U.S. patent application number 15/785775 was filed with the patent office on 2018-06-07 for multimeric constructs.
This patent application is currently assigned to Genzyme Corporation. The applicant listed for this patent is Genzyme Corporation. Invention is credited to Peter Pechan, Abraham Scaria, Samuel Wadsworth.
Application Number | 20180155417 15/785775 |
Document ID | / |
Family ID | 36060586 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180155417 |
Kind Code |
A1 |
Scaria; Abraham ; et
al. |
June 7, 2018 |
MULTIMERIC CONSTRUCTS
Abstract
Multimeric fusion proteins of an Ig-like domain of Flt-1 are
rendered functional by inclusion of a linker moiety. Vectors
encoding the fusion proteins and host cells expressing the fusion
proteins can be used therapeutically to block neovascularization in
individuals with pathological conditions related to
neovascularization. Such conditions include age-related macular
degeneration, cancer, psoriasis, proliferative diabetic
retinopathy, asthma, uveitis, osteoarthritis, and rheumatoid
arthritis. The same means of multimerization used for an Ig like
domain of Flt-1, i.e., a linker and a multimerization domain, can
be used for other polypeptides, including extracellular receptors,
antibody variable regions, cytokines, chemokines, and growth
factors.
Inventors: |
Scaria; Abraham; (Cambridge,
MA) ; Pechan; Peter; (Cambridge, MA) ;
Wadsworth; Samuel; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Assignee: |
Genzyme Corporation
Cambridge
MA
|
Family ID: |
36060586 |
Appl. No.: |
15/785775 |
Filed: |
October 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14170257 |
Jan 31, 2014 |
9815892 |
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15785775 |
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13019432 |
Feb 2, 2011 |
8658602 |
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14170257 |
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11716794 |
Mar 12, 2007 |
7928072 |
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13019432 |
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PCT/US05/32320 |
Sep 13, 2005 |
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11716794 |
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60608887 |
Sep 13, 2004 |
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60658209 |
Mar 4, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/705 20130101;
A61P 3/10 20180101; A61P 11/06 20180101; A61P 27/02 20180101; A61P
29/00 20180101; A61P 19/02 20180101; A61P 9/10 20180101; A61P 19/08
20180101; C07K 2319/32 20130101; C07K 2319/735 20130101; A61P 35/00
20180101; C12P 21/00 20130101; C07K 14/475 20130101; C07K 14/52
20130101; C07K 2319/30 20130101; C07K 16/22 20130101; A61K 38/00
20130101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; C12P 21/00 20060101 C12P021/00; C07K 14/475 20060101
C07K014/475; C07K 14/52 20060101 C07K014/52; C07K 14/705 20060101
C07K014/705 |
Claims
1-57. (canceled)
58. A method comprising: delivering a nucleic acid encoding a
fusion protein of the formula X-Y-Z to a mammal, wherein X
comprises a polypeptide that is an extracellular receptor, an
antibody variable region, a cytokine, a chemokine, and a growth
factor, wherein Y consists essentially of a 5-25 amino acid residue
polypeptide, and wherein Z is an Fc portion, and whereby said
fusion protein is expressed in the mammal.
59. The method of claim 58 wherein the fusion protein comprises a
sequence selected from the group consisting of SEQ ID NO: 2, 8, 21,
23, and 25.
60. The method of claim 58, wherein the mammal has wet age-related
macular degeneration or proliferative diabetic retinopathy.
61. The method of claim 58, wherein the mammal has cancer.
62. The method of claim 58, wherein the mammal has rheumatoid
arthritis.
63. The method of claim 58, wherein the mammal has asthma.
64. The method of claim 58, wherein the mammal has
osteoarthritis.
65-71. (canceled)
72. The fusion protein of claim 58, wherein X comprises an
extracellular receptor and said receptor is selected from the group
consisting of a tyrosine kinase receptor and a serine threonine
kinase receptor.
73. The method of claim 58, wherein the extracellular receptor is a
VEGF receptor.
74. The method of claim 58, wherein X is the IgG-like domain 2 of
VEGF-R1 (FLT-1).
75. The method of claim 58, wherein the polypeptide Y is
flexible.
76. The method of claim 58, wherein the polypeptide Y is selected
from the group consisting of gly9 (SEQ ID NO: 27), glu9 (SEQ ID NO:
28), ser9 (SEQ ID NO: 29), gly5cyspro2cys (SEQ ID NO: 30),
(gly4ser)3 (SEQ ID NO: 31), SerCysValProLeuMetArgCysGlyGlyCysCysAsn
(SEQ ID NO: 32), Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys
Cys Asn (SEQ ID NO: 13), Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys (SEQ
ID NO: 26), and Gly9ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ
ID NO: 34).
77. The method of claim 58, wherein the Fc is an IgG1 Fc.
78. The method of claim 58, wherein Z is an IgG1 CH3 region.
79. The method of claim 58, wherein Z is an IgG2 CH3 region.
80. The method of claim 58, wherein the nucleic acid is in a
vector.
81. The method of claim 80, wherein the vector is a viral vector or
a plasmid vector.
82. The method of claim 81, wherein the viral vector is an
adeno-associated virus (AAV) vector.
83. The method of claim 58, wherein the nucleic is delivered by
intravitreal injection to the mammal.
84. The method of claim 58, wherein the mammal is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
14/170,257, filed Jan. 31, 2014, which is a divisional of U.S. Ser.
No. 13/019,432, filed Feb. 2, 2011, now U.S. Pat. No. 8,658,602,
which is a continuation of U.S. Ser. No. 11/716,794, filed Mar. 12,
2007, now U.S. Pat. No. 7,928,072, which is a 35 U.S.C. .sctn. 371
filing of PCT/US05/32320, filed Sep. 13, 2005, from which
applications priority is claimed under 35 U.S.C. .sctn. 120, which
application claims the benefit under 35 U.S.C. .sctn. 119(e)(1) to
U.S. Provisional Application Ser. Nos. 60/608,887, filed Sep. 13,
2004 and 60/658,209, filed Mar. 4, 2005, all of which applications
are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to recombinantly constructed proteins
useful for treating pathological neovascularization, e.g., asthma,
arthritis, cancer, and macular degeneration.
BACKGROUND OF THE INVENTION
[0003] Pathological neovascularization is a key component of
diseases like wet age-related macular degeneration (AMD),
proliferative diabetic retinopathy, rheumatoid arthritis,
osteoarthritis, and asthma. It also plays an important role in
growth and spread of tumors. Neovascularization is regulated by an
exquisite balance of pro- and anti-angiogenic factors.
[0004] Vascular endothelial growth factor (VEGF) is known to be
necessary for neovascularization. Inhibition of VEGF activity has
been shown to inhibit neovascularization in animal models of AMD,
arthritis and in various tumor models. Methods used to inhibit VEGF
activity include antibodies, receptor fusion proteins, peptides and
small molecules.
[0005] VEGF-R1 (Flt-1) and VEGF-R2 (KDR) proteins have been shown
to bind VEGF with high affinity. Both Flt-1 and KDR have seven
Ig-like domains in their extracellular region. Domain 2 has been
shown to be essential for VEGF binding. Fusions of each of the
full-length, soluble receptor (domains 1-7) and domains 1-3 to IgG
Fc bind VEGF efficiently. IgG Fc fusions to Ig-like domain 2 alone
was, however, incapable of binding VEGF, as was a combination of
Ig-like domain 1 and 2. Davis-Smyth, 1996. Therefore, Ig-like
domains 1 and 3 seem to be required along with domain 2 for
efficient VEGF binding.
BRIEF SUMMARY OF THE INVENTION
[0006] According to one embodiment of the invention a fusion
protein is provided. The fusion protein has the formula X-Y-Z. X
comprises a polypeptide selected from the group consisting of an
extracellular receptor, an antibody variable region, a cytokine, a
chemokine, and a growth factor. Y consists essentially of a 5-25
amino acid residue polypeptide. Z is a CH3 region of an IgG heavy
chain molecule.
[0007] Another embodiment of the invention is a polypeptide of the
formula X-Y-Z. X comprises a polypeptide selected from the group
consisting of an extracellular receptor, an antibody variable
region, a cytokine, a chemokine, and a growth factor. Y consists
essentially of a linker moiety which provides the spatial
separation of 5-25 amino acid residues. Z is a CH3 region of an IgG
heavy chain molecule.
[0008] Yet another aspect of the invention is a fusion protein of
the formula X-Y-Z. X comprises a polypeptide selected from the
group consisting of an extracellular receptor, an antibody variable
region, a cytokine, a chemokine, and a growth factor. Y consists
essentially of a 5-25 amino acid residue polypeptide. Z is an Fc
portion of an antibody molecule.
[0009] A fusion protein of the formula X-Y-Z is also provided. X
comprises a polypeptide selected from the group consisting of an
extracellular receptor, an antibody variable region, a cytokine, a
chemokine, and a growth factor. Y consists essentially of a linker
moiety which provides the spatial separation of 5-25 amino acid
residues. Z is an Fc portion of an antibody molecule.
[0010] Still another aspect of the invention is a method of
multimerizing a polypeptide X. A polypeptide X is linked to a
polypeptide Z via a polypeptide Y to form polypeptide XYZ. X
comprises a polypeptide selected from the group consisting of an
extracellular receptor, an antibody variable region, a cytokine, a
chemokine, and a growth factor. Y consists essentially of a 5-25
amino acid residue polypeptide. Z is a CH3 region of an IgG heavy
chain molecule. Polypeptide XYZ which is formed multimerizes.
[0011] Yet another embodiment of the invention provides a method of
multimerizing a polypeptide X. Polypeptide X is linked to a
polypeptide Z via a moiety Y to form polymer XYZ. X comprises a
polypeptide selected from the group consisting of an extracellular
receptor, an antibody variable region, a cytokine, a chemokine, and
a growth factor. Y consists essentially of a linker moiety which
provides the spatial separation of 5-25 amino acid residues. Z is a
CH3 region of an IgG heavy chain molecule. Polypeptide XYZ which is
so formed multimerizes.
[0012] In one embodiment of the invention a nucleic acid molecule
is provided. The nucleic acid molecule encodes a fusion protein
which comprises an Ig-like domain 2 of VEGF-R1 (Flt-1); a linker;
and a multimerization domain. The fusion protein comprises a
sequence selected from the group consisting of SEQ ID NO: 2, 8, 21,
23, and 25.
[0013] In another embodiment of the invention a fusion protein is
provided. The fusion protein comprises an Ig-like domain 2 of
VEGF-R1 (Flt-1), a linker, and a multimerization domain. The fusion
protein comprises a sequence selected from the group consisting of
SEQ ID NO: 2, 8, 21, 23, and 25.
[0014] In another embodiment of the invention an in vitro method is
provided. A nucleic acid molecule is delivered to an isolated
mammalian cell. The nucleic acid molecule encodes a fusion protein
which comprises an Ig-like domain 2 of VEGF-R1 (Flt-1; a linker;
and a multimerization domain. The fusion protein comprises a
sequence selected from the group consisting of SEQ ID NO: 2, 8, 21,
23, and 25. Expression of the fusion protein is controlled by a
promoter. A cell is formed which expresses a fusion protein.
[0015] Still another embodiment of the invention is a method for
delivering a fusion protein to a mammal. A mammalian cell which
expresses the fusion protein is delivered to a mammal. The cell
expresses and secretes the fusion protein thereby supplying the
fusion protein to the mammal. The fusion protein comprises an
Ig-like domain 2 of VEGF-R1 (Flt-1), a linker, and a
multimerization domain. The fusion protein comprises a sequence
selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and
25.
[0016] Another aspect of the invention is a method for supplying a
fusion protein to a mammal. A fusion protein which comprises an
Ig-like domain 2 of VEGF-R1 (Flt-1), a linker, and a
multimerization domain is delivered to a mammal. The fusion protein
comprises a sequence selected from the group consisting of SEQ ID
NO: 2, 8, 21, 23, and 25. Alternatively, a nucleic acid construct
which encodes said fusion protein can be delivered to the mammal,
whereby the fusion protein is expressed by the mammal.
[0017] These and other embodiments of the invention which will be
described in more detail below provide the art with methods and
agents for treating disease related to vascular proliferation and
inflammation. The agents may provide increased stability and
bioavailability relative to natural forms of the proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Flexible region of 9-Gly linker in D2-9Gly-Fc
construct. The predicted relative flexibility by Karpus and Schultz
(1985) method shows the polyglycine 9-mer (9-Gly) linker (aa 94 to
103) in D2-9Gly-Fc protein as a region with greater flexibility
than the average (>1) as compared to D2-Fc construct that does
not contain 9-Gly linker. Both fusion proteins contain identical
amino acid sequences enclosed in boxes: sp--signal peptide (aa--24
to -1), Flt-1 domain 2 (aa 1 to 93) and IgGI-Fc residues (244 aa).
The arrow represents the signal peptidase cleavage site as
predicted using the SignalP V2.0 program (Nielsen et al.,
1997).
[0019] FIG. 2. Biological activity of D2-9Gly-Fc vs. D2-Fc. 293
cells were grown in the starvation media (M199+5% FCS) and
transfected with plasmids containing D29Gly-Fc and D2-Fc expression
cassettes under control of CMV promoter. Conditioned media (CM) was
collected 72 h later. HUVECs were seeded into 96 well plate (2E3
cells/well) in starvation media+VEGF (10 ng/mL) and 50 ul CM plus
VEGF (10 ng/mL) was added 24 h later. The controls (+/-VEGF) were
incubated with CM from the control pEGFP (Clontech; pEGFP carries a
red-shifted variant of wild-type green fluorescent protein (GFP)
which has been optimized for brighter fluorescence and higher
expression in mammalian cells) plasmid transfection. The positive
control was treated with 50 ng of Flt-1-IgG recombinant protein
(R&D Systems). The HUVECs were assayed for proliferation 3 days
post treatment using CellTiter 96.RTM. AQ.sub.ueous reagent
(Promega). The data represent the means of the average values of
OD.sub.490 of two experiments each assayed in triplicates.
[0020] FIG. 3. Western blot analysis of D2-9Gly-Fc and D2-Fc. The
size of both D2-9Gly-Fc and D2-Fc proteins appears to be twice as
large while migrating in non-reducing gel as compared to migration
in reducing gel. The proteins were loaded from the conditioned
media following 293 cell transfection of plasmids expressing
D2-9Gly-Fc and D2-Fc were separated by SDS-electrophoresis and
transferred to PVDF membrane. The blot was probed with goat
anti-human anti-IgG1 Fc and rabbit anti-goat IgG-HRP
antibodies.
[0021] FIG. 4. sFlt-1 hybrid proteins containing 9Gly linker and
VEGF Ex3. Structure comparison of D2-9Gly-Ex3/CH3 to previously
constructed proteins. All three proteins contain identical amino
acid sequence of Flt-1 domain 2, consisting of 24 aa of Flt-1
signal peptide and 93 aa of Flt-1 domain 2. D2-9Gly-Ex3/CH3
contains 9 aa of 9Gly linker, 14 aa of VEGF Ex3 and 120 aa of the
CH3 region of human IgG1 heavy chain Fc.
[0022] FIG. 5. Biological activity of D2-9Gly-Ex3/CH3 vs.
D2-9Gly-Fc. Protein D2-9GlyEx3/CH3, where domain 2 is connected to
the CH3 region through 9Gly linker and VEGF Ex 3, is also
efficiently inhibiting VEGF-dependent HUVECs proliferation as
compared to control proteins D2-9Gly-Fc and D2-Fc. 50 ng of the
recombinant Flt-1-IgG (R&D Systems) was used as a control.
[0023] FIG. 6. HUVECs proliferation assay comparing
D2-(Gly.sub.4Ser).sub.3-Fc protein activity with D2-9Gly-Fc and
D2-9Gly-Ex3/CH3.
[0024] FIG. 7. Western blot. Proteins (non-reduced and reduced)
from conditioned medium of transfected 293 cells (15 ul of CM) with
plasmids expressing (1): D2-9Gly-Fc; (2): D2-(G.sub.4S).sub.3-Fc
and (3)--EGFP proteins were separated by SDS-electrophoresis and
transferred to PVDF membrane. The blot was probed with goat
anti-human IgG1 Fe and rabbit anti-goat IgG-HRP antibodies.
[0025] FIG. 8. Combinations of proteins with/without 9Gly linker or
VEGF Ex3. Structure comparison of three novel proteins with or
without 9Gly linker and/or VEGF Ex3, D2-9Gly-CH3, D2-CH3 and
D2-Ex3/CH3.
[0026] FIG. 9. HUVECs proliferation assay with the Flt-1(D2)
constructs with 9Gly, Ex3 and CH3 combinations. Conditioned media
from 293 cells (5 ul) containing proteins D2-Ex3/CH3, D2-9Gly-CH3
and D2-CH3 were compared to D2-9Gly-Fc and D2-9Gly-Ex3/CH3.
[0027] FIG. 10 Western blot. 293 cells were transfected with
plasmids expressing: (1) D2-9Gly-Fc; (2) D2-9Gly-CH3 (52/26 kDa);
and (3) D2-CH3 (50/25 kDa). Proteins from 293 cells conditioned
medium (15 ul of CM non-reduced and/or reduced) were separated by
SDS-electrophoresis and transferred to PVDF membrane. The blot was
probed with anti-human VEGF-R1 HRP conjugate (R&D Systems).
[0028] FIG. 11. VEGF "in vitro" binding assay. Conditioned media
from 293 cells containing known concentrations of both D2-9Gly-Fc
and Flt-1 D(1-3) control soluble receptors (ranging in
concentrations from 0.29-150 pM) were serially diluted and mixed
with 10 pM VEGF. The amount of unbound VEGF was then measured by
ELISA. D2-9Gly-Fc binds VEGF with higher affinity than all other
constructs. "n" represents the number of independent experiments
(transfection and binding assay).
[0029] FIG. 12. The binding kinetics of the soluble Flt-1
constructs were measured by surface plasmon resonance with a
BIAcore instrument. sFlt-1 constructs were immobilized onto a
sensor chip, and VEGF165 was injected at concentrations ranging
from 0.2 to 15 nM. The sensorgrams were evaluated using the BIA
Evaluation program, the rate constants Ka and Kd were determined
and the dissociation constant (KD) calculated from the ratio of
Kd/Ka=KD. The lower KD value means better affinity.
[0030] FIG. 13A-13C. FIG. 13A shows expression levels of Flt-1
constructs having various linkers. FIG. 13B shows dimerization or
multimerization of Flt-1 constructs having various linkers and a
CH3 moiety of Fc of IgG 1. The difference between the non-reduced
and the reduced conditions indicates that the proteins had
multimerized. FIG. 13C shows the inhibitory bioactivity of
indicated Flt-1 constructs present in condition medium in a HUVEC
proliferation assay in the presence of VEGF. Each of the constructs
demonstrated inhibitory activity approaching proliferation levels
of the HUVEC in the absence of VEGF.
[0031] FIG. 14. Using a murine oxygen-induced retinopathy (OIR)
model of retinal neovascularization (NV), one of the Flt-1
constructs was administered to the mouse eyes and
neovascularization was determined. The mice were exposed to
hyperoxic conditions. The number of neovascular events was
determined in the treated eyes compared to the events in the
untreated eyes of the same animals. The animal was considered a
"responder" if there was a greater than 50% reduction in
neovascular events.
DETAILED DESCRIPTION OF THE INVENTION
[0032] It is a discovery of the present inventors that a Flt-1
Ig-like domain 2 without domains 1 and 3 is capable of efficiently
binding VEGF and inhibiting VEGF-dependent endothelial cell
proliferation. Domain 2 can be covalently linked to a
multimerization domain via a linker. Linkers are typically
polypeptide chains. The length of the chain may be 6, 7, 9, 11, 13,
15 or more amino acid residues, but typically is between 5 and 25
residues. Depending upon the length and side chain composition, a
linker may have but need not have greater than average flexibility.
Flexibility can be calculated using algorithms known in the art.
Multimerization domains are those portions of multimeric proteins
which promote the association of subunits to form, for example,
dimers, trimers, tetramers, etc. Suitable recombinant proteins for
efficiently binding VEGF and/or inhibiting VEGF-dependent
endothelial cell proliferation are selected from the group
consisting of SEQ ID NO: 2, 8, 21, 23, and 25.
[0033] 1321 Moreover, the inventors have found that the
multimerization domains and linkers can be used with a variety of
other proteins or portions of proteins to induce multimerization.
Such proteins may be those which bind to ligand or receptor only
when multimerized; or may be those whose binding affinity is
enhanced when multimerized. Suitable proteins for multimerization
include extracellular receptors (which include portions thereof),
antibody variable regions, cytokines, chemokines, and growth
factors. Suitable proteins include tyrosine kinase receptors and
senile thereonine kinase receptors. Specific examples of
extracellular receptors include EGF-receptor, G protein coupled
receptors, FGF receptor, Fc receptors, T cell receptors, etc.
Examples of antibody variable regions include Fab, F(ab')2, and
ScFv. Examples of cytokines include GM-CSF, IL-1a, IL-113, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-18, IL-21,
1L-23, IFN-a, IFN-13, MIP-1a, MIP-113, TGF-13, TNFa, and TNF-13.
Examples of chemokines include BCA-1/BLC, BRAK, Chemokine CC-2,
CTACK, CXCL-16, ELC, ENA, ENA-70, ENA-74 , ENA-78, Eotaxin,
Exodus-2, Fractalkine, GCP-2, GRO, GRO alpha (MGSA), GRO-beta,
GRO-gamma, HCC-1, HCC-4, 1-309, IP-10, I-TAC, LAG-1, LD78-beta,
LEC/NCC-4, LL-37, Lymphotactin, MCP, MCAF (MCP-1), MCP-2, MCP-3,
MCP-4, MDC, MDC, MDC-2, MDC-4, MEC/CCL28, MIG, MIP, MIP-1 alpha,
MIP-1 beta, MIP-1 delta, MIP-3/MPIF-1, MIP-3 alpha, MIP-3 bet ,
MIP-4 (PARC), MIP-5, NAP-2 , PARC, PF-4, RANTES, RANTES-2, SDF-1
alpha, SDF-1 beta, TARC, and TECK. Examples of growth factors
include Human Amphiregulin, Human Angiogenesis Proteins, Human ACE,
Human Angiogenin, Human Angiopoietin, Human Angiostatin, Human
Betacellulin, Human BMP, Human BMP-13/CDMP-2, Human BMP-14/CDMP-1,
Human BMP-2, Human BMP-3, Human BMP-4, Human BMP-5, Human BMP-6,
Human BMP-7, Human BMP-8, Human BMP-9, Human Colony Stimulating
Factors, Human flt3-Ligand, Human GCSF, Human GM-CSF, Human M-CSF,
Human Connective Tissue Growth Factor, Human Cripto-1, Human
Cryptic, Human ECGF, Human EGF, Human EG-VEGF, Human
Erythropoietin, Human Fetuin, Human FGF, Human FGF-1, Human FGF 10,
Human FGF-16, Human FGF-17, Human FGF-18, Human FGF-19, Human FGF2,
Human FGF-20, Human FGF-3, Human FGF-4, Human FGF-5, Human FGF-6,
Human FGF-7/KGF, Human FGF-8, Human FGF-9, Human FGF-acidic, Human
FGF-basic, Human GDF-11, Human GDF-15, Human Growth Hormone
Releasing Factor, Human HB-EGF, Human Heregulin, Human HGF, Human
IGF, Human IGF-I, Human IGF-II, Human Inhibin, Human KGF, Human
LCGF, Human LIF, Human Miscellaneous Growth Factors, Human MSP,
Human Myostatin, Human Myostatin Propeptide, Human Nerve Growth
Factor, Human Oncostatin M, Human PD-ECGF, Human PDGF, Human PDGF
(AA Homodimer), Human PDGF (AB Heterodimer), Human PDGF (BB
Homodimer), Human PDGF (CC Homodimer), Human PIGF, Human PIGF,
Human PIGF-1, Human PIGF-2, Human SCF, Human SMDF, Human Stem Cell
Growth Factor, Human SCGF-alpha, Human SCGF-beta, Human
Thrombopoietin, Human Transforming Growth Factor, Human TGF-alpha,
Human TGF-beta, and Human VEGF.
[0034] Flt-1 receptor protein has an extracellular portion which
comprises seven Ig-like domains. These are located at residue
numbers 32 . . . 123, 151 . . . 214, 230 . . . 327, 335 . . . 421,
428 . . . 553, 556 . . . 654, and 661 . . . 747 of Genbank
accession no. P17948, see also SEQ ID NO: 15. Residue numbers 1-26
comprise a signal sequence. Flt-1 protein is encoded by the DNA
sequence shown at Genbank accession no. NM_002019 (SEQ ID NO:
14).
[0035] Multimerization domains can be used as are known in the art.
Sequences of the Fc portion of IgG1 or IgG2 lambda heavy chain can
be used, for example, CH3 alone (aa 371-477) or both of CH2 and CH3
domains (aa 247-477). Fc portion of Ig molecules is that which is
obtained by cleavage of whole antibody molecules with the enzyme
papain. Other means can be used to obtain these portions. For the
IgG1 lambda heavy chain protein sequence, see Genbank accession no
Y14737and SEQ ID NO: 10. Other Fc regions can be used for example
from other IgG types and from IgA, IgM, IgD, or IgE antibodies. The
multimerization region of VEGF can also be used. A DNA sequence
encoding VEGF is shown at Genbank accession no. NM003376and SEQ ID
NO: 11. An amino acid sequence of VEGF is shown at Genbank
accession no. CAC19513and SEQ ID NO: 12. The multimerization region
of VEGF (SEQ ID NO: 13), encoded by VEGF exon 3 (VEGF Ex3), is at
about amino acid residues 7588 of VEGF protein (SEQ ID NO: 12).
Multimerization domains will cause at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 85%, 90%, or 95% of the monomeric fusion
proteins to migrate on a non-denaturing polyacrylamide gel at a
rate appropriate for a multimer. Glycosylation can affect the
migration of a protein in a gel. Although particular sequences are
shown here, variants such as allelic variants can be used as well.
Typically such variants will have at least 85%, 90%, 95%, 97%, 98%,
or 99% identity with the disclosed sequence.
[0036] Multimerization can be assayed, for example, using reducing
and non-reducing gels, as demonstrated herein. Multimerization can
also be assayed by detection of increased binding affinity of a
protein for its ligand/receptor. BiaCore.TM. surface plasmon
resonance assays can be used in this regard. These assays detect
changes in mass by measuring changes in refractive index in an
aqueous layer close to a sensor chip surface. Any method known in
the art can be used to detect multimerization.
[0037] Linker moieties according to the invention can be comprised
of for example 5-100 amino acid residues, 5-75 amino acid residues,
5-50 amino acid residues, 5-25 amino acid residues, 5-20 amino acid
residues, 5-15 amino acid residues, 5-10 amino acid residues, 5-9
amino acid residues. Examples of useful linkers include: gly9 (SEQ
ID NO: 27), glu9 (SEQ ID NO: 28), ser9 (SEQ ID NO: 29),
gly5cyspro2cys (SEQ ID NO: 30), (gly4ser)3 (SEQ ID NO: 31), Ser Cys
Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn (SEQ ID NO: 32), Pro
Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn (SEQ ID NO:
13), Gly Asp Leu Ile Tyr Arg Asn Gl{dot over (n)} Lys (SEQ ID NO:
26), and Gly9ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID NO:
34). Other polypeptide linkers which can be used include a
polyglycine of different lengths, including of 5, 7, or 30
residues. Additionally, other portions of Flt-1 can be used as a
linker, for example domain 3 of Flt-1. See SEQ ID NO: 15. Linker
moieties can also be made from other polymers, such as polyethylene
glycol. Such linkers can have from 10 to 1000, 10-500, 10-250,
10-100, or 10-50 ethylene glycol monomer units. Suitable polymers
should be of a size similar to the size occupied by the appropriate
range of amino acid residues. A typical sized polymer would provide
a spacing of from about 10-25 angstroms.
[0038] Fusion proteins according to the invention can be made by
any means known in the art. While such proteins can be made
synthetically, or by linking portions which are made, recombinant
production can also be used. A fused gene sequence can be produced
using the standard tools of recombinant DNA. The fused gene
sequence can be inserted into a vector, for example a viral or
plasmid vector, for replicating the fused gene sequence. A promoter
sequence which is functional in the ultimate recipient cell can be
introduced upstream of the fused gene sequence. Promoters used can
be constitutive, inducible or repressible. Examples of each type
are well-known in the art. The vector can be introduced into a host
cell or mammal by any means known in the art. Suitable vectors
which can be used include adenovirus, adeno-associated virus,
retrovirus, lentivirus, and plasmids. If the vector is in a viral
vector and the vector has been packaged, then the virions can be
used to infect cells. If naked DNA is used, then transfection or
transformation procedures as are appropriate for the particular
host cells can be used. Formulations of naked DNA utilizing
polymers, liposomes, or nanospheres can be used for fusion gene
delivery. Cells which can be transformed or transfected with
recombinant constructs according to the invention may be any which
are convenient to the artisan. Exemplary cell types which may be
used include bacteria, yeast, insects, and mammalian cells. Among
mammalian cells, cells of many tissue types may be chosen, as is
convenient. Exemplary cells which may be used are fibroblasts,
hepatocytes, endothelial cells, stem cells, hematopoietic cells,
epithelial cells, myocytes, neuronal cells, and keratinocytes.
These cells can be used to produce protein in vitro, or can be
delivered to mammals including humans to produce the encoded
proteins in vivo. This means of delivery is an alternative to
delivering nucleic acid to a mammal, delivering viral vector to a
mammal, and delivering fusion protein to a mammal.
[0039] Compositions of protein or nucleic acids can be in carriers,
such as buffers, aqueous or lipophilic carriers, sterile or
non-sterile, pyrogenic or non-pyrogenic vehicles. Non-pyrogenic
vehicles are useful for injectible formulations. Formulations can
be liquid or solid, for example, lyophilized. Formulations can also
be administered as aerosols. Compositions may contain one or more
fusion proteins or one or more nucleic acids, or both fusion
proteins and nucleic acids. The fusion proteins and or nucleic
acids in a composition may be homogeneous, in which case
homomultimer proteins will form, or they may be heterogeneous in
the composition, in which case heteromultimer proteins will form.
In the case of heteromultimers, typically the X moiety will vary
between fusion proteins, but the Z moiety will be the same between
fusion proteins.
[0040] Fusion proteins can be provided to a cell or mammalian host
by any means known in the art. Protein can be delivered to the cell
or host. Nucleic acid can be administered to the cell or host.
Transformed or transfected cells can be administered to the cell or
host. In the latter case, cells of the same genetic background are
desired to reduce transplantation rejection.
[0041] Suitable cells for delivery to mammalian host animals
include any mammalian cell type from any organ, tumor, or cell
line. For example, human, murine, goat, ovine, bovine, dog, cat,
and porcine cells can be used. Suitable cell types for use include
without limitation, fibroblasts, hepatocytes, endothelial cells,
keratinocytes, hematopoietic cells, epithelial cells, myocytes,
neuronal cells, and stem cells.
[0042] Means of delivery of fusion proteins or nucleic acids
encoding fusion proteins include delivery of cells expressing the
fusion proteins, delivery of the fusion proteins, and delivery of
nucleic acids encoding the fusion proteins. Fusion proteins, cells,
or nucleic acids can be delivered directly to the desired organ or
tumor, for example by injection, catheterization, or endoscopy.
They can also be delivered intravenously, intrabronchially,
intra-tumorally, intrathecally, intramuscularly, intraocularly,
topically, subcutaneously, transdermally or per os. Patients who
can be effectively treated include those with wet age-related
macular degeneration, proliferative diabetic retinopathy,
rheumatoid arthritis, osteoarthritis, uveitis, asthma, and cancer.
The treatments will improve symptoms and/or markers of disease
and/or disease severity.
[0043] Nucleic acids can be delivered to mammals, and in particular
to humans, in any desired vector. These include viral or non-viral
vectors, including adenovirus vectors, adeno-associated virus
vectors, retrovirus vectors, lentivirus vectors, and plasmid
vectors. Exemplary types of viruses include HSV (herpes simplex
virus), adenovirus, AAV (adeno associated virus), HIV (human
immunodeficiency virus), BIV (bovine immunodeficiency virus), and
MLV (murine leukemia virus). Nucleic acids can be administered in
any desired format that provides sufficiently efficient delivery
levels, including in virus particles, in liposomes, in
nanoparticles, and complexed to polymers.
[0044] Combinations of protein and nucleic acid treatments can be
used. For example, a fusion protein according to the invention can
be administered to a patient. If a favorable response is observed,
then a nucleic acid molecule encoding the fusion protein can be
administered for a long term effect. Alternatively, the protein and
nucleic acid can be administered simultaneously or approximately
simultaneously. In another alternative, an antibody or fusion
protein for a ligand can be administered followed by or
concomitantly with an antibody or fusion partner for a receptor.
Another option employs a combination of nucleic acids in which one
encodes an antibody and another encodes a fusion protein. Some
antibodies that can be employed in combination with the Flt-1
constructs of the present invention (whether in the protein or
nucleic acid form) are bevacizumab and ranibizumab, both directed
to VEGF. These are particularly useful for treating cancer and
macular degeneration, respectively.
[0045] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et
al., 1989); Current Protocols In Molecular Biology (F. M. Ausubel
et al., eds., 1987); Oligonucleotide Synthesis (M. J. Gait, ed.,
1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods In
Enzymology (Academic Press, Inc.); Handbook Of Experimental
Immunology (D. M. Wei & C. C. Blackwell, eds.); Gene Transfer
Vectors For Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,
1994); Current Protocols In Immunology (J. E. Coligan et al., eds.,
1991); Antibodies: A Laboratory Manual (E. Harlow and D. Lane eds.
(1988)); and PCR 2: A Practical Approach (M. J. MacPherson, B. D.
Hames and G. R. Taylor eds. (1995)).
[0046] A gene delivery vehicle is any molecule that can carry
inserted polynucleotides into a host cell. Examples of gene
delivery vehicles are liposomes, biocompatible polymers, including
natural polymers and synthetic polymers; lipoproteins;
polypeptides; polysaccharides; lipopolysaccharides; artificial
viral envelopes; metal particles; and bacteria, viruses, such as
baculovirus, adenovirus and retrovirus, bacteriophage, cosmid,
plasmid, fungal vectors and other recombination vehicles typically
used in the art which have been described for expression in a
variety of eukaryotic and prokaryotic hosts, and may be used for
gene therapy as well as for simple protein expression.
[0047] Gene delivery, gene transfer, and the like as used herein,
are terms referring to the introduction of an exogenous
polynucleotide (sometimes referred to as a "transgene") into a host
cell, irrespective of the method used for the introduction. Such
methods include a variety of well-known techniques such as
vector-mediated gene transfer (by, e.g., viral
infection/transfection, or various other protein-based or
lipid-based gene delivery complexes) as well as techniques
facilitating the delivery of "naked" polynucleotides (such as
electroporation, "gene gun" delivery and various other techniques
used for the introduction of polynucleotides). The introduced
polynucleotide may be stably or transiently maintained in the host
cell. Stable maintenance typically requires that the introduced
polynucleotide either contains an origin of replication compatible
with the host cell or integrates into a replicon of the host cell
such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear
or mitochondrial chromosome. A number of vectors are known to be
capable of mediating transfer of genes to mammalian cells, as is
known in the art and described herein.
[0048] The exogenous polynucleotide is inserted into a vector such
as adenovirus, partially-deleted adenovirus, fully-deleted
adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus,
naked plasmid, plasmid/liposome complex, etc. for delivery to the
host via intravenous, intramuscular, intraportal or other route of
administration. Expression vectors which can be used in the methods
and compositions of the present invention include, for example,
viral vectors. One of the most frequently used methods of
administration of gene therapy, both in vivo and ex vivo, is the
use of viral vectors for delivery of the gene. Many species of
virus are known, and many have been studied for gene therapy
purposes. The most commonly used viral vectors include those
derived from adenoviruses, adeno-associated viruses (AAV) and
retroviruses, including lentiviruses, such as human
immunodeficiency virus (HIV).
[0049] Adenovirus is a non-enveloped, nuclear DNA virus with a
genome of about 36 kb, which has been well-characterized through
studies in classical genetics and molecular biology (Hurwitz, M.
S., Adenoviruses Virology, 3rd edition, Fields et al., eds., Raven
Press, New York, 1996; Hitt, M. M. et al., Adenovirus Vectors, The
Development of Human Gene Therapy, Friedman, T. ed., Cold Spring
Harbor Laboratory Press, New York 1999). The viral genes are
classified into early (designated E1 -E4) and late (designated
L1-L5) transcriptional units, referring to the generation of two
temporal classes of viral proteins. The demarcation of these events
is viral DNA replication. The human adenoviruses are divided into
numerous serotypes (approximately 47, numbered accordingly and
classified into 6 groups: A, B, C, D, E and F), based upon
properties including hemaglutination of red blood cells,
oncogenicity, DNA and protein amino acid compositions and
homologies, and antigenic relationships.
[0050] Recombinant adenoviral vectors have several advantages for
use as gene delivery vehicles, including tropism for both dividing
and non-dividing cells, minimal pathogenic potential, ability to
replicate to high titer for preparation of vector stocks, and the
potential to carry large inserts (Berkner, K. L., Curr. Top. Micro.
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64
1994). Adenoviral vectors with deletions of various adenoviral gene
sequences, such as pseudoadenoviral vectors (PAVs) and
partially-deleted adenoviral (termed "DeAd"), have been designed to
take advantage of the desirable features of adenovirus which render
it a suitable vehicle for delivery of nucleic acids to recipient
cells.
[0051] In particular, pseudoadenoviral vectors (PAVs), also known
as `gutless adenovirus` or mini-adenoviral vectors, are adenoviral
vectors derived from the genome of an adenovirus that contain
minimal cis-acting nucleotide sequences required for the
replication and packaging of the vector genome and which can
contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which
covers pseudoadenoviral vectors (PAV) and methods for producing
PAV, incorporated herein by reference). PAVs have been designed to
take advantage of the desirable features of adenovirus which render
it a suitable vehicle for gene delivery. While adenoviral vectors
can generally carry inserts of up to 8 kb in size by the deletion
of regions which are dispensable for viral growth, maximal carrying
capacity can be achieved with the use of adenoviral vectors
containing deletions of most viral coding sequences, including
PAVs. See U.S. Pat. No. 5,882,877 of Gregory et al.; Kochanek et
al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Parks et al.,
Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996; Lieber et al., J.
Virol. 70:8944-8960, 1996; Fisher et al., Virology 217:11-22, 1996;
U.S. Pat. No. 5,670,488; PCT Publication No. WO96/33280, published
Oct. 24, 1996; PCT Publication No. WO96/40955, published Dec. 19,
1996; PCT Publication No. WO97/25446, published Jul. 19, 1997; PCT
Publication No. WO95/29993, published Nov. 9, 1995; PCT Publication
No. WO97/00326, published Jan. 3, 1997; Morral et al., Hum. Gene
Ther. 10:2709-2716, 1998. Such PAVs, which can accommodate up to
about 36 kb of foreign nucleic acid, are advantageous because the
carrying capacity of the vector is optimized, while the potential
for host immune responses to the vector or the generation of
replication-competent viruses is reduced. PAV vectors contain the
5' inverted terminal repeat (ITR) and the 3' ITR nucleotide
sequences that contain the origin of replication, and the
cis-acting nucleotide sequence required for packaging of the PAV
genome, and can accommodate one or more transgenes with appropriate
regulatory elements, e.g. promoter, enhancers, etc.
[0052] Other, partially deleted adenoviral vectors provide a
partially-deleted adenoviral (termed "DeAd") vector in which the
majority of adenoviral early genes required for virus replication
are deleted from the vector and placed within a producer cell
chromosome under the control of a conditional promoter. The
deletable adenoviral genes that are placed in the producer cell may
include E1A/E1B, E2, E4 (only ORF6 and ORF6/7 need be placed into
the cell), pIX and pIVa2. E3 may also be deleted from the vector,
but since it is not required for vector production, it can be
omitted from the producer cell. The adenoviral late genes, normally
under the control of the major late promoter (MLP), are present in
the vector, but the MLP may be replaced by a conditional
promoter.
[0053] Conditional promoters suitable for use in DeAd vectors and
producer cell lines include those with the following
characteristics: low basal expression in the uninduced state, such
that cytotoxic or cytostatic adenovirus genes are not expressed at
levels harmful to the cell; and high level expression in the
induced state, such that sufficient amounts of viral proteins are
produced to support vector replication and assembly. Preferred
conditional promoters suitable for use in DeAd vectors and producer
cell lines include the dimerizer gene control system, based on the
immunosuppressive agents FK506 and rapamycin, the ecdysone gene
control system and the tetracycline gene control system. Also
useful in the present invention may be the GeneSwitch technology
(Valentis, Inc., Woodlands, Tex.) described in Abruzzese et al.,
Hum. Gene Ther. 1999 10:1499-507, the disclosure of which is hereby
incorporated herein by reference. The partially deleted adenoviral
expression system is further described in WO99/57296, the
disclosure of which is hereby incorporated by reference herein.
[0054] Adeno-associated virus (AAV) is a single-stranded human DNA
parvovirus whose genome has a size of 4.6 kb. The AAV genome
contains two major genes: the rep gene, which codes for the rep
proteins (Rep 76, Rep 68, Rep 52, and Rep 40) and the cap gene,
which codes for AAV replication, rescue, transcription and
integration, while the cap proteins form the AAV viral particle.
AAV derives its name from its dependence on an adenovirus or other
helper virus (e.g., herpesvirus) to supply essential gene products
that allow AAV to undergo a productive infection, i.e., reproduce
itself in the host cell. In the absence of helper virus, AAV
integrates as a provirus into the host cell's chromosome, until it
is rescued by superinfection of the host cell with a helper virus,
usually adenovirus (Muzyczka, Curr. Top. Micor. lmmunol.
158:97-127, 1992).
[0055] Interest in AAV as a gene transfer vector results from
several unique features of its biology. At both ends of the AAV
genome is a nucleotide sequence known as an inverted terminal
repeat (ITR), which contains the cis-acting nucleotide sequences
required for virus replication, rescue, packaging and integration.
The integration function of the ITR mediated by the rep protein in
trans permits the AAV genome to integrate into a cellular
chromosome after infection, in the absence of helper virus. This
unique property of the virus has relevance to the use of AAV in
gene transfer, as it allows for a integration of a recombinant AAV
containing a gene of interest into the cellular genome. Therefore,
stable genetic transformation, ideal for many of the goals of gene
transfer, may be achieved by use of rAAV vectors. Furthermore, the
site of integration for AAV is well-established and has been
localized to chromosome 19 of humans (Kotin et al., Proc. Natl.
Acad. Sci. 87:2211-2215, 1990). This predictability of integration
site reduces the danger of random insertional events into the
cellular genome that may activate or inactivate host genes or
interrupt coding sequences, consequences that can limit the use of
vectors whose integration of AAV, removal of this gene in the
design of rAAV vectors may result in the altered integration
patterns that have been observed with rAAV vectors (Ponnazhagan et
al., Hum Gene Titer. 8:275-284, 1997).
[0056] There are other advantages to the use of AAV for gene
transfer. The host range of AAV is broad. Moreover, unlike
retroviruses, AAV can infect both quiescent and dividing cells. In
addition, AAV has not been associated with human disease, obviating
many of the concerns that have been raised with retrovirus-derived
gene transfer vectors.
[0057] Standard approaches to the generation of recombinant rAAV
vectors have required the coordination of a series of intracellular
events: transfection of the host cell with an rAAV vector genome
containing a transgene of interest flanked by the AAV ITR
sequences, transfection of the host cell by a plasmid encoding the
genes for the AAV rep and cap proteins which are required in trans,
and infection of the transfected cell with a helper virus to supply
the non-AAV helper functions required in trans (Muzyczka, N., Curr.
Top. Micor. Immunol. 158:97-129, 1992). The adenoviral (or other
helper virus) proteins activate. transcription of the AAV rep gene,
and the rep proteins then activate transcription of the AAV cap
genes. The cap proteins then utilize the ITR sequences to package
the rAAV genome into an rAAV viral particle. Therefore, the
efficiency of packaging is determined, in part, by the availability
of adequate amounts of the structural proteins, as well as the
accessibility of any cis-acting packaging sequences required in the
rAAV vector genome.
[0058] Retrovirus vectors are a common tool for gene delivery
(Miller, Nature (1992) 357:455-460). The ability of retrovirus
vectors to deliver an unrearranged, single copy gene into a broad
range of rodent, primate and human somatic cells makes retroviral
vectors well suited for transferring genes to a cell.
[0059] Retroviruses are RNA viruses wherein the viral genome is
RNA. When a host cell is infected with a retrovirus, the genomic
RNA is reverse transcribed into a DNA intermediate which is
integrated very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to as a
provirus. Transcription of the provirus and assembly into
infectious virus occurs in the presence of an appropriate helper
virus or in a cell line containing appropriate sequences enabling
encapsidation without coincident production of a contaminating
helper virus. A helper virus is not required for the production of
the recombinant retrovirus if the sequences for encapsidation are
provided by co-transfection with appropriate vectors.
[0060] The retroviral genome and the proviral DNA have three genes:
the gag, the pol, and the env, which are flanked by two long
terminal repeat (LTR) sequences. The gag gene encodes the internal
structural (matrix, capsid, and nucleocapsid) proteins; the pol
gene encodes the RNA-directed DNA polymerase (reverse
transcriptase) and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of the virion RNAs. The LTR contains all other
cis-acting sequences necessary for viral replication. Lentiviruses
have additional genes including vit vpr, tat, rev, vpu, nef, and
vpx (in HD/-1, HIV-2 and/or SIV). Adjacent to the 5' LTR are
sequences necessary for reverse transcription of the genome (the
tRNA primer binding site) and for efficient encapsidation of viral
RNA into particles (the Psi site). If the sequences necessary for
encapsidation (or packaging of retroviral RNA into infectious
virions) are missing from the viral genome, the result is a cis
defect which prevents encapsidation of genomic RNA. However, the
resulting mutant is still capable of directing the synthesis of all
varion proteins.
[0061] Lentiviruses are complex retroviruses which, in addition to
the common retroviral genes gag, pol and env, contain other genes
with regulatory or structural function. The higher complexity
enables the lentivirus to modulate the life cycle thereof, as in
the course of latent infection. A typical lentivirus is the human
immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo,
HIV can infect terminally differentiated cells that rarely divide,
such as lymphocytes and macrophages. In vitro, HIV can infect
primary cultures of monocyte-derived macrophages (MDM) as well as
HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by
treatment with aphidicolin or gamma irradiation. Infection of cells
is dependent on the active nuclear import of HIV preintegration
complexes through the nuclear pores of the target cells. That
occurs by the interaction of multiple, partly redundant, molecular
determinants in the complex with the nuclear import machinery of
the target cell. Identified determinants include a functional
nuclear localization signal (NLS) in the gag matrix (MA) protein,
the karyophilic virion-associated protein, vpr, and a C-terminal
phosphotyrosine residue in the gag MA protein. The use of
retroviruses for gene therapy is described, for example, in U.S.
Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, the disclosures of
which are hereby incorporated herein by reference.
[0062] Other methods for delivery of DNA to cells do not use
viruses for delivery. For example, cationic amphiphilic compounds
can be used to deliver the nucleic acid of the present invention.
Because compounds designed to facilitate intracellular delivery of
biologically active molecules must interact with both non-polar and
polar environments (in or on, for example, the plasma membrane,
tissue fluids, compartments within the cell, and the biologically
active molecular itself), such compounds are designed typically to
contain both polar and non-polar domains. Compounds having both
such domains may be termed amphiphiles, and many lipids and
synthetic lipids that have been disclosed for use in facilitating
such intracellular delivery (whether for in vitro or in vivo
application) meet this definition. One particularly important class
of such amphiphiles is the cationic amphiphiles. In general,
cationic amphiphiles have polar groups that are capable of being
positively charged at or around physiological pH, and this property
is understood in the art to be important in defining how the
amphiphiles interact with the many types of biologically active
(therapeutic) molecules including, for example, negatively charged
polynucleotides such as DNA.
[0063] The use of compositions comprising cationic amphiphilic
compounds for gene delivery is described, for example, in U.S. Pat.
No. 5,049,386; U.S. Pat. No. 5,279,833; U.S. Pat. No. 5,650,096;
U.S. Pat. No. 5,747,471; U.S. Pat. No. 5,767,099; U.S. Pat. No.
5,910,487; U.S. Pat. No. 5,719,131; U.S. Pat. No. 5,840,710; U.S.
Pat. No. 5,783,565; U.S. Pat. No. 5,925,628; U.S. Pat. No.
5,912,239; U.S. Pat. No. 5,942,634; U.S. Pat. No. 5,948,925; U.S.
Pat. No. 6,022,874; U.S. Pat. No. 5,994,317; U.S. Pat. No.
5,861,397; U.S. Pat. No. 5,952,916; U.S. Pat. No. 5,948,767; U.S.
Pat. No. 5,939,401; and U.S. Pat. No. 5,935,936, the disclosures of
which are hereby incorporated herein by reference.
[0064] In addition, nucleic acid of the present invention can be
delivered using "naked DNA." Methods for delivering a
non-infectious, non-integrating DNA sequence encoding a desired
polypeptide or peptide operably linked to a promoter, free from
association with transfection-facilitating proteins, viral
particles, liposomal formulations, charged lipids and calcium
phosphate precipitating agents are described in U.S. Pat. No.
5,580,859; U.S. Pat. No. 5,963,622; U.S. Pat. No. 5,910,488; the
disclosures of which are hereby incorporated herein by
reference.
[0065] Gene transfer systems that combine viral and nonviral
components have also been reported. Cristiano et al., (1993) Proc.
Natl. Acad. Sci. USA 90:11548; Wu et al. (1994) J. Biol. Chem.
269:11542; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099;
Yoshimura et al. (1993) J. Biol. Chem. 268:2300; Curiel et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8850; Kupfer et al. (1994)
Human Gene Ther. 5:1437; and Gottschalk et al. (1994) Gene Ther.
1:185. In most cases, adenovirus has been incorporated into the
gene delivery systems to take advantage of its endosomolytic
properties. The reported combinations of viral and nonviral
components generally involve either covalent attachment of the
adenovirus to a gene delivery complex or co-internalization of
unbound adenovirus with cationic lipid: DNA complexes.
[0066] For delivery of DNA and protein to the eye, administration
will typically be local. This has the advantage of limiting the
amount of DNA that needs to be administered and limiting systemic
side-effects. Many possible modes of delivery can be used,
including, but not limited to: topical administration on the cornea
by a gene gun; subconjunctival injection, intracameral injection,
via eye drops to the cornea, injection into the anterior chamber
via the termporal limbus, intrastromal injection, corneal
application combined with electrical pulses, intracorneal
injection, subretinal injection, intravitreal injection, and
intraocular injection. Alternatively cells can be transfected or
transduced ex vivo and delivered by intraocular implantation. See,
Auricchio, Mol. Ther. 6: 490-494, 2002; Bennett, Nature Med. 2:
649-654, 1996; Borras, Experimental Eye Research 76: 643-652, 2003;
Chaum, Survey of Ophthalmology 47: 449-469, 2002; Campochiaro,
Expert Opinions in Biological Therapy 2: 537-544 (2002); Lai, Gene
Therapy 9: 804 813, 2002; Pleyer, Progress in Retinal and Eye
Research, 22: 277-293, 2003.
[0067] The effects of various proposed therapeutic agents and
administrations can be tested in suitable animal models for
particular diseases. For example, retinopathy of prematurity can be
tested in an oxygen-induced retinopathy model in the mouse as
described in Smith, Investigative Ophthalmology & Visual
Science, 35: 101-111, 1994. Laser-induced choroidal
neovascularization in a mouse can be used as a model for human
choroidal neovascularization (CNV) occurs in diseases such as
age-related macular degeneration. Tobe, American Journal of
Pathology 153: 1641-1646, 1998. Other models of CNV have been
developed in primates, rats, minipigs, and rabbits. Mouse models of
age-related macular degeneration have been developed in
genetically-deficient mice. Mice deficient in either monocyte
chemoattractant protein-1 or C-C chemokine receptor-2 develop
features of age-related macular degeneration. Ambati, Nature Med.
9: 1390-1397, 2003.
[0068] While the invention has been described with respect to
specific examples including presently preferred modes of carrying
out the invention, those skilled in the art will appreciate that
there are numerous variations and permutations of the above
described systems and techniques that fall within the spirit and
scope of the invention as set forth in the appended claims.
EXAMPLES
Example 1
[0069] Two constructs were generated: the first, D2-9Gly-Fc,
containing a polyglycine 9-mer (9Gly) linker and the second, D2-Fc,
with the same sequence except the 9Gly linker (FIG. 1).
[0070] We analyzed the amino acid sequences of D2-9Gly-Fc and D2-Fc
proteins using the Protein Analysis Toolbox of the sequence
analysis program MacVector 6.5.1. (IBI, New Haven, Conn.). The
polyglycine 9-mer linker in the D2-9Gly-Fc sequence was identified
as a region with higher than average flexibility by the flexibility
prediction method of Karpus and Schultz (1985) Naturwiss, 72:
212-213. No such region was detected in the D2-Fc sequence (FIG.
1).
Example 2
[0071] We tested an isolated Flt-1 Flt-1 Ig-like domain 2 connected
to the IgG1 Fc region by a flexible polyglycine 9-mer linker
(D2-9Gly-Fc). The D2-9Gly-Fc fusion protein is capable of
efficiently binding VEGF and of inhibiting VEGF-dependent human
umbilical vein endothelial cell (HUVEC) proliferation. See FIG. 2.
In contrast, when Flt-1 Ig-like domain 2 is linked directly to the
IgG1 heavy chain (Fc) to form D2-Fc, only minimal VEGF binding was
observed. See FIG. 2. Both the dimerization via IgG1 Fc and the
insertion of a flexible linker appear to facilitate VEGF binding to
Flt-1 domain 2. The presence of dimeric forms in both D2-9Gly-Fc
and D2-Fc were confirmed by the Western blot analysis. See FIG.
3.
Example 3
[0072] An intravitreal injection of AAV vector (1.times.108 to
1.times.109 particles in a volume of 0.0005 mL) is administered to
newborn (P0) or 1 day old (P1) C57BL/6 mice. Retinal
neovascularization (NV) is induced in C57BL/6 mice by exposing P7
pups and their nursing dam to hyperoxia for 5 days. The pups are
returned to room air on P12 and are euthanized at P17 (time of peak
NV). (Smith L E H, Weslowski E, McLellan A, Kostyk S K, D'Amato R,
Sullivan and D'Amore P A. Oxygen-Induced Retinopathy in the Mouse.
Invest Opth Vis Sci. 1994;35:101-111.) Entire paraffm embedded eyes
are serially cross sectioned at 5 micron intervals. The degree of
NV is determined by counting the number of endothelial cell nuclei
internal to the inner limiting membrane in sections taken every 100
microns.
[0073] Cohorts of animals treated with the AAV vectors coding for
the anti-angiogenic agents are compared to cohorts treated with
vectors coding for irrelevant transgenes or with vectors that do
not code for a transgene. The average number of endothelial cell
nuclei in each treated eye is compared to each animal's untreated
fellow eye.
Example 4
Generation of D2-9Gly-Ex3/CH3
[0074] Domain 2 of Flt-1 has been shown to be essential for VEGF165
binding. However, it was demonstrated that Flt-1 domain 2 alone was
incapable of binding VEGF A. (Davis-Smyth et al., 1996.) VEGF A,
when present as a dimer, binds to Flt-1 through acidic residues
(amino acids 63-67 of the mature protein) that allows a possible
mechanism for ligand-induced dimerization of receptor (Keyt et al.,
1996).
[0075] Therefore, a dimerization of domain 2 of Flt-1 was used as a
strategy to restore the binding of domain 2 of Flt-1 to VEGF A.
Fusion with a fragment of IgG heavy chain can be used for
dimerization of proteins (Davis-Smyth et al., 1996). Here we
demonstrate that amino acids 75-88 (i.e., PSCVPLMRCGGCCN; SEQ ID
NO: 13) of VEGF A (SEQ ID NO: 12) increase the biological activity
of sFlt-1 hybrid proteins.
[0076] Initially, three hybrid proteins were engineered:
D2-9Gly-Fc, D2-Fc and D2-9GlyEx3/CH3 (FIG. 4). All three hybrid
proteins contain the same Flt-1 domain D2 as D29Gly-Fc. No VEGF
binding was observed with D2-Fc, which does not contain the
polyglycine 9-mer (9Gly) linker. The third protein,
D2-9Gly-Ex3/CH3, contains the polyglycine 9-mer (9Gly) linker and
the multimerization domain of VEGF (aa PSCVPLMRCGGCCN; SEQ ID NO:
13; VEGF Ex3), but it also contains the CH3 region of human IgG1
heavy chain Fc (aa 371-477 of the SEQ ID NO: 10).
[0077] The protein D2-Fc did not show efficient inhibitory activity
in the HUVEC proliferation assay (FIG. 5) and by implication did
not bind to VEGF165 efficiently. However, the third hybrid protein,
D2-9Gly-Ex3/CH3, which comprises domain 2 of Flt-1 fused to the CH3
region via both the 9Gly linker and the dimerization region of
VEGF165 (Ex 3), did demonstrate inhibitory activity in a
VEGF-dependent HUVECs proliferation assay (FIG. 5). This implies
that this hybrid protein binds to VEGF165 efficiently.
Example 5
Using Linker (Gly4Ser)3 in Flt-1 D2 Construct
[0078] The use of several polyglycine linkers has been previously
described for improvement of protein features (Mouz et al., 1996;
Qiu et al., 1998). For the next construct we have used another type
of linker, the 15-mer (Gly-Gly-Gly-Gly-Ser)3 (Huston et al., 1988).
D2-(Gly4Ser)3-Fc protein was generated and it contains Flt-1 domain
2, (Gly4Ser)3 linker and the Fc region of human IgG1 heavy
chain.
[0079] D2-(Gly4Ser)3-Fc was further characterized in HUVECs
proliferation assay. Biological activity of D2-(Gly4Ser)3-Fc as
measured by inhibition of HUVEC proliferation was similar to that
of D2-9Gly-Fc and D2-9Gly-Ex3/CH3 (FIG. 6).
[0080] The D2-(Gly4Ser)3-Fc construct was further characterized by
Western blot and compared to D2-9Gly-Fc (FIG. 9). Both constructs
are present mostly in a dimer form and the monomer forms were
detected after separation of reduced samples.
Example 6
Role of 9Gly or VEGF Ex3 in Flt-1 (D2) Constructs
[0081] In order to investigate the role of 9Gly linker or VEGF
dimerizing sequence Ex3 on soluble receptor VEGF binding, three
other constructs were generated: D2-9Gly-CH3, D2-CH3 and D2-Ex3/CH3
(FIG. 8). All three constructs were generated and like all the
previous constructs were also put under control of CMV promoter.
Their VEGF blocking activity was assessed in HUVECs proliferation
assay (FIG. 9).
[0082] The HUVEC proliferation assay of proteins containing the CH3
region of IgG1 has shown that D2-9Gly-CH3 (without Ex3) and protein
D2-Ex3/CH3 (without 9Gly linker) had similar VEGF blocking potency
as compared to the parental D2-9GlyEx3/CH3 protein. However,
protein D2-CH3 appeared to be the weakest VEGF inhibitor from all
of them (FIG. 9).
[0083] The Flt-1 ELISA data of conditioned media from transfected
293 cells has shown similar Flt-1 levels for D2-9Gly-Ex3/CH3,
D2-9Gly-CH3 and D2-Ex3/CH3 and D2CH3 (70-90 ng/ml) and a little
higher (-150 ng/ml) for the least active form of D2CH3. Western
blot of D2-9Gly-CH3 and D2-CH3 constructs (FIG. 10) is showing a
prevalence of dimer forms in non-reduced conditions.
Example 7
D2-9Gly-Fc Binds VEGF Better than all Constructs
[0084] VEGF binding assay allows us to compare the relative VEGF
binding affinities of our soluble VEGF receptors in a cell free
system.
[0085] Briefly, conditioned media containing known concentrations
of soluble receptor (ranging in concentrations from 0.29-150 pM)
were serially diluted and mixed with 10 pM VEGF. The amount of
unbound VEGF was then measured by ELISA. D2-9Gly-Fc binds VEGF with
higher affinity to bind VEGF at receptor concentrations from 0.001
to -0.2 pM than all other constructs. D2-CH3 has the lowest
affinity to bind VEGF (FIG. 11).
REFERENCES
[0086] Davis-Smyth, et al., EMBO J., 15, 1996, 4919 [0087] Huston,
J. S., et al. (1991) Methods Enzymol. 203, 46-88 [0088] Huston, J.
S., et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 5879-5883. [0089]
Johnson, S., et al. (1991) Methods Enzymol. 203, 88-98 Karpus, P.
A., et al. (1985) Naturwiss., 72, 212-213. [0090] Keyt, B. A., et
al. (1996) J. Biol. Chem. 271: 5638 -5646. Korff, A. A., et at.
(1997) Protein Engng, 10, 423-433. Lee, Y-L., et al. (1998) Human
Gene Therapy, 9, 457-465 [0091] Mouz N., et al. (1996) Proc. Nati
Acad. Sci. USA, 93, 9414-9419. [0092] Nielsen, et al. (1997)
Protein Eng., 10, 1 [0093] Qiu, H., et al. (1998) J. Biol. Chem.
273: 11173-11176.
Sequence CWU 1
1
3411077DNAArtificial SequenceRecombinant fusion protein or sequence
encoding same 1atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg tacagtgaaa
tccccgaaat tatacacatg 120actgaaggaa gggagctcgt cattccctgc
cgggttacgt cacctaacat cactgttact 180ttaaaaaagt ttccacttga
cactttgatc cctgatggaa aacgcataat ctgggacagt 240agaaagggct
tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa
300gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca
aaccggtgga 360ggtggaggtg gaggtggagg tcctaaatct tgtgacaaaa
ctcacacatg cccaccgtgc 420ccagcacctg aactcctggg gggaccgtca
gtcttcctct tccccccaaa acccaaggac 480accctcatga tctcccggac
ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 540gaccctgagg
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
600aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct
caccgtcctg 660caccaggact ggctgaatgg caaggagtac aagtgcaagg
tctccaacaa agccctccca 720gcccccatcg agaaaaccat ctccaaagcc
aaagggcagc cccgagaacc acaggtgtac 780accctgcccc catcccggga
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 840aaaggcttct
atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac
900aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct
ctacagcaag 960ctcaccgtgg acaagagcag gtggcagcag gggaacgtct
tctcatgctc cgtgatgcat 1020gaggctctgc acaaccacta cacgcagaag
agcctctccc tgtctccggg taaatag 10772358PRTArtificial
SequenceRecombinant fusion protein or sequence encoding same 2Met
Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10
15 Cys Leu Leu Leu Thr Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser
20 25 30 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu
Val Ile 35 40 45 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr
Leu Lys Lys Phe 50 55 60 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg Lys Gly Phe Ile Ile Ser Asn
Ala Thr Tyr Lys Glu Ile Gly Leu 85 90 95 Leu Thr Cys Glu Ala Thr
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 100 105 110 Leu Thr His Arg
Gln Thr Gly Gly Gly Gly Gly Gly Gly Gly Gly Pro 115 120 125 Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 130 135 140
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 145
150 155 160 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 165 170 175 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 180 185 190 Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn 195 200 205 Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp 210 215 220 Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 225 230 235 240 Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 245 250 255 Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 260 265
270 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 290 295 300 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 305 310 315 320 Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 325 330 335 Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 340 345 350 Ser Leu Ser Pro Gly
Lys 355 31050DNAArtificial SequenceRecombinant fusion protein or
sequence encoding same 3atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg tacagtgaaa
tccccgaaat tatacacatg 120actgaaggaa gggagctcgt cattccctgc
cgggttacgt cacctaacat cactgttact 180ttaaaaaagt ttccacttga
cactttgatc cctgatggaa aacgcataat ctgggacagt 240agaaagggct
tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa
300gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca
aacccctaaa 360tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 420tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 480gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 540gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
600acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 660tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 720gccaaagggc agccccgaga accacaggtg
tacaccctgc ccccatcccg ggatgagctg 780accaagaacc aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 840gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
900gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 960caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 1020aagagcctct ccctgtctcc gggtaaatag
10504349PRTArtificial SequenceRecombinant fusion protein or
sequence encoding same 4Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu
Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Gly Arg
Pro Phe Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu Ile Ile His
Met Thr Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys Arg Val Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60 Pro Leu Asp
Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg
Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 85 90
95 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr
100 105 110 Leu Thr His Arg Gln Thr Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 115 120 125 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu 130 135 140 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu 145 150 155 160 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 165 170 175 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 180 185 190 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 195 200 205 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 210 215
220 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
225 230 235 240 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 245 250 255 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 260 265 270 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 275 280 285 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 290 295 300 Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 305 310 315 320 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345
5426DNAArtificial SequenceRecombinant fusion protein or sequence
encoding same 5atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg tacagtgaaa
tccccgaaat tatacacatg 120actgaaggaa gggagctcgt cattccctgc
cgggttacgt cacctaacat cactgttact 180ttaaaaaagt ttccacttga
cactttgatc cctgatggaa aacgcataat ctgggacagt 240agaaagggct
tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa
300gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca
aaccggtgga 360ggtggaggtg gaggtggagg tccttcctgt gtgcccctga
tgcgatgcgg gggctgctgc 420aattag 4266141PRTArtificial
SequenceRecombinant fusion protein or sequence encoding same 6Met
Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10
15 Cys Leu Leu Leu Thr Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser
20 25 30 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu
Val Ile 35 40 45 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr
Leu Lys Lys Phe 50 55 60 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg Lys Gly Phe Ile Ile Ser Asn
Ala Thr Tyr Lys Glu Ile Gly Leu 85 90 95 Leu Thr Cys Glu Ala Thr
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 100 105 110 Leu Thr His Arg
Gln Thr Gly Gly Gly Gly Gly Gly Gly Gly Gly Pro 115 120 125 Ser Cys
Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn 130 135 140
7744DNAArtificial SequenceRecombinant fusion protein or sequence
encoding same 7atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg tacagtgaaa
tccccgaaat tatacacatg 120actgaaggaa gggagctcgt cattccctgc
cgggttacgt cacctaacat cactgttact 180ttaaaaaagt ttccacttga
cactttgatc cctgatggaa aacgcataat ctgggacagt 240agaaagggct
tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa
300gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca
aaccggtgga 360ggtggaggtg gaggtggagg tccttcctgt gtgcccctga
tgcgatgcgg gggctgctgc 420aatcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag 480aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 540tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
600gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg
gcagcagggg 660aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 720ctctccctgt ctccgggtaa atag
7448247PRTArtificial SequenceRecombinant fusion protein or sequence
encoding same 8Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala
Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Gly Arg Pro Phe
Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu Ile Ile His Met Thr
Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys Arg Val Thr Ser Pro
Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60 Pro Leu Asp Thr Leu
Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg Lys Gly
Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 85 90 95 Leu
Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 100 105
110 Leu Thr His Arg Gln Thr Gly Gly Gly Gly Gly Gly Gly Gly Gly Pro
115 120 125 Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Gln
Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230
235 240 Leu Ser Leu Ser Pro Gly Lys 245 91434DNAHomo sapiens
9atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag
60gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc
120tgtgcagcgt ctggattcac cttcagtaat tatggcatgc actgggtccg
ccaggctcca 180ggcaaggggc tggagtgggt ggcagctata tggtatgatg
gaagtaataa atactatgca 240gactccgtga agggccgatt caccatctcc
agagacaatt ccaagaacac gttgtatatg 300caaatgaaca gcctgagagc
cgaggacacg gctgtgtatt attgtgcgag agagggtcgg 360tgggtacgat
atactacggt gactactatc ggatactact ttgactactg gggccaggga
420accctggtca ccgtctcctc agcctccacc aagggcccat cggtcttccc
cctggcaccc 480tcctccaaga gcacctctgg gggcacagcg gccctgggct
gcctggtcaa ggactacttc 540cccgaaccgg tgacggtgtc gtggaactca
ggcgccctga ccagcggcgt gcacaccttc 600ccggctgtcc tacagtcctc
aggactctac tccctcagca gcgtggtgac cgtgccctcc 660agcagcttgg
gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag
720gtggacaaga gagttgagcc caaatcttgt gacaaaactc acacatgccc
accgtgccca 780gcacctgaac tcctgggggg accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc 840ctcatgatct cccggacccc tgaggtcaca
tgcgtggtgg tggacgtgag ccacgaagac 900cctgaggtca agttcaactg
gtacgtggac ggcgtggagg tgcataatgc caagacaaag 960ccgcgggagg
agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac
1020caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc
cctcccagcc 1080cccatcgaga aaaccatctc caaagccaaa gggcagcccc
gagaaccaca ggtgtacacc 1140ctgcccccat cccgggagga gatgaccaag
aaccaggtca gcctgacctg cctggtcaaa 1200ggcttctatc ccagcgacat
cgccgtggag tgggagagca atgggcagcc ggagaacaac 1260tacaagacca
cgcctcccgt gctggactcc gacggctcct tcttcctcta tagcaagctc
1320accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt
gatgcatgag 1380gctctgcaca accactacac gcagaagagc ctctccctgt
ccccgggtaa atga 143410477PRTHomo sapiens 10Met Glu Phe Gly Leu Ser
Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly
Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45
Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50
55 60 Glu Trp Val Ala Ala Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn 85 90 95 Thr Leu Tyr Met Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Gly Arg Trp
Val Arg Tyr Thr Thr Val Thr 115 120 125 Thr Ile Gly Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 130 135 140 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 165 170 175
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180
185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly 195 200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly 210 215 220 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys 245 250 255 Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 275 280 285 Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295 300
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305
310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys 340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys 355
360 365 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser 370 375 380 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 405 410 415 Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly 420 425 430 Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445 Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460 His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
11648DNAHomo sapiens 11atgaactttc tgctgtcttg ggtgcattgg agccttgcct
tgctgctcta cctccaccat 60gccaagtggt cccaggctgc acccatggca gaaggaggag
ggcagaatca tcacgaagtg 120gtgaagttca tggatgtcta tcagcgcagc
tactgccatc caatcgagac cctggtggac 180atcttccagg agtaccctga
tgagatcgag tacatcttca agccatcctg tgtgcccctg 240atgcgatgcg
ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc
300aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat
aggagagatg 360agcttcctac agcacaacaa atgtgaatgc agaccaaaga
aagatagagc aagacaagaa 420aaaaaatcag ttcgaggaaa gggaaagggg
caaaaacgaa agcgcaagaa atcccggtat 480aagtcctgga gcgttccctg
tgggccttgc tcagagcgga gaaagcattt gtttgtacaa 540gatccgcaga
cgtgtaaatg ttcctgcaaa aacacagact cgcgttgcaa ggcgaggcag
600cttgagttaa acgaacgtac ttgcagatgt gacaagccga ggcggtga
64812215PRTHomo sapiens 12Met Asn Phe Leu Leu Ser Trp Val His Trp
Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser
Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His
Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys
His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro
Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85
90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro
His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His
Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln
Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg
Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Pro
Cys Gly Pro Cys Ser Glu Arg Arg Lys His 165 170 175 Leu Phe Val Gln
Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr 180 185 190 Asp Ser
Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys 195 200 205
Arg Cys Asp Lys Pro Arg Arg 210 215 1314PRTHomo sapiens 13Pro Ser
Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn 1 5 10
145777DNAHomo sapiens 14gcggacactc ctctcggctc ctccccggca gcggcggcgg
ctcggagcgg gctccggggc 60tcgggtgcag cggccagcgg gcctggcggc gaggattacc
cggggaagtg gttgtctcct 120ggctggagcc gcgagacggg cgctcagggc
gcggggccgg cggcggcgaa cgagaggacg 180gactctggcg gccgggtcgt
tggccggggg agcgcgggca ccgggcgagc aggccgcgtc 240gcgctcacca
tggtcagcta ctgggacacc ggggtcctgc tgtgcgcgct gctcagctgt
300ctgcttctca caggatctag ttcaggttca aaattaaaag atcctgaact
gagtttaaaa 360ggcacccagc acatcatgca agcaggccag acactgcatc
tccaatgcag gggggaagca 420gcccataaat ggtctttgcc tgaaatggtg
agtaaggaaa gcgaaaggct gagcataact 480aaatctgcct gtggaagaaa
tggcaaacaa ttctgcagta ctttaacctt gaacacagct 540caagcaaacc
acactggctt ctacagctgc aaatatctag ctgtacctac ttcaaagaag
600aaggaaacag aatctgcaat ctatatattt attagtgata caggtagacc
tttcgtagag 660atgtacagtg aaatccccga aattatacac atgactgaag
gaagggagct cgtcattccc 720tgccgggtta cgtcacctaa catcactgtt
actttaaaaa agtttccact tgacactttg 780atccctgatg gaaaacgcat
aatctgggac agtagaaagg gcttcatcat atcaaatgca 840acgtacaaag
aaatagggct tctgacctgt gaagcaacag tcaatgggca tttgtataag
900acaaactatc tcacacatcg acaaaccaat acaatcatag atgtccaaat
aagcacacca 960cgcccagtca aattacttag aggccatact cttgtcctca
attgtactgc taccactccc 1020ttgaacacga gagttcaaat gacctggagt
taccctgatg aaaaaaataa gagagcttcc 1080gtaaggcgac gaattgacca
aagcaattcc catgccaaca tattctacag tgttcttact 1140attgacaaaa
tgcagaacaa agacaaagga ctttatactt gtcgtgtaag gagtggacca
1200tcattcaaat ctgttaacac ctcagtgcat atatatgata aagcattcat
cactgtgaaa 1260catcgaaaac agcaggtgct tgaaaccgta gctggcaagc
ggtcttaccg gctctctatg 1320aaagtgaagg catttccctc gccggaagtt
gtatggttaa aagatgggtt acctgcgact 1380gagaaatctg ctcgctattt
gactcgtggc tactcgttaa ttatcaagga cgtaactgaa 1440gaggatgcag
ggaattatac aatcttgctg agcataaaac agtcaaatgt gtttaaaaac
1500ctcactgcca ctctaattgt caatgtgaaa ccccagattt acgaaaaggc
cgtgtcatcg 1560tttccagacc cggctctcta cccactgggc agcagacaaa
tcctgacttg taccgcatat 1620ggtatccctc aacctacaat caagtggttc
tggcacccct gtaaccataa tcattccgaa 1680gcaaggtgtg acttttgttc
caataatgaa gagtccttta tcctggatgc tgacagcaac 1740atgggaaaca
gaattgagag catcactcag cgcatggcaa taatagaagg aaagaataag
1800atggctagca ccttggttgt ggctgactct agaatttctg gaatctacat
ttgcatagct 1860tccaataaag ttgggactgt gggaagaaac ataagctttt
atatcacaga tgtgccaaat 1920gggtttcatg ttaacttgga aaaaatgccg
acggaaggag aggacctgaa actgtcttgc 1980acagttaaca agttcttata
cagagacgtt acttggattt tactgcggac agttaataac 2040agaacaatgc
actacagtat tagcaagcaa aaaatggcca tcactaagga gcactccatc
2100actcttaatc ttaccatcat gaatgtttcc ctgcaagatt caggcaccta
tgcctgcaga 2160gccaggaatg tatacacagg ggaagaaatc ctccagaaga
aagaaattac aatcagagat 2220caggaagcac catacctcct gcgaaacctc
agtgatcaca cagtggccat cagcagttcc 2280accactttag actgtcatgc
taatggtgtc cccgagcctc agatcacttg gtttaaaaac 2340aaccacaaaa
tacaacaaga gcctggaatt attttaggac caggaagcag cacgctgttt
2400attgaaagag tcacagaaga ggatgaaggt gtctatcact gcaaagccac
caaccagaag 2460ggctctgtgg aaagttcagc atacctcact gttcaaggaa
cctcggacaa gtctaatctg 2520gagctgatca ctctaacatg cacctgtgtg
gctgcgactc tcttctggct cctattaacc 2580ctccttatcc gaaaaatgaa
aaggtcttct tctgaaataa agactgacta cctatcaatt 2640ataatggacc
cagatgaagt tcctttggat gagcagtgtg agcggctccc ttatgatgcc
2700agcaagtggg agtttgcccg ggagagactt aaactgggca aatcacttgg
aagaggggct 2760tttggaaaag tggttcaagc atcagcattt ggcattaaga
aatcacctac gtgccggact 2820gtggctgtga aaatgctgaa agagggggcc
acggccagcg agtacaaagc tctgatgact 2880gagctaaaaa tcttgaccca
cattggccac catctgaacg tggttaacct gctgggagcc 2940tgcaccaagc
aaggagggcc tctgatggtg attgttgaat actgcaaata tggaaatctc
3000tccaactacc tcaagagcaa acgtgactta ttttttctca acaaggatgc
agcactacac 3060atggagccta agaaagaaaa aatggagcca ggcctggaac
aaggcaagaa accaagacta 3120gatagcgtca ccagcagcga aagctttgcg
agctccggct ttcaggaaga taaaagtctg 3180agtgatgttg aggaagagga
ggattctgac ggtttctaca aggagcccat cactatggaa 3240gatctgattt
cttacagttt tcaagtggcc agaggcatgg agttcctgtc ttccagaaag
3300tgcattcatc gggacctggc agcgagaaac attcttttat ctgagaacaa
cgtggtgaag 3360atttgtgatt ttggccttgc ccgggatatt tataagaacc
ccgattatgt gagaaaagga 3420gatactcgac ttcctctgaa atggatggct
cccgaatcta tctttgacaa aatctacagc 3480accaagagcg acgtgtggtc
ttacggagta ttgctgtggg aaatcttctc cttaggtggg 3540tctccatacc
caggagtaca aatggatgag gacttttgca gtcgcctgag ggaaggcatg
3600aggatgagag ctcctgagta ctctactcct gaaatctatc agatcatgct
ggactgctgg 3660cacagagacc caaaagaaag gccaagattt gcagaacttg
tggaaaaact aggtgatttg 3720cttcaagcaa atgtacaaca ggatggtaaa
gactacatcc caatcaatgc catactgaca 3780ggaaatagtg ggtttacata
ctcaactcct gccttctctg aggacttctt caaggaaagt 3840atttcagctc
cgaagtttaa ttcaggaagc tctgatgatg tcagatatgt aaatgctttc
3900aagttcatga gcctggaaag aatcaaaacc tttgaagaac ttttaccgaa
tgccacctcc 3960atgtttgatg actaccaggg cgacagcagc actctgttgg
cctctcccat gctgaagcgc 4020ttcacctgga ctgacagcaa acccaaggcc
tcgctcaaga ttgacttgag agtaaccagt 4080aaaagtaagg agtcggggct
gtctgatgtc agcaggccca gtttctgcca ttccagctgt 4140gggcacgtca
gcgaaggcaa gcgcaggttc acctacgacc acgctgagct ggaaaggaaa
4200atcgcgtgct gctccccgcc cccagactac aactcggtgg tcctgtactc
caccccaccc 4260atctagagtt tgacacgaag ccttatttct agaagcacat
gtgtatttat acccccagga 4320aactagcttt tgccagtatt atgcatatat
aagtttacac ctttatcttt ccatgggagc 4380cagctgcttt ttgtgatttt
tttaatagtg cttttttttt ttgactaaca agaatgtaac 4440tccagataga
gaaatagtga caagtgaaga acactactgc taaatcctca tgttactcag
4500tgttagagaa atccttccta aacccaatga cttccctgct ccaacccccg
ccacctcagg 4560gcacgcagga ccagtttgat tgaggagctg cactgatcac
ccaatgcatc acgtacccca 4620ctgggccagc cctgcagccc aaaacccagg
gcaacaagcc cgttagcccc aggggatcac 4680tggctggcct gagcaacatc
tcgggagtcc tctagcaggc ctaagacatg tgaggaggaa 4740aaggaaaaaa
agcaaaaagc aagggagaaa agagaaaccg ggagaaggca tgagaaagaa
4800tttgagacgc accatgtggg cacggagggg gacggggctc agcaatgcca
tttcagtggc 4860ttcccagctc tgacccttct acatttgagg gcccagccag
gagcagatgg acagcgatga 4920ggggacattt tctggattct gggaggcaag
aaaaggacaa atatcttttt tggaactaaa 4980gcaaatttta gacctttacc
tatggaagtg gttctatgtc cattctcatt cgtggcatgt 5040tttgatttgt
agcactgagg gtggcactca actctgagcc catacttttg gctcctctag
5100taagatgcac tgaaaactta gccagagtta ggttgtctcc aggccatgat
ggccttacac 5160tgaaaatgtc acattctatt ttgggtatta atatatagtc
cagacactta actcaatttc 5220ttggtattat tctgttttgc acagttagtt
gtgaaagaaa gctgagaaga atgaaaatgc 5280agtcctgagg agagttttct
ccatatcaaa acgagggctg atggaggaaa aaggtcaata 5340aggtcaaggg
aagaccccgt ctctatacca accaaaccaa ttcaccaaca cagttgggac
5400ccaaaacaca ggaagtcagt cacgtttcct tttcatttaa tggggattcc
actatctcac 5460actaatctga aaggatgtgg aagagcatta gctggcgcat
attaagcact ttaagctcct 5520tgagtaaaaa ggtggtatgt aatttatgca
aggtatttct ccagttggga ctcaggatat 5580tagttaatga gccatcacta
gaagaaaagc ccattttcaa ctgctttgaa acttgcctgg 5640ggtctgagca
tgatgggaat agggagacag ggtaggaaag ggcgcctact cttcagggtc
5700taaagatcaa gtgggccttg gatcgctaag ctggctctgt ttgatgctat
ttatgcaagt 5760tagggtctat gtattta 5777151338PRTHomo
sapiensDOMAIN(235)..(336)domain 3 15Met Val Ser Tyr Trp Asp Thr Gly
Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly
Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu
Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His
Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60
Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65
70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu
Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys
Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser
Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val
Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu
Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185
190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr
His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr
Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val
Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln
Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser
Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile
Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp
Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310
315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr
Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly
Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro
Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr
Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile
Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr
Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu
Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430
Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435
440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr
Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu
Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe
Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser
Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met
Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile
Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg
Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555
560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser
565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile
Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile
Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr
Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly
Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu
Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu
Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala
Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680
685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu
690 695 700 Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile
Glu Arg 705 710 715 720 Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys
Lys Ala Thr Asn Gln 725 730 735 Lys Gly Ser Val Glu Ser Ser Ala Tyr
Leu Thr Val Gln Gly Thr Ser 740 745 750 Asp Lys Ser Asn Leu Glu Leu
Ile Thr Leu Thr Cys Thr Cys Val Ala 755 760 765 Ala Thr Leu Phe Trp
Leu Leu Leu Thr Leu Leu Ile Arg Lys Met Lys 770 775 780 Arg Ser Ser
Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp 785 790 795 800
Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp 805
810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly Lys
Ser 820 825 830 Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser
Ala Phe Gly 835 840 845 Ile Lys Lys Ser Pro Thr Cys Arg Thr Val Ala
Val Lys Met Leu Lys 850 855 860 Glu Gly Ala Thr Ala Ser Glu Tyr Lys
Ala Leu Met Thr Glu Leu Lys 865 870 875 880 Ile Leu Thr His Ile Gly
His His Leu Asn Val Val Asn Leu Leu Gly 885 890 895 Ala Cys Thr Lys
Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905 910 Lys Tyr
Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915 920 925
Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys 930
935 940 Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu
Asp Ser Val 945 950 955 960 Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly
Phe Gln Glu Asp Lys Ser 965 970 975 Leu Ser Asp Val Glu Glu Glu Glu
Asp Ser Asp Gly Phe Tyr Lys Glu 980 985 990 Pro Ile Thr Met Glu Asp
Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005 Gly Met Glu
Phe Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020 Ala
Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025 1030
1035 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr
1040 1045 1050 Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met
Ala Pro 1055 1060 1065 Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys
Ser Asp Val Trp 1070 1075 1080 Ser Tyr Gly Val Leu Leu Trp Glu Ile
Phe Ser Leu Gly Gly Ser 1085 1090 1095 Pro Tyr Pro Gly Val Gln Met
Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110 Arg Glu Gly Met Arg
Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125 Ile Tyr Gln
Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140 Arg
Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145 1150
1155 Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn
1160 1165 1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr
Pro Ala 1175 1180 1185 Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser
Ala Pro Lys Phe 1190 1195 1200 Asn Ser Gly Ser Ser Asp Asp Val Arg
Tyr Val Asn Ala Phe Lys 1205 1210 1215 Phe Met Ser Leu Glu Arg Ile
Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230 Asn Ala Thr Ser Met
Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245 Leu Leu Ala
Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260 Lys
Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265 1270
1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys
1280 1285 1290 His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg
Phe Thr 1295 1300 1305 Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala
Cys Cys Ser Pro 1310 1315 1320 Pro Pro Asp Tyr Asn Ser Val Val Leu
Tyr Ser Thr Pro Pro Ile 1325 1330 1335 165830DNAHomo sapiens
16actgagtccc gggaccccgg gagagcggtc agtgtgtggt cgctgcgttt cctctgcctg
60cgccgggcat cacttgcgcg ccgcagaaag tccgtctggc agcctggata tcctctccta
120ccggcacccg cagacgcccc tgcagccgcc ggtcggcgcc cgggctccct
agccctgtgc 180gctcaactgt cctgcgctgc ggggtgccgc gagttccacc
tccgcgcctc cttctctaga 240caggcgctgg gagaaagaac cggctcccga
gttctgggca tttcgcccgg ctcgaggtgc 300aggatgcaga gcaaggtgct
gctggccgtc gccctgtggc tctgcgtgga gacccgggcc 360gcctctgtgg
gtttgcctag tgtttctctt gatctgccca ggctcagcat acaaaaagac
420atacttacaa ttaaggctaa tacaactctt caaattactt gcaggggaca
gagggacttg 480gactggcttt ggcccaataa tcagagtggc agtgagcaaa
gggtggaggt gactgagtgc 540agcgatggcc tcttctgtaa gacactcaca
attccaaaag tgatcggaaa tgacactgga 600gcctacaagt gcttctaccg
ggaaactgac ttggcctcgg tcatttatgt ctatgttcaa 660gattacagat
ctccatttat tgcttctgtt agtgaccaac atggagtcgt gtacattact
720gagaacaaaa acaaaactgt ggtgattcca tgtctcgggt ccatttcaaa
tctcaacgtg 780tcactttgtg caagataccc agaaaagaga tttgttcctg
atggtaacag aatttcctgg 840gacagcaaga agggctttac tattcccagc
tacatgatca gctatgctgg catggtcttc 900tgtgaagcaa aaattaatga
tgaaagttac cagtctatta tgtacatagt tgtcgttgta 960gggtatagga
tttatgatgt ggttctgagt ccgtctcatg gaattgaact atctgttgga
1020gaaaagcttg tcttaaattg tacagcaaga actgaactaa atgtggggat
tgacttcaac 1080tgggaatacc cttcttcgaa gcatcagcat aagaaacttg
taaaccgaga cctaaaaacc 1140cagtctggga gtgagatgaa gaaatttttg
agcaccttaa ctatagatgg tgtaacccgg 1200agtgaccaag gattgtacac
ctgtgcagca tccagtgggc tgatgaccaa gaagaacagc 1260acatttgtca
gggtccatga aaaacctttt gttgcttttg gaagtggcat ggaatctctg
1320gtggaagcca cggtggggga gcgtgtcaga atccctgcga agtaccttgg
ttacccaccc 1380ccagaaataa aatggtataa aaatggaata ccccttgagt
ccaatcacac aattaaagcg 1440gggcatgtac tgacgattat ggaagtgagt
gaaagagaca caggaaatta cactgtcatc 1500cttaccaatc ccatttcaaa
ggagaagcag agccatgtgg tctctctggt tgtgtatgtc 1560ccaccccaga
ttggtgagaa atctctaatc tctcctgtgg attcctacca gtacggcacc
1620actcaaacgc tgacatgtac ggtctatgcc attcctcccc cgcatcacat
ccactggtat 1680tggcagttgg aggaagagtg cgccaacgag cccagccaag
ctgtctcagt gacaaaccca 1740tacccttgtg aagaatggag aagtgtggag
gacttccagg gaggaaataa aattgaagtt 1800aataaaaatc aatttgctct
aattgaagga aaaaacaaaa ctgtaagtac ccttgttatc 1860caagcggcaa
atgtgtcagc tttgtacaaa tgtgaagcgg tcaacaaagt cgggagagga
1920gagagggtga tctccttcca cgtgaccagg ggtcctgaaa ttactttgca
acctgacatg 1980cagcccactg agcaggagag cgtgtctttg tggtgcactg
cagacagatc tacgtttgag 2040aacctcacat ggtacaagct tggcccacag
cctctgccaa tccatgtggg agagttgccc 2100acacctgttt gcaagaactt
ggatactctt tggaaattga atgccaccat gttctctaat 2160agcacaaatg
acattttgat catggagctt aagaatgcat ccttgcagga ccaaggagac
2220tatgtctgcc ttgctcaaga caggaagacc aagaaaagac attgcgtggt
caggcagctc 2280acagtcctag agcgtgtggc acccacgatc acaggaaacc
tggagaatca gacgacaagt 2340attggggaaa gcatcgaagt ctcatgcacg
gcatctggga atccccctcc acagatcatg 2400tggtttaaag ataatgagac
ccttgtagaa gactcaggca ttgtattgaa ggatgggaac 2460cggaacctca
ctatccgcag agtgaggaag gaggacgaag gcctctacac ctgccaggca
2520tgcagtgttc ttggctgtgc aaaagtggag gcatttttca taatagaagg
tgcccaggaa 2580aagacgaact tggaaatcat tattctagta ggcacggcgg
tgattgccat gttcttctgg 2640ctacttcttg tcatcatcct acggaccgtt
aagcgggcca atggagggga actgaagaca 2700ggctacttgt ccatcgtcat
ggatccagat gaactcccat tggatgaaca ttgtgaacga 2760ctgccttatg
atgccagcaa atgggaattc cccagagacc ggctgaagct aggtaagcct
2820cttggccgtg gtgcctttgg ccaagtgatt gaagcagatg cctttggaat
tgacaagaca 2880gcaacttgca ggacagtagc agtcaaaatg ttgaaagaag
gagcaacaca cagtgagcat 2940cgagctctca tgtctgaact caagatcctc
attcatattg gtcaccatct caatgtggtc 3000aaccttctag gtgcctgtac
caagccagga gggccactca tggtgattgt ggaattctgc 3060aaatttggaa
acctgtccac ttacctgagg agcaagagaa atgaatttgt cccctacaag
3120accaaagggg cacgattccg tcaagggaaa gactacgttg gagcaatccc
tgtggatctg 3180aaacggcgct tggacagcat caccagtagc cagagctcag
ccagctctgg atttgtggag 3240gagaagtccc tcagtgatgt agaagaagag
gaagctcctg aagatctgta taaggacttc 3300ctgaccttgg agcatctcat
ctgttacagc ttccaagtgg ctaagggcat ggagttcttg 3360gcatcgcgaa
agtgtatcca cagggacctg gcggcacgaa atatcctctt atcggagaag
3420aacgtggtta aaatctgtga ctttggcttg gcccgggata tttataaaga
tccagattat 3480gtcagaaaag gagatgctcg cctccctttg aaatggatgg
ccccagaaac aatttttgac 3540agagtgtaca caatccagag tgacgtctgg
tcttttggtg ttttgctgtg ggaaatattt 3600tccttaggtg cttctccata
tcctggggta aagattgatg aagaattttg taggcgattg 3660aaagaaggaa
ctagaatgag ggcccctgat tatactacac cagaaatgta ccagaccatg
3720ctggactgct ggcacgggga gcccagtcag agacccacgt tttcagagtt
ggtggaacat 3780ttgggaaatc tcttgcaagc taatgctcag caggatggca
aagactacat tgttcttccg 3840atatcagaga ctttgagcat ggaagaggat
tctggactct ctctgcctac ctcacctgtt 3900tcctgtatgg aggaggagga
agtatgtgac cccaaattcc attatgacaa cacagcagga 3960atcagtcagt
atctgcagaa cagtaagcga aagagccggc ctgtgagtgt aaaaacattt
4020gaagatatcc cgttagaaga accagaagta aaagtaatcc cagatgacaa
ccagacggac 4080agtggtatgg ttcttgcctc agaagagctg aaaactttgg
aagacagaac caaattatct 4140ccatcttttg gtggaatggt gcccagcaaa
agcagggagt ctgtggcatc tgaaggctca 4200aaccagacaa gcggctacca
gtccggatat cactccgatg acacagacac caccgtgtac 4260tccagtgagg
aagcagaact tttaaagctg atagagattg gagtgcaaac cggtagcaca
4320gcccagattc tccagcctga ctcggggacc acactgagct ctcctcctgt
ttaaaaggaa 4380gcatccacac cccaactccc ggacatcaca tgagaggtct
gctcagattt tgaagtgttg 4440ttctttccac cagcaggaag tagccgcatt
tgattttcat ttcgacaaca gaaaaaggac 4500ctcggactgc agggagccag
tcttctaggc atatcctgga agaggcttgt gacccaagaa 4560tgtgtctgtg
tcttctccca gtgttgacct gatcctcttt tttcattcat ttaaaaagca
4620ttatcatgcc cctgctgcgg gtctcaccat gggtttagaa caaagagctt
caagcaatgg 4680ccccatcctc aaagaagtag cagtacctgg ggagctgaca
cttctgtaaa actagaagat 4740aaaccaggca acgtaagtgt tcgaggtgtt
gaagatggga aggatttgca gggctgagtc 4800tatccaagag gctttgttta
ggacgtgggt cccaagccaa gccttaagtg tggaattcgg 4860attgatagaa
aggaagacta acgttacctt gctttggaga gtactggagc ctgcaaatgc
4920attgtgtttg ctctggtgga ggtgggcatg gggtctgttc tgaaatgtaa
agggttcaga 4980cggggtttct ggttttagaa ggttgcgtgt tcttcgagtt
gggctaaagt agagttcgtt 5040gtgctgtttc tgactcctaa tgagagttcc
ttccagaccg ttagctgtct ccttgccaag 5100ccccaggaag aaaatgatgc
agctctggct ccttgtctcc caggctgatc ctttattcag 5160aataccacaa
agaaaggaca ttcagctcaa ggctccctgc cgtgttgaag agttctgact
5220gcacaaacca gcttctggtt tcttctggaa tgaataccct catatctgtc
ctgatgtgat 5280atgtctgaga ctgaatgcgg gaggttcaat gtgaagctgt
gtgtggtgtc aaagtttcag 5340gaaggatttt acccttttgt tcttccccct
gtccccaacc cactctcacc ccgcaaccca 5400tcagtatttt agttatttgg
cctctactcc agtaaacctg attgggtttg ttcactctct 5460gaatgattat
tagccagact tcaaaattat tttatagccc aaattataac atctattgta
5520ttatttagac ttttaacata tagagctatt tctactgatt tttgcccttg
ttctgtcctt 5580tttttcaaaa aagaaaatgt gttttttgtt tggtaccata
gtgtgaaatg ctgggaacaa 5640tgactataag acatgctatg gcacatatat
ttatagtctg tttatgtaga aacaaatgta 5700atatattaaa gccttatata
taatgaactt tgtactattc acattttgta tcagtattat 5760gtagcataac
aaaggtcata atgctttcag caattgatgt cattttatta aagaacattg
5820aaaaacttga 5830171356PRTHomo sapiens 17Met Gln Ser Lys Val Leu
Leu Ala Val Ala Leu Trp Leu Cys Val Glu 1 5 10 15 Thr Arg Ala Ala
Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro 20 25 30 Arg Leu
Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr 35 40 45
Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro 50
55 60 Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys
Ser 65 70 75 80 Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val
Ile Gly Asn 85 90 95 Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu
Thr Asp Leu Ala Ser 100 105 110 Val Ile Tyr Val Tyr Val Gln Asp Tyr
Arg Ser Pro Phe Ile Ala Ser 115 120 125 Val Ser Asp Gln His Gly Val
Val Tyr Ile Thr Glu Asn Lys Asn Lys 130 135 140 Thr Val Val Ile Pro
Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser 145 150 155 160 Leu Cys
Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg 165 170 175
Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile 180
185 190 Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu
Ser 195 200 205 Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr
Arg Ile Tyr 210 215 220 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu
Leu Ser Val Gly Glu 225 230 235 240 Lys Leu Val Leu Asn Cys Thr Ala
Arg Thr Glu Leu Asn Val Gly Ile 245 250 255 Asp Phe Asn Trp Glu Tyr
Pro Ser Ser Lys His Gln His Lys Lys Leu 260 265 270 Val Asn Arg Asp
Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 275 280 285 Leu Ser
Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 290 295 300
Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 305
310 315 320 Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser
Gly Met 325 330 335 Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val
Arg Ile Pro Ala 340 345 350 Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile
Lys Trp Tyr Lys Asn Gly 355 360 365 Ile Pro Leu Glu Ser Asn His Thr
Ile Lys Ala Gly His Val Leu Thr 370 375 380 Ile Met Glu Val Ser Glu
Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu 385 390 395 400 Thr Asn Pro
Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val 405 410 415 Val
Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val 420 425
430 Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr
435 440 445 Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu
Glu Glu 450 455 460 Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val
Thr Asn Pro Tyr 465 470 475 480 Pro Cys Glu Glu Trp Arg Ser Val Glu
Asp Phe Gln Gly Gly Asn Lys 485 490 495 Ile Glu Val Asn Lys Asn Gln
Phe Ala Leu Ile Glu Gly Lys Asn Lys 500 505 510 Thr Val Ser Thr Leu
Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr 515 520 525 Lys Cys Glu
Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser 530 535 540 Phe
His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln 545 550
555 560 Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg
Ser 565 570 575 Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln
Pro Leu Pro 580 585 590 Ile His Val Gly Glu Leu Pro Thr Pro Val Cys
Lys Asn Leu Asp Thr 595 600 605 Leu Trp Lys Leu Asn Ala Thr Met Phe
Ser Asn Ser Thr Asn Asp Ile 610 615 620 Leu Ile Met Glu Leu Lys Asn
Ala Ser Leu Gln Asp Gln Gly Asp Tyr 625 630 635 640 Val Cys Leu Ala
Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val 645 650 655 Arg Gln
Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn 660 665 670
Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys 675
680 685 Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp
Asn 690 695 700 Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp
Gly Asn Arg 705 710 715 720 Asn Leu Thr Ile Arg Arg Val Arg Lys Glu
Asp Glu Gly Leu Tyr Thr 725 730 735 Cys Gln Ala Cys Ser Val Leu Gly
Cys Ala Lys Val Glu Ala Phe Phe 740 745 750 Ile Ile Glu Gly Ala Gln
Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu 755 760 765 Val Gly Thr Ala
Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile 770 775 780 Ile Leu
Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly 785 790 795
800 Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His
805 810 815 Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro
Arg Asp 820 825 830 Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala
Phe Gly Gln Val 835 840 845 Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys
Thr Ala Thr Cys Arg Thr 850 855 860 Val Ala Val Lys Met Leu Lys Glu
Gly Ala Thr His Ser Glu His Arg 865 870 875 880 Ala Leu Met Ser Glu
Leu Lys Ile Leu Ile His Ile Gly His His Leu 885 890 895 Asn Val Val
Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu 900 905 910 Met
Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu 915 920
925 Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg
930 935 940 Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp
Leu Lys 945 950 955 960 Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser
Ser Ala Ser Ser Gly 965 970 975 Phe Val Glu Glu Lys Ser Leu Ser Asp
Val Glu Glu Glu Glu Ala Pro 980 985 990 Glu Asp Leu
Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr 995 1000 1005
Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys 1010
1015 1020 Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser
Glu 1025 1030 1035 Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala
Arg Asp Ile 1040 1045 1050 Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly
Asp Ala Arg Leu Pro 1055 1060 1065 Leu Lys Trp Met Ala Pro Glu Thr
Ile Phe Asp Arg Val Tyr Thr 1070 1075 1080 Ile Gln Ser Asp Val Trp
Ser Phe Gly Val Leu Leu Trp Glu Ile 1085 1090 1095 Phe Ser Leu Gly
Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu 1100 1105 1110 Glu Phe
Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro 1115 1120 1125
Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp 1130
1135 1140 His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser Glu Leu Val
Glu 1145 1150 1155 His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln
Asp Gly Lys 1160 1165 1170 Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr
Leu Ser Met Glu Glu 1175 1180 1185 Asp Ser Gly Leu Ser Leu Pro Thr
Ser Pro Val Ser Cys Met Glu 1190 1195 1200 Glu Glu Glu Val Cys Asp
Pro Lys Phe His Tyr Asp Asn Thr Ala 1205 1210 1215 Gly Ile Ser Gln
Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro 1220 1225 1230 Val Ser
Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu 1235 1240 1245
Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val 1250
1255 1260 Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys
Leu 1265 1270 1275 Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys Ser
Arg Glu Ser 1280 1285 1290 Val Ala Ser Glu Gly Ser Asn Gln Thr Ser
Gly Tyr Gln Ser Gly 1295 1300 1305 Tyr His Ser Asp Asp Thr Asp Thr
Thr Val Tyr Ser Ser Glu Glu 1310 1315 1320 Ala Glu Leu Leu Lys Leu
Ile Glu Ile Gly Val Gln Thr Gly Ser 1325 1330 1335 Thr Ala Gln Ile
Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser 1340 1345 1350 Pro Pro
Val 1355 18675DNAArtificial SequenceRecombinant fusion protein or
sequence encoding same 18atggtcagct actgggacac cggggtcctg
ctgtgcgcgc tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg
tacagtgaaa tccccgaaat tatacacatg 120actgaaggaa gggagctcgt
cattccctgc cgggttacgt cacctaacat cactgttact 180ttaaaaaagt
ttccacttga cactttgatc cctgatggaa aacgcataat ctgggacagt
240agaaagggct tcatcatatc aaatgcaacg tacaaagaaa tagggcttct
gacctgtgaa 300gcaacagtca atgggcattt gtataagaca aactatctca
cacatcgaca aacccagccc 360cgagaaccac aggtgtacac cctgccccca
tcccgggatg agctgaccaa gaaccaggtc 420agcctgacct gcctggtcaa
aggcttctat cccagcgaca tcgccgtgga gtgggagagc 480aatgggcagc
cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc
540ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg
gaacgtcttc 600tcatgctccg tgatgcatga ggctctgcac aaccactaca
cgcagaagag cctctccctg 660tctccgggta aatag 67519224PRTArtificial
SequenceRecombinant fusion protein or sequence encoding same 19Met
Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10
15 Cys Leu Leu Leu Thr Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser
20 25 30 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu
Val Ile 35 40 45 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr
Leu Lys Lys Phe 50 55 60 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg Lys Gly Phe Ile Ile Ser Asn
Ala Thr Tyr Lys Glu Ile Gly Leu 85 90 95 Leu Thr Cys Glu Ala Thr
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 100 105 110 Leu Thr His Arg
Gln Thr Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145
150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 20717DNAArtificial
SequenceRecombinant fusion protein or sequence encoding same
20atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc
60acaggatctg gtagaccttt cgtagagatg tacagtgaaa tccccgaaat tatacacatg
120actgaaggaa gggagctcgt cattccctgc cgggttacgt cacctaacat
cactgttact 180ttaaaaaagt ttccacttga cactttgatc cctgatggaa
aacgcataat ctgggacagt 240agaaagggct tcatcatatc aaatgcaacg
tacaaagaaa tagggcttct gacctgtgaa 300gcaacagtca atgggcattt
gtataagaca aactatctca cacatcgaca aaccccttcc 360tgtgtgcccc
tgatgcgatg cgggggctgc tgcaatcagc cccgagaacc acaggtgtac
420accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac
ctgcctggtc 480aaaggcttct atcccagcga catcgccgtg gagtgggaga
gcaatgggca gccggagaac 540aactacaaga ccacgcctcc cgtgctggac
tccgacggct ccttcttcct ctacagcaag 600ctcaccgtgg acaagagcag
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 660gaggctctgc
acaaccacta cacgcagaag agcctctccc tgtctccggg taaatag
71721238PRTArtificial SequenceRecombinant fusion protein or
sequence encoding same 21Met Val Ser Tyr Trp Asp Thr Gly Val Leu
Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Gly
Arg Pro Phe Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu Ile Ile
His Met Thr Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys Arg Val
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60 Pro Leu
Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65 70 75 80
Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 85
90 95 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr 100 105 110 Leu Thr His Arg Gln Thr Pro Ser Cys Val Pro Leu Met
Arg Cys Gly 115 120 125 Gly Cys Cys Asn Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 130 135 140 Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val 145 150 155 160 Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 165 170 175 Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 180 185 190 Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 195 200 205
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 210
215 220 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225
230 235 22702DNAArtificial SequenceRecombinant fusion protein or
sequence encoding same 22atggtcagct actgggacac cggggtcctg
ctgtgcgcgc tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg
tacagtgaaa tccccgaaat tatacacatg 120actgaaggaa gggagctcgt
cattccctgc cgggttacgt cacctaacat cactgttact 180ttaaaaaagt
ttccacttga cactttgatc cctgatggaa aacgcataat ctgggacagt
240agaaagggct tcatcatatc aaatgcaacg tacaaagaaa tagggcttct
gacctgtgaa 300gcaacagtca atgggcattt gtataagaca aactatctca
cacatcgaca aaccggtgga 360ggtggaggtg gaggtggagg tcagccccga
gaaccacagg tgtacaccct gcccccatcc 420cgggatgagc tgaccaagaa
ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 480agcgacatcg
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
540cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac
cgtggacaag 600agcaggtggc agcaggggaa cgtcttctca tgctccgtga
tgcatgaggc tctgcacaac 660cactacacgc agaagagcct ctccctgtct
ccgggtaaat ag 70223233PRTArtificial SequenceRecombinant fusion
protein or sequence encoding same 23Met Val Ser Tyr Trp Asp Thr Gly
Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly
Ser Gly Arg Pro Phe Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys
Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60
Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65
70 75 80 Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile
Gly Leu 85 90 95 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr
Lys Thr Asn Tyr 100 105 110 Leu Thr His Arg Gln Thr Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gln 115 120 125 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 165 170 175 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 180 185
190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230
241095DNAArtificial SequenceRecombinant fusion protein or sequence
encoding same 24atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatctg gtagaccttt cgtagagatg tacagtgaaa
tccccgaaat tatacacatg 120actgaaggaa gggagctcgt cattccctgc
cgggttacgt cacctaacat cactgttact 180ttaaaaaagt ttccacttga
cactttgatc cctgatggaa aacgcataat ctgggacagt 240agaaagggct
tcatcatatc aaatgcaacg tacaaagaaa tagggcttct gacctgtgaa
300gcaacagtca atgggcattt gtataagaca aactatctca cacatcgaca
aaccggtgga 360ggtggatcgg gtggaggtgg atcgggtgga ggtggatcgc
ctaagagctg cgacaaaact 420cacacatgcc caccgtgccc agcacctgaa
ctcctggggg gaccgtcagt cttcctcttc 480cccccaaaac ccaaggacac
cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 540gtggacgtga
gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
600gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc 660agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 720tccaacaaag ccctcccagc ccccatcgag
aaaaccatct ccaaagccaa agggcagccc 780cgagaaccac aggtgtacac
cctgccccca tcccgggatg agctgaccaa gaaccaggtc 840agcctgacct
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc
900aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggctcc 960ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 1020tcatgctccg tgatgcatga ggctctgcac
aaccactaca cgcagaagag cctctccctg 1080tctccgggta aatag
109525364PRTArtificial SequenceRecombinant fusion protein or
sequence encoding same 25Met Val Ser Tyr Trp Asp Thr Gly Val Leu
Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Gly
Arg Pro Phe Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu Ile Ile
His Met Thr Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys Arg Val
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60 Pro Leu
Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65 70 75 80
Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 85
90 95 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr 100 105 110 Leu Thr His Arg Gln Thr Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro 130 135 140 Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe 145 150 155 160 Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 165 170 175 Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 180 185 190 Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 195 200 205
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 210
215 220 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val 225 230 235 240 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala 245 250 255 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 260 265 270 Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly 275 280 285 Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 290 295 300 Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 305 310 315 320 Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 325 330
335 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
340 345 350 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360
269PRTArtificial Sequencelinker of fusion protein 26Gly Asp Leu Ile
Tyr Arg Asn Gln Lys 1 5 279PRTArtificial Sequencelinker moiety for
fusion proteins 27Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5
289PRTArtificial Sequencelinker moiety for fusion proteins 28Glu
Glu Glu Glu Glu Glu Glu Glu Glu 1 5 299PRTArtificial Sequencelinker
moiety for fusion proteins 29Ser Ser Ser Ser Ser Ser Ser Ser Ser 1
5 309PRTArtificial Sequencelinker moiety for fusion proteins 30Gly
Gly Gly Gly Gly Cys Pro Pro Cys 1 5 3115PRTArtificial
Sequencelinker moiety for fusion proteins 31Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 3213PRTArtificial
Sequencelinker moiety for fusion proteins 32Ser Cys Val Pro Leu Met
Arg Cys Gly Gly Cys Cys Asn 1 5 10 339PRTArtificial Sequencelinker
moiety for fusion proteins 33Gly Asp Leu Ile Tyr Arg Asn Gln Lys 1
5 3423PRTArtificial Sequencelinker moiety for fusion proteins 34Gly
Gly Gly Gly Gly Gly Gly Gly Gly Pro Ser Cys Val Pro Leu Met 1 5 10
15 Arg Cys Gly Gly Cys Cys Asn 20
* * * * *