly Val Ser 245 250 255Leu Gln Thr
Tyr Asp Asp Leu Val Ala Arg Gly Cys His Cys Ala 260 265
27011348DNAMus musculus 11agcgcgcatg cgcacccaag agactcgtgc
ccactgggtc cggggcgctg ctgtcacttg 60gagactgtgc aggcaactct tgaagacttg
ggctggagcg actgggtgct gtccccgcgc 120cagctgcagc tgagcatgtg
cgtgggcgag tgtccccacc tgtatcgctc cgcgaacacg 180catgcgcaga
tcaaagcacg cctgcatggc ctgcagcctg acaaggtgcc tgccccgtgc
240tgtgtcccct ccagctacac cccggtggtt cttatgcaca ggacagacag
tggtgtgtca 300ctgcagactt atgatgacct ggtggcccgg ggctgccact gcgcttga
34812115PRTMus musculus 12Ser Ala His Ala His Pro Arg Asp Ser Cys
Pro Leu Gly Pro Gly Arg1 5 10 15Cys Cys His Leu Glu Thr Val Gln Ala
Thr Leu Glu Asp Leu Gly Trp 20 25 30Ser Asp Trp Val Leu Ser Pro Arg
Gln Leu Gln Leu Ser Met Cys Val 35 40 45Gly Glu Cys Pro His Leu Tyr
Arg Ser Ala Asn Thr His Ala Gln Ile 50 55 60Lys Ala Arg Leu His Gly
Leu Gln Pro Asp Lys Val Pro Ala Pro Cys65 70 75 80Cys Val Pro Ser
Ser Tyr Thr Pro Val Val Leu Met His Arg Thr Asp 85 90 95Ser Gly Val
Ser Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg Gly Cys 100 105 110His
Cys Ala 11513109PRTArtificial SequenceSynthetic Polypeptide 13Gly
Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr1 5 10
15Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser
20 25 30Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
Gln 35 40 45Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu
His Arg 50 55 60Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
Ala Ser Tyr65 70 75 80Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val Ser Leu Gln 85 90 95Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys
His Cys Ile 100 10514112PRTArtificial SequenceSynthetic Polypeptide
14Ala Arg Gln Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg1
5 10 15Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
Trp 20 25 30Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
Ala Cys 35 40 45Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile
Lys Thr Ser 50 55 60Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro
Cys Cys Val Pro65 70 75 80Ala Ser Tyr Asn Pro Met Val Leu Ile Gln
Lys Thr Asp Thr Gly Val 85 90 95Ser Leu Gln Thr Tyr Asp Asp Leu Leu
Ala Lys Asp Cys His Cys Ile 100 105 11015112PRTArtificial
SequenceSynthetic Polypeptide 15Ala Arg Asp Gly Asp His Cys Pro Leu
Gly Pro Gly Arg Cys Cys Arg1 5 10 15Leu His Thr Val Arg Ala Ser Leu
Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30Val Leu Ser Pro Arg Glu Val
Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45Pro Ser Gln Phe Arg Ala
Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60Leu His Arg Leu Lys
Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro65 70 75 80Ala Ser Tyr
Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95Ser Leu
Gln Thr Tyr Asp Asp Leu Leu Ala Lys
Asp Cys His Cys Ile 100 105 11016112PRTArtificial SequenceSynthetic
Polypeptide 16Ala Arg Asn Gly Glu His Cys Pro Leu Gly Pro Gly Arg
Cys Cys Arg1 5 10 15Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly
Trp Ala Asp Trp 20 25 30Val Leu Ser Pro Arg Glu Val Gln Val Thr Met
Cys Ile Gly Ala Cys 35 40 45Pro Ser Gln Phe Arg Ala Ala Asn Met His
Ala Gln Ile Lys Thr Ser 50 55 60Leu His Arg Leu Lys Pro Asp Thr Val
Pro Ala Pro Cys Cys Val Pro65 70 75 80Ala Ser Tyr Asn Pro Met Val
Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105
11017109PRTArtificial SequenceSynthetic Polypeptide 17Gly Glu His
Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr1 5 10 15Val Arg
Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser 20 25 30Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln 35 40
45Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg
50 55 60Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
Tyr65 70 75 80Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
Ser Leu Gln 85 90 95Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
Ile 100 10518112PRTArtificial SequenceSynthetic Polypeptide 18Ala
Arg Gln Gly Glu His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg1 5 10
15Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
20 25 30Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
Cys 35 40 45Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys
Thr Ser 50 55 60Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys
Cys Val Pro65 70 75 80Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys
Thr Asp Thr Gly Val 85 90 95Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala
Lys Asp Cys His Cys Ile 100 105 110195PRTArtificial
SequenceSynthetic Polypeptide 19Gly Gly Gly Gly Ser1
52010PRTArtificial SequenceSynthetic Polypeptide 20Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 102120PRTArtificial SequenceSynthetic
Polypeptide 21Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly1 5 10 15Gly Gly Gly Ser 202240PRTArtificial
SequenceSynthetic Polypeptide 22Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser Gly Gly Gly Gly
Ser 35 40234PRTArtificial SequenceSynthetic Polypeptide 23Gly Gly
Gly Gly1245PRTArtificial SequenceSynthetic Polypeptide 24Gly Gly
Gly Gly Gln1 52520PRTArtificial SequenceSynthetic Polypeptide 25Gly
Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly1 5 10
15Gly Gly Gly Gln 2026216PRTArtificial SequenceSynthetic
Polypeptide 26Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 165 170 175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser
Pro Gly 210 21527216PRTArtificial SequenceSynthetic Polypeptide
27Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1
5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Lys Glu Met 115 120 125Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155
160Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21528216PRTArtificial SequenceSynthetic Polypeptide 28Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr
Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21529210PRTArtificial SequenceSynthetic Polypeptide 29Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40
45His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
50 55 60Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 100 105 110Cys Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185
190His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
195 200 205Pro Gly 21030210PRTArtificial SequenceSynthetic
Polypeptide 30Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 115 120 125Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 130 135
140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro
Pro145 150 155 160Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Asp Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly
21031216PRTArtificial SequenceSynthetic Polypeptide 31Ala Pro Glu
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr
Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21532217PRTArtificial SequenceSynthetic Polypeptide 32Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Lys Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr
Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210
21533217PRTArtificial SequenceSynthetic Polypeptide 33Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr
Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210
21534217PRTArtificial SequenceSynthetic Polypeptide 34Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys Arg Lys Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr
Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr
Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu
Ser Leu Ser Pro Gly Lys 210 21535211PRTArtificial SequenceSynthetic
Polypeptide 35Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro
Pro Cys Arg Lys Glu Met Thr Lys Asn Gln Val Ser 115 120 125Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 130 135
140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro145 150 155 160Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly Lys
21036211PRTArtificial SequenceSynthetic Polypeptide 36Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40
45His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
50 55 60Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met
Thr Lys Asn Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro145 150 155 160Val Leu Lys
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 165 170 175Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185
190His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
195 200 205Pro Gly Lys 21037217PRTArtificial SequenceSynthetic
Polypeptide 37Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Met 115 120 125Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly
Ser Phe Phe Leu 165 170 175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser
Pro Gly Lys 210 21538630PRTArtificial SequenceFusion Protein 38Gly
Gly Gly Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro Ala1 5 10
15Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 35 40 45Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe65 70 75 80Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170
175Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
180 185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser225 230 235 240Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Arg 260 265 270Lys Ser Ser
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 275 280 285Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 290 295
300Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu305 310 315 320Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 325 330 335Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg 340 345 350Val Val Ser Val Leu Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys 355 360 365Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu 370 375 380Lys Thr Ile Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr385 390 395 400Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 405 410
415Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
420 425 430Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met 435 440 445Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 450 455 460Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His465 470 475 480Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 485 490 495Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 500 505 510Ser Gly Gly
Gly Gly Ser Ala Arg Asn Gly Asp His Cys Pro Leu Gly 515 520 525Pro
Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp 530 535
540Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val
Thr545 550 555 560Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala
Ala Asn Met His 565 570 575Ala Gln Ile Lys Thr Ser Leu His Arg Leu
Lys Pro Asp Thr Val Pro 580 585 590Ala Pro Cys Cys Val Pro Ala Ser
Tyr Asn Pro Met Val Leu Ile Gln 595 600 605Lys Thr Asp Thr Gly Val
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala 610 615 620Lys Asp Cys His
Cys Ile625 63039348PRTArtificial SequenceFusion Protein 39Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly
Ser Gly Gly Gly 210 215 220Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala Arg Asn Gly225 230 235 240Asp His Cys Pro Leu Gly Pro
Gly Arg Cys Cys Arg Leu His Thr Val 245 250 255Arg Ala Ser Leu Glu
Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro 260 265 270Arg Glu Val
Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe 275 280 285Arg
Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu 290 295
300Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr
Asn305 310 315 320Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
Ser Leu Gln Thr 325 330 335Tyr Asp Asp Leu Leu Ala Lys Asp Cys His
Cys Ile 340 34540332PRTArtificial SequenceFusion Protein 40Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Lys Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly
Ala Arg Asn Gly 210 215 220Asp His Cys Pro Leu Gly Pro Gly Arg Cys
Cys Arg Leu His Thr Val225 230 235 240Arg Ala Ser Leu Glu Asp Leu
Gly Trp Ala Asp Trp Val Leu Ser Pro 245 250 255Arg Glu Val Gln Val
Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe 260 265 270Arg Ala Ala
Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu 275 280 285Lys
Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn 290 295
300Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln
Thr305 310 315 320Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
325 33041325PRTArtificial SequenceFusion Protein 41Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Asp
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Gly Asp His Cys Pro Leu Gly Pro
210 215 220Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu
Asp Leu225 230 235 240Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu
Val Gln Val Thr Met 245 250 255Cys Ile Gly Ala Cys Pro Ser Gln Phe
Arg Ala Ala Asn Met His Ala 260 265 270Gln Ile Lys Thr Ser Leu His
Arg Leu Lys Pro Asp Thr Val Pro Ala 275 280 285Pro Cys Cys Val Pro
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys 290 295 300Thr Asp Thr
Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys305 310 315
320Asp Cys His Cys Ile 32542328PRTArtificial SequenceFusion Protein
42Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1
5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155
160Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Ala Arg
Asp Gly Asp His Cys Pro 210 215 220Leu Gly Pro Gly Arg Cys Cys Arg
Leu His Thr Val Arg Ala Ser Leu225 230 235 240Glu Asp Leu Gly Trp
Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln 245 250 255Val Thr Met
Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn 260 265 270Met
His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr 275 280
285Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu
290 295 300Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu305 310 315 320Leu Ala Lys Asp Cys His Cys Ile
32543325PRTArtificial SequenceFusion Protein 43Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln 100 105 110Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Asp Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Gly Asp His Cys Pro Leu Gly Pro
210 215 220Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu
Asp Leu225 230 235 240Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu
Val Gln Val Thr Met 245 250 255Cys Ile Gly Ala Cys Pro Ser Gln Phe
Arg Ala Ala Asn Met His Ala 260 265 270Gln Ile Lys Thr Ser Leu His
Arg Leu Lys Pro Asp Thr Val Pro Ala 275 280 285Pro Cys Cys Val Pro
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys 290 295 300Thr Asp Thr
Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys305 310 315
320Asp Cys His Cys Ile 32544328PRTArtificial SequenceFusion Protein
44Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1
5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Cys
Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155
160Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Ala Arg
Asp Gly Asp His Cys Pro 210 215 220Leu Gly Pro Gly Arg Cys Cys Arg
Leu His Thr Val Arg Ala Ser Leu225 230 235 240Glu Asp Leu Gly Trp
Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln 245 250 255Val Thr Met
Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn 260 265 270Met
His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr 275 280
285Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu
290 295 300Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu305 310 315 320Leu Ala Lys Asp Cys His Cys Ile
32545319PRTArtificial SequenceFusion Protein 45Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75
80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 100 105 110Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185 190His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200
205Pro Gly Gly Glu His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
210 215 220His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
Trp Val225 230 235 240Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys
Ile Gly Ala Cys Pro 245 250 255Ser Gln Phe Arg Ala Ala Asn Met His
Ala Gln Ile Lys Thr Ser Leu 260 265 270His Arg Leu Lys Pro Asp Thr
Val Pro Ala Pro Cys Cys Val Pro Ala 275 280 285Ser Tyr Asn Pro Met
Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser 290 295 300Leu Gln Thr
Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile305 310
31546322PRTArtificial SequenceFusion Protein 46Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75
80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 100 105 110Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185 190His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200
205Pro Gly Ala Arg Gln Gly Glu His Cys Pro Leu Gly Pro Gly Arg Cys
210 215 220Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly
Trp Ala225 230 235 240Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val
Thr Met Cys Ile Gly 245 250 255Ala Cys Pro Ser Gln Phe Arg Ala Ala
Asn Met His Ala Gln Ile Lys 260 265 270Thr Ser Leu His Arg Leu Lys
Pro Asp Thr Val Pro Ala Pro Cys Cys 275 280 285Val Pro Ala Ser Tyr
Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr 290 295 300Gly Val Ser
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His305 310 315
320Cys Ile47322PRTArtificial SequenceFusion Protein 47Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40
45His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
50 55 60Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185
190His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
195 200 205Pro Gly Ala Arg Gln Gly Glu His Cys Pro Leu Gly Pro Gly
Arg Cys 210 215 220Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp
Leu Gly Trp Ala225 230 235 240Asp Trp Val Leu Ser Pro Arg Glu Val
Gln Val Thr Met Cys Ile Gly 245 250 255Ala Cys Pro Ser Gln Phe Arg
Ala Ala Asn Met His Ala Gln Ile Lys 260 265 270Thr Ser Leu His Arg
Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys 275 280 285Val Pro Ala
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr 290 295 300Gly
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His305 310
315 320Cys Ile48342PRTArtificial SequenceFusion Protein 48Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25
30Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
35 40 45His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 50 55 60Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170
175Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
180 185 190His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 195 200 205Pro Gly Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln
Gly Gly Gly Gly 210 215 220Gln Gly Gly Gly Gly Gln Ala Arg Asn Gly
Asp His Cys Pro Leu Gly225 230 235 240Pro Gly Arg Cys Cys Arg Leu
His Thr Val Arg Ala Ser Leu Glu Asp 245 250 255Leu Gly Trp Ala Asp
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr 260 265 270Met Cys Ile
Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His 275 280 285Ala
Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro 290 295
300Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile
Gln305 310 315 320Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala 325 330 335Lys Asp Cys His Cys Ile
34049342PRTArtificial SequenceFusion Protein 49Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75
80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185 190His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200
205Pro Gly Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly
210 215 220Gln Gly Gly Gly Gly Gln Ala Arg Gln Gly Asp His Cys Pro
Leu Gly225 230 235 240Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg
Ala Ser Leu Glu Asp 245 250 255Leu Gly Trp Ala Asp Trp Val Leu Ser
Pro Arg Glu Val Gln Val Thr 260
265 270Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met
His 275 280 285Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp
Thr Val Pro 290 295 300Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro
Met Val Leu Ile Gln305 310 315 320Lys Thr Asp Thr Gly Val Ser Leu
Gln Thr Tyr Asp Asp Leu Leu Ala 325 330 335Lys Asp Cys His Cys Ile
34050342PRTArtificial SequenceFusion Protein 50Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75
80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185 190His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200
205Pro Gly Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly
210 215 220Gln Gly Gly Gly Gly Gln Ala Arg Gln Gly Glu His Cys Pro
Leu Gly225 230 235 240Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg
Ala Ser Leu Glu Asp 245 250 255Leu Gly Trp Ala Asp Trp Val Leu Ser
Pro Arg Glu Val Gln Val Thr 260 265 270Met Cys Ile Gly Ala Cys Pro
Ser Gln Phe Arg Ala Ala Asn Met His 275 280 285Ala Gln Ile Lys Thr
Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro 290 295 300Ala Pro Cys
Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln305 310 315
320Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala
325 330 335Lys Asp Cys His Cys Ile 34051332PRTArtificial
SequenceFusion Protein 51Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 115 120
125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr
Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Arg Gln Gly 210 215 220Asp His Cys
Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val225 230 235
240Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro
245 250 255Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
Gln Phe 260 265 270Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
Leu His Arg Leu 275 280 285Lys Pro Asp Thr Val Pro Ala Pro Cys Cys
Val Pro Ala Ser Tyr Asn 290 295 300Pro Met Val Leu Ile Gln Lys Thr
Asp Thr Gly Val Ser Leu Gln Thr305 310 315 320Tyr Asp Asp Leu Leu
Ala Lys Asp Cys His Cys Ile 325 33052332PRTArtificial
SequenceFusion Protein 52Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 115 120
125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr
Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Arg Gln Gly 210 215 220Glu His Cys
Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val225 230 235
240Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro
245 250 255Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
Gln Phe 260 265 270Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
Leu His Arg Leu 275 280 285Lys Pro Asp Thr Val Pro Ala Pro Cys Cys
Val Pro Ala Ser Tyr Asn 290 295 300Pro Met Val Leu Ile Gln Lys Thr
Asp Thr Gly Val Ser Leu Gln Thr305 310 315 320Tyr Asp Asp Leu Leu
Ala Lys Asp Cys His Cys Ile 325 33053327PRTArtificial
SequenceFusion Protein 53Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 115 120
125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr
Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly Gly Gly Gly
Gly Ser Ala Arg Gln Gly Asp His Cys Pro Leu 210 215 220Gly Pro Gly
Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu225 230 235
240Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val
245 250 255Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala
Asn Met 260 265 270His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys
Pro Asp Thr Val 275 280 285Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr
Asn Pro Met Val Leu Ile 290 295 300Gln Lys Thr Asp Thr Gly Val Ser
Leu Gln Thr Tyr Asp Asp Leu Leu305 310 315 320Ala Lys Asp Cys His
Cys Ile 32554327PRTArtificial SequenceFusion Protein 54Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 20 25 30Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 35 40
45His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
50 55 60Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly65 70 75 80Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 85 90 95Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 100 105 110Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 115 120 125Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 130 135 140Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro145 150 155 160Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 165 170 175Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 180 185
190His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
195 200 205Pro Gly Gly Gly Gly Gly Ser Ala Arg Gln Gly Glu His Cys
Pro Leu 210 215 220Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg
Ala Ser Leu Glu225 230 235 240Asp Leu Gly Trp Ala Asp Trp Val Leu
Ser Pro Arg Glu Val Gln Val 245 250 255Thr Met Cys Ile Gly Ala Cys
Pro Ser Gln Phe Arg Ala Ala Asn Met 260 265 270His Ala Gln Ile Lys
Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val 275 280 285Pro Ala Pro
Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile 290 295 300Gln
Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu305 310
315 320Ala Lys Asp Cys His Cys Ile 32555322PRTArtificial
SequenceFusion Protein 55Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met1 5 10 15Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 20 25 30Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 35 40 45His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 50 55 60Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly65 70 75 80Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 85 90 95Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 100 105 110Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 115 120
125Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
130 135 140Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr
Pro Pro145 150 155 160Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Asp Leu Thr Val 165 170 175Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 180 185 190His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 195 200 205Pro Gly Ala Arg Gln
Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys 210 215 220Cys Arg Leu
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala225 230 235
240Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
245 250 255Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln
Ile Lys 260 265 270Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro
Ala Pro Cys Cys 275 280 285Val Pro Ala Ser Tyr Asn Pro Met Val Leu
Ile Gln Lys Thr Asp Thr 290 295 300Gly Val Ser Leu Gln Thr Tyr Asp
Asp Leu Leu Ala Lys Asp Cys His305 310 315 320Cys
Ile56348PRTArtificial SequenceFusion Protein 56Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Asp Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Asp Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Gln Gly Gly Gly
210 215 220Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Ala Arg
Gln Gly225 230 235 240Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys
Arg Leu His Thr Val 245 250 255Arg Ala Ser Leu Glu Asp Leu Gly Trp
Ala Asp Trp Val Leu Ser Pro 260 265 270Arg Glu Val Gln Val Thr Met
Cys Ile Gly Ala Cys Pro Ser Gln Phe 275 280 285Arg Ala Ala Asn Met
His Ala Gln Ile Lys Thr Ser Leu His Arg Leu 290 295 300Lys Pro Asp
Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn305 310 315
320Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr
325 330 335Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 340
34557348PRTArtificial SequenceFusion Protein 57Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 165 170 175Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser
Pro Gly Gly Gly Gly Gly Gln Gly Gly Gly 210 215 220Gly Gln Gly Gly
Gly Gly Gln Gly Gly Gly Gly Gln Ala Arg Gln Gly225 230 235 240Glu
His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val 245 250
255Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro
260 265 270Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
Gln Phe 275 280 285Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
Leu His Arg Leu 290 295 300Lys Pro Asp Thr Val Pro Ala Pro Cys Cys
Val Pro Ala Ser Tyr Asn305 310 315 320Pro Met Val Leu Ile Gln Lys
Thr Asp Thr Gly Val Ser Leu Gln Thr 325 330 335Tyr Asp Asp Leu Leu
Ala Lys Asp Cys His Cys Ile 340 345585PRTArtificial
SequenceSynthetic Polypeptide 58Gly Gly Gly Gly Ala1 5
* * * * *