U.S. patent application number 16/795450 was filed with the patent office on 2020-08-20 for dual pdgf/vegf antagonists.
This patent application is currently assigned to KODIAK SCIENCES INC.. The applicant listed for this patent is KODIAK SCIENCES INC.. Invention is credited to James Aggen, Didier Benoit, Stephen A. Charles, Justin Cohen, Tetsuya Ishino, Laura Lin, Lidia Mosyak, D. Victor Perlroth, William Somers, Wayne To.
Application Number | 20200262905 16/795450 |
Document ID | 20200262905 / US20200262905 |
Family ID | 1000004812677 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200262905 |
Kind Code |
A1 |
Perlroth; D. Victor ; et
al. |
August 20, 2020 |
DUAL PDGF/VEGF ANTAGONISTS
Abstract
The invention provides a dual VEGF/PDGF antagonist comprising a
VEGF antagonist linked to a PDGF antagonist. The VEGF antagonist is
an antibody to a VEGF or VEGFR or is a VEGFR extracellular trap
segment (i.e., a segment from the extracellular region of one or
more VEGFR receptors that inhibits binding of at least one VEGFR to
at least one VEGF). The PDGF antagonist is an antibody to a PDGF or
PDGFR or is a PDGFR extracellular trap segment (i.e., segment from
the extracellular region of one or more PDGFRs, which inhibits
binding of at least one PDGFR and at least one PDGF). The dual
antagonist is preferably conjugated to a half-life extending
moiety, such as a HEMA-PC polymer. The dual antagonist is
particularly useful for treating wet aged related macular
degeneration.
Inventors: |
Perlroth; D. Victor; (Palo
Alto, CA) ; Charles; Stephen A.; (Ravenna, OH)
; Aggen; James; (Westwood, MA) ; Benoit;
Didier; (San Jose, CA) ; To; Wayne; (San
Mateo, CA) ; Mosyak; Lidia; (Newton, MA) ;
Lin; Laura; (Weston, MA) ; Cohen; Justin;
(Quincy, MA) ; Ishino; Tetsuya; (Boston, MA)
; Somers; William; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KODIAK SCIENCES INC. |
PALO ALTO |
CA |
US |
|
|
Assignee: |
KODIAK SCIENCES INC.
PALO ALTO
CA
|
Family ID: |
1000004812677 |
Appl. No.: |
16/795450 |
Filed: |
February 19, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15820325 |
Nov 21, 2017 |
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16795450 |
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14753824 |
Jun 29, 2015 |
9840553 |
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15820325 |
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PCT/US2015/038203 |
Jun 28, 2015 |
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14753824 |
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62018579 |
Jun 28, 2014 |
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62018579 |
Jun 28, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/31 20130101;
C07K 2317/92 20130101; C07K 2319/036 20130101; A61K 38/1866
20130101; C07K 2317/76 20130101; C07K 2317/71 20130101; C07K 16/22
20130101; C07K 2317/526 20130101; C07K 2317/524 20130101; C07K
2317/565 20130101; C07K 16/28 20130101; C07K 16/2863 20130101; A61K
38/1858 20130101; C07K 14/71 20130101; A61K 38/179 20130101; C07K
2317/72 20130101; C07K 2318/10 20130101; C07K 2319/30 20130101;
C07K 2317/55 20130101; C07K 2317/522 20130101; A61K 2039/505
20130101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; A61K 38/17 20060101 A61K038/17; A61K 38/18 20060101
A61K038/18; C07K 14/71 20060101 C07K014/71; C07K 16/28 20060101
C07K016/28 |
Claims
1. A fusion protein comprising a vascular endothelial growth factor
(hereinafter "VEGF") antagonist linked to a platelet-derived growth
factor (hereinafter "PDGF") antagonist, wherein the VEGF antagonist
is an anti-VEGF antibody, and the PDGF antagonist is a PDGF
receptor (hereinafter "PDGFR") extracellular trap segment, wherein
the PDGFR extracellular trap segment comprises domains D1-D3 of
PDGFR-.beta..
2.-61. (canceled)
Description
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
[0001] Any and all applications for which a foreign or domestic
priority claim is identified in the Application Data Sheet as filed
with the present application are hereby incorporated by reference
under 37 CFR 1.57.
[0002] This application is a continuation of patent application
Ser. No. 15/820,325, filed Nov. 21, 2017, which is a divisional of
patent application Ser. No. 14/753,824, filed Jun. 29, 2015, the
entirety of which is incorporated herein by reference. Patent
application Ser. No. 14/753,824 is a continuation of Patent
Application Serial No PCT/US2015/038203, filed Jun. 28, 2015, all
of which claim full priority benefit of U.S. Provisional
Application Ser. No. 62/018,579 filed Jun. 28, 2014, which is
incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0003] A Sequence Listing submitted as an ASCII text file via
EFS-Web is hereby incorporated by reference in accordance with 35
U.S.C. .sctn. 1.52(e). The name of the ASCII text file for the
Sequence Listing is 32236617_1.TXT, the date of creation of the
ASCII text file is Feb. 19, 2020, and the size of the ASCII text
file is 219 KB.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] Angiogenesis (the formation of blood vessels) occurs
throughout an organism's development. Indeed, the first organ in an
embryo is a blood vessel. Angiogenesis is also crucial for wound
healing, restoring blood flow to damaged tissue. However, improper
or dysregulated angiogenesis contributes to or causes many diseases
including cancer, psoriasis, arthritis and blindness. Carmeliet P.
2003. Angiogenesis in health and disease. Nature Med
9(6):653-660.
Description of the Related Art
[0005] Age related macular degeneration (AMD) is a leading cause of
vision loss and blindness in the elderly. About ten million
Americans are afflicted with AMD. The prevalence of AMD in the
population increases steadily with age: at 40 years of age only
about 2% of the population is affected by AMD but by the age of 80
it is about 25%. Friedman, D. S. et al. 2004. Arch. Ophthalmol.
122:564-572. There are generally two types of AMD: dry and wet.
[0006] Dry AMD is the most common form of the disease. In dry AMD,
there is a depletion of the layer of the retinal pigment epithelial
cells in the macula. Dry AMD is chronic and generally causes some
loss of vision. In severe cases of dry AMD, patients can develop
near total blindness. Wet AMD develops in some 10-15% of patients
with dry AMD. Wet AMD is characterized by angiogenesis,
specifically choroidal neovascularization (CNV). CNV is
characterized by the presence of new immature blood vessels which
grow towards the outer retina from the choroid. These immature
blood vessels leak fluid below and in the retina, causing vision
loss and blindness. Wet AMD blindness is typically acute.
[0007] Angiogenesis also plays a crucial role in cancer and tumor
formation and maintenance. The recruitment of new blood vessels is
an essential component of the metastatic pathway. For many tumors,
the vascular density can provide a prognostic indicator of
metastatic potential: highly vascular tumors have a higher
incidence of metastasis than less vascular tumors.
[0008] Angiogenesis is the result of a complex interplay between
growth factors, vascular endothelial cells, extracellular matrix
molecules, chemokines and cell signaling molecules. Factors
identified as mediators of angiogenesis include: basic and acidic
fibroblast growth factor, transforming growth factors .alpha. and
.beta. platelet-derived growth factor (PDGF), angiogenin,
platelet-derived endothelial cell growth factor, IL8, and vascular
endothelial growth factor (VEGF). The role of VEGF in angiogenesis
has been extensively reported on.
[0009] It has been shown that VEGF signaling presents a crucial
rate limiting step in physiological angiogenesis. VEGF also plays a
central role in pathological angiogenesis (e.g., tumor growth).
Ferrara N and Davis-Smyth T. 1997. The biology of vascular
endothelial growth factor. Endocr. Rev. 18: 4-25. VEGF is also
known to induce vascular leakage. Bates D O and Curry F E. 1997.
Vascular endothelial growth factor increases microvascular
permeability via a Ca (2+)-dependent pathway. Am J Physiol. 273:
H687-H694; Roberts W G and Palade G E. 1995. Increased
microvascular permeability and endothelial fenestration induced by
vascular endothelial growth factor. J Cell Sci. 108:2369-2379.
[0010] Anti-VEGF therapeutics have been successfully used to treat
wet AMD and cancer. Genentech's anti-VEGF monoclonal antibody
bevacizumab (Avastin.RTM.) received FDA approval in 2004 for the
treatment of cancer. Anti-VEGF agents have been approved for the
treatment of wet AMD. In 2004, the FDA approved Eyetech/Pfizer
Macugen.RTM.. Genentech's Lucentis.RTM. was approved in 2006 for
wet AMD. Bevacizumab is also used off label for the treatment of
wet AMD. In 2011, Regeneron's Eylea.RTM. was approved for treatment
of wet AMD.
[0011] Despite the success of anti-VEGF therapeutics, none of them
causes regression in the pathological neovascular (NV) tissue.
Hence, NV tissue remains despite continued anti-VEGF treatment and
can prevent significant vision gain for treated patients. The NV
tissue consists of endothelial cells, pericytes and inflammatory
cells (i.e., occasional macrophages). The presence of pericytes on
capillaries not only leads to NV support and stabilization but
promotes endothelial cell survival through chemical signaling and
physical interactions including pericyte production of VEGF. This
endothelial survival signaling by integrated pericytes is critical
and may explain the resistance of the NV tissue to VEGF withdrawal,
i.e., lack of NV regression to monotherapy anti-VEGF treatment. In
addition, over time the pathological NV tissue can lead to fibrosis
and scarring.
[0012] Subretinal scarring develops in nearly half of treated eyes
within two years of anti-VEGF therapy. Daniel E, Toth C A, Grunwald
J E. 2014. Risk of scar in the comparison of age-related macular
degeneration in clinical settings. Retina 32: 1480-1485. Subretinal
fibrosis formation can cause permanent dysfunction of the macular
system; it causes destruction of photoreceptors, retinal pigment
epithelium and choroidal vessels. Ishikawa K, Ram K, Hinton D R.
2015. Molecular mechanisms of subretinal fibrosis in age-related
macular degeneration. Eye Res. xxx: 1-7. While anti-VEGF therapy
generally stabilizes or improves visual acuity, scar formation has
been identified as one of the causes of loss of visual acuity after
treatment. Cohen S Y, Oubraham H, Uzzan J, et al. 2012. Causes of
unsuccessful ranibizumab treatment in exudative age-related macular
degeneration in clinical settings. Retina 32: 1480-1485.
[0013] PDGF has been reported to play a role in pericyte
recruitment, maturation and resistance to anti-VEGF mediated
regression. Corneal and choroidal neovascularization animal models
have been reported to have demonstrated that administration of
agents that block the PDGF-B/PDGFR-.beta. interaction leads to
pericyte stripping from the pathological neovasculature. Jo N,
Mailhos C, Ju M, et al. 2006. Inhibition of Platelet-Derived Growth
Factor B Signaling Enhances the Efficacy of Anti-Vascular
Endothelial Growth Factor Therapy in Multiple Models of Ocular
Neovascularization. American J Path. 168(6):2036-2053.
[0014] To target both pathways, clinical trials are currently
underway in which patients receive two medications: Lucentis.RTM.
(an anti-VEGF Fab) and Fovista.TM. a PEGYlated aptamer directed
against PDGF by Ophthotech. Fovista is directed against only a
single PDGF ligand: PDGF-BB. However, there are many other PDGF
ligands: PDGF-AA, PDGF-CC and PDGF-DD. PDGF-DD, for example, has
been shown to play a crucial role in ocular angiogenesis. Kumar A,
Hou X, Chunsik L, et al. 2010. Platelet-derived Growth Factor-DD
Targeting Arrests Pathological Angiogenesis by Modulating Glycogen
Synthase Kinase-313 Phosphorylation. J Biol Chem
285(20):15500-15510. Yet Fovista does not interact with PDGF-DD.
There is a need in the art for broader based anti-PDGF
therapies.
[0015] In addition, aptamer based therapeutics in general have poor
pharmacokinetic properties in that aptamers are subject to renal
filtration and to serum digestion. While these problems can be
somewhat overcome with PEGylation, PEGylation tends to reduce
binding to target. Aptamers typically bind with much lower affinity
to targets than their antibody counterparts. PEGylation will tend
to reduce binding even further. There is, thus, a need in the art
for non-aptamer based anti-PDGF therapeutics.
[0016] Current clinical plans for Fovista double the number of
injections patients must receive for treatment relative to the
currently approved anti-VEGF therapies. Fovista is formulated
separately from the anti-VEGF agent so patients must be given two
injections instead of one. Moreover the injections cannot be at the
same time because of build-up in intraocular pressure caused by a
single injection.
[0017] From the view point of both patients and treating
physicians, intravitreal injections are not trivial. Many patients
experience pain and discomfort from the injection and patient
compliance is a serious issue. Common side effects of intravitreal
injections include conjunctiva! hemorrhage, eye pain, vitreous
floaters, increased intraocular pressure, and intraocular
inflammation. Intravitreal injections are associated with
relatively rare serious adverse events, including endophthalmitis,
retinal detachment and traumatic cataracts.
[0018] There is thus a need in the art for therapies that do not
increase the number of intravitreal injections that patients must
endure. In addition, current anti-VEGF therapies often require once
a month injections. There is also a need for therapies which are
needed less frequently than once a month.
SUMMARY OF THE INVENTION
[0019] The invention provides a dual VEGF/PDGF antagonist
comprising a VEGF antagonist linked to a PDGF antagonist, wherein
the VEGF antagonist (a) is an antibody to a VEGF or VEGFR or (b) is
a VEGFR extracellular trap segment and the PDGF antagonist (a) is
an antibody to a PDGF or PDGFR or (b) is a PDGFR extracellular trap
segment, provided that the VEGF and PDGF antagonists are not both
antibodies. Optionally, the VEGF antagonist is an antibody
comprising a heavy chain and a light chain and the PDGF antagonist
is the PDGFR extracellular trap segment, and the heavy chain of the
antibody is fused via a linker to the C-terminus of the PDGFR
extracellular trap segment, and the light chain is complexed with
the heavy chain. Optionally, the antibody is a Fab fragment.
Optionally, the antibody is an intact antibody. Optionally, the
PDGF antagonist is an extracellular trap segment of a PDGFR-.alpha.
or PDGFR-.beta. receptor and the VEGF antagonist is an antibody to
a VEGF. Optionally, the PDGFR extracellular trap segment comprises
one or more of domains D1-D5 of PDGFR-.beta.. Optionally, the PDGFR
extracellular trap segment comprises domains D1-D3 of PDGFR-.beta..
Optionally, the PDGFR extracellular trap segment comprises amino
acids 33 to 314 of SEQ ID NO. 11. Optionally, the VEGF antagonist
comprises an anti-VEGF antibody. Optionally, the anti-VEGF antibody
is an anti-VEGF-A antibody. Optionally, the PDGFR extracellular
trap segment is located C-terminal of the heavy or light chain.
Optionally, the PDGFR extracellular trap segment is located
N-terminal of the heavy or light chain.
[0020] Optionally, the dual VEGF/PDGF antagonist of further
comprising a linker which is located between the PDGFR trap and the
anti-VEGF antibody heavy chain. Optionally the linker is
GGGGSGGGGS, GG, or GGGGSGGGGSGGGGSGGGGSG.
[0021] Optionally, the anti-VEGF antibody heavy chain comprises
CDR.sub.H1: GYDFTHYGMN, CDR.sub.H2: WINTYTGEPTYAADFKR, and
CDR.sub.H3: YPYYYGTSHWYFDV. Optionally, the anti-VEGF light chain
comprises CDR.sub.L1: SASQDISNYLN, CDR.sub.L2: FTSSLHS and
CDR.sub.L3: QQYSTVPWT.
[0022] Optionally, the anti-VEGF heavy chain isotype is IgG
comprising a CH.sub.1, hinge, CH.sub.2 and CH.sub.3 domains and the
light chain isotype is kappa. Optionally the IgG 1 constant domain
has the sequence set forth in SEQ ID NO. 17 and the light chain
constant region has the sequence set forth in SEQ ID NO. 18.
[0023] Optionally, the IgG 1 constant domain has one or more
mutations to reduce effector function. Optionally the mutations are
to one or more of the following amino acid positions (EU
numbering): E233, L234, L235, G236, G237, A327, A330, and P331.
Optionally, the mutations are selected from the group consisting
of: E233P, L234V, L234A, L235A, G237A, A327G, A330S and P331S.
Optionally, mutations are L234A, L235A and G237A.
[0024] Optionally, the dual VEGF/PDGF antagonist comprises a heavy
chain further comprising a cysteine residue added by recombinant
DNA technology. Optionally, the cysteine residue is selected from
the group consisting of (EU numbering) Q347C and L443C.
[0025] Optionally, the dual VEGF/PDGF antagonist has a heavy chain
comprising the amino acid sequence off SEQ ID NO. 9 and the light
chain has an amino acid sequence of SEQ ID NO. 10.
[0026] Optionally, the dual VEGF/PDGF antagonist comprises a PDGFR
trap extracellular segment comprising one or more of domains D1-D5
of PDGFR-.beta.. Optionally, the PDGFR trap extracellular segment
comprises domains D1-D3 of PDGFR-.beta.. Optionally, the PDGFR trap
extracellular segment comprises amino acids 33 to 314 of SEQ ID NO.
11.
[0027] Optionally, the dual VEGF/PDGF antagonist comprises a VEGF
antagonist, which is an anti-VEGF antibody. Optionally, the
antibody is an anti-VEGF-A Fab fragment. Optionally, the PDGFR
extracellular trap segment is located C-terminal of the Fab heavy
or light chain. Optionally, the PDGFR extracellular trap segment is
located N-terminal of the Fab heavy or light chain.
[0028] Optionally, the dual VEGF/PDGF comprises a heavy chain
comprising an anti-VEGF-A Fab fragment heavy chain and a light
chain comprising an anti-VEGF-A light chain. Optionally, the dual
antagonist further comprises a linker which is located between the
PDGFR trap and the anti-VEGF Fab fragment heavy chain. Optionally,
the linker is selected from group consisting of GGGGSGGGGS, GG, and
GGGGSGGGGSGGGGSGGGGSG. Optionally, the anti-VEGF Fab fragment heavy
chain comprises CDR.sub.H1: GYDFTHYGMN, CDR.sub.H2:
WINTYTGEPTYAADFKR, and CDR.sub.H3: YPYYYGTSHWYFDV. Optionally, the
anti-VEGF light chain comprises CDRd: SASQDISNYLN, CDRr2: FTSSLHS
and CDRL3: QQYSTVPWT. Optionally, the anti-VEGF heavy chain isotype
is IgG 1 comprising a CH.sub.1 domain and the light chain isotype
is kappa.
[0029] Any of the dual VEGF/PDGF antagonists can further comprise a
half-life extending moiety. Optionally, the half-life extending
moiety comprises a polymer, which is PEG or a zwitterionic polymer.
Optionally, the zwitterionic polymer comprises a monomer comprising
phosphorylcholine. Optionally, the monomer comprises
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate.
Optionally, the monomer comprises
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate
(HEMA-PC). Optionally, the polymer has 3 or more arms. Optionally,
the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms.
Optionally, the polymer has 3, 6 or 9 arms. Optionally, the polymer
has 9 arms. Optionally, the polymer portion of the conjugate has a
peak molecular weight of between 300,000 and 1,750,000 Da.
Optionally, the polymer portion of the conjugate has a peak
molecular weight between 500,000 and 1,000,000 Da. Optionally, the
polymer portion of the conjugate has a peak molecular weight
between 600,000 to 800,000 Da. Optionally, the dual VEGF/PDGF
antagonist is covalently bonded to the polymer. Optionally, the
polymer is covalently bonded to at least one of an amino group, a
hydroxyl group, a sulfhydryl group and a carboxyl group.
Optionally, the sulfhydryl group is from a naturally occurring
cysteine residue. Optionally, the sulfhydryl group is from a
cysteine residue added by recombinant DNA technology. Optionally,
the polymer is covalently bonded to the cysteine residue at
position 731 of SEQ ID NO. 9.
[0030] Optionally, the VEGF antagonist comprises a VEGFR
extracellular trap segment comprising one or more extracellular
segments of VEGFR-1, VEGFR-2 and VEGFR-3 and the PDGF antagonist is
an anti-PDGF antibody. Optionally, the extracellular segment of
VEGFR comprises one or more of domains D1-D7. Optionally, the
extracellular segment comprises D2 from VEGFR-1 and D3 from
VEGFR-2. Optionally, the D2 is N-terminal to the D3 and further
comprises a linker between the domains. Optionally, the PDGF
antagonist is an intact antibody. Optionally, the PDGF antagonist
is a Fab fragment. Optionally, the anti-PDGFR antibody is humanized
2A 1E2, HuM4 Ts.22, humanized 1B3, humanized 2C5, anti-PDGF-BB,
anti-PDGF-DD, anti-PDGF-BB or anti-PDGF-AB. Optionally, the heavy
chain is IgG 1 and the light chain is kappa. Optionally, the heavy
chain sequence has a cysteine added via recombinant DNA technology
the cysteine selected from the groups consisting of Q347C or a
L443C. Optionally, the dual VEGF/PDGF antagonist further comprises
a half-life extending moiety conjugated to the cysteine.
Optionally, the dual VEGF/PDGF antagonist protein has a half-life
extending moiety comprising a zwitterionic polymer, the polymer
comprising one or more monomer units and wherein at least one
monomer unit comprises a zwitterionic group, such as
phosphorylcholine. Optionally, the monomer comprises
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate.
Optionally, the monomer comprises 2-(methacryloyloxyethyl)-2'
(trimethylammoniumethyl) phosphate (HEMA-PC). Optionally, the
polymer has 3 or more arms. Optionally, the polymer has 2, 3, 4, 5,
6, 7, 8, 9, 10, 11 or 12 arms. Optionally, the polymer has 3, 6 or
9 arms. Optionally, the polymer has 9 arms. Optionally, the polymer
portion of the conjugate has a peak molecular weight of between
300,000 and 1,750,000 Da. Optionally, the polymer portion of the
conjugate has a peak molecular weight between 500,000 and 1,000,000
Da. Optionally, the polymer portion of the conjugate has a peak
molecular weight between 600,000 to 800,000 Da.
[0031] In some dual VEGF/PDGF antagonists the PDGF antagonist
comprises a PDGF extracellular trap segment comprising one or more
extracellular segments of a PDGFR selected from the group
consisting of PDGFR-.alpha. and PDGFR-.beta. and the VEGF
antagonist is a VEGF extracellular trap segment comprising one or
more extracellular segments of a VEGFR selected from the group
consisting of VEGFR-1, VEGFR-2 and VEGFR-3. Optionally, the
extracellular trap segment of VEGFR comprises one or more of
domains D1-D7. Optionally, the extracellular trap segment comprises
D2 from VEGFR-1 and D3 from VEGFR-2. Optionally, the D2 is
N-terminal to the D3 and further comprises a linker between the
domains. Optionally, the PDGFR trap comprises one or more of
domains D1-D5 of PDGFR-.beta.. Optionally, the PDGFR trap comprises
domains D1-D3 of PDGFR-. Optionally, the PDGFR trap comprises amino
acids 33 to 314 of SEQ ID NO. 11. Optionally, the dual VEGF/PDGF
antagonist further comprises a linker sequence between the VEGF
antagonist and the PDGF antagonist. Optionally, the dual VEGF/PDGF
antagonist further comprises a half-life extending moiety.
Optionally, the half-life extending moiety comprises a polymer
selected from the group consisting of PEG and a zwitterionic
polymer. Optionally, the half-life extending moiety comprises a
zwitterionic polymer. Optionally, the zwitterionic polymer
comprises a monomer comprising phosphorylcholine. Optionally, the
monomer comprises 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)
phosphate. Optionally, the monomer comprises
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate
(HEMA-PC). Optionally, the polymer has 3 or more arms. Optionally,
the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms.
Optionally, the polymer has 3, 6 or 9 arms. Optionally, the polymer
portion of the conjugate has a peak molecular weight of between
300,000 and 1,750,000 Da. Optionally, the polymer portion of the
conjugate has a peak molecular weight between 500,000 and 1,000,000
Da. Optionally, the polymer portion of the conjugate has a peak
molecular weight between 600,000 to 800,000 Da. Optionally, the
polymer has 9 arms. Optionally, the dual VEGF/PDGF antagonist is
covalently bonded to the polymer. Optionally, the polymer is
covalently bonded to at least one of an amino group, a hydroxyl
group, a sulfhydryl group and a carboxyl group. Optionally, the
sulfhydryl group is from a naturally occurring cysteine residue.
Optionally, the sulfhydryl group is from a cysteine residue added
by recombinant DNA technology.
[0032] Any dual VEGF/PDGF antagonist as described above can be used
in treatment or prophylaxis of disease, particularly a neovascular
disorder, optionally an ocular neovascular disorder, such as wet
age related macular degeneration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1: Protein Sequence of human PDGFR-.beta..
[0034] FIG. 2: Protein Sequence of VEGFR-1.
[0035] FIG. 3: Protein Sequence of VEGFR-2.
[0036] FIG. 4: Protein Sequence of VEGFR-3.
[0037] FIG. 5: bevacizumab sequence (DrugBank DBOO 112)
[0038] FIG. 6: ranibizumab (published by Novartis).
[0039] FIGS. 7A, B: Protein Sequence of A. PDGFR.beta.-GS
10-anti-VEGF-A light chain and B. anti-VEGF-A heavy chain.
[0040] FIGS. 8A, B: A. Protein Sequence of
PDGFR.beta.-GG-anti-VEGF-A light chain and B. anti-VEGF-A heavy
chain.
[0041] FIGS. 9A, B. Protein Sequence of A. PDGFR.beta.-GS
10-anti-VEGF-A heavy chain (wild type Fe) and B. anti-VEGF-A light
chain.
[0042] FIGS. 10A, B. Protein Sequence of A.
PDGFR.beta.-GG-anti-VEGF-A heavy chain (wild type Fe) and B.
anti-VEGF-A light chain.
[0043] FIGS. 11A, B. Protein Sequence of A. anti-VEGF-A heavy chain
(wild type Fc)-GS21-PDGFR.beta. and B. anti-VEGF-A light chain.
[0044] FIGS. 12A, B. Protein Sequence of A.
PDGFR-.beta.-GS21-anti-VEGF-A heavy chain (Q347C) and B.
anti-VEGF-A light chain (TAF347).
[0045] FIGS. 13A, B. Protein Sequence of A.
PDGFR-.beta.-GS21-anti-VEGF-A heavy chain (L443C) and B.
anti-VEGF-A light chain (TAF443).
[0046] FIGS. 14A, B. Protein Sequence of A. PDGFR.beta.-GS
10-anti-VEGF-A light chain and B. anti-VEGF-A Fab.
[0047] FIGS. 15A, B. Protein Sequence of A. PDGFR-GG-anti-VEGF-A
light chain and B. anti-VEGF-A Fab.
[0048] FIGS. 16A, B. Protein Sequence of A. PDGFR.beta.-GS
10-anti-VEGF-A Fab and B. anti-VEGF-A light chain.
[0049] FIGS. 17A, B. Protein Sequence of A.
PDGFR.beta.-GG-anti-VEGF-A Fab and B. anti-VEGF-A light chain.
[0050] FIGS. 18A, B. Protein Sequence of A. anti-VEGF-A
Fab-GS21-PDGFR.beta. and B. anti-VEGF-A light chain.
[0051] FIGS. 19A, B. Protein Sequence of A. PDGFR.beta.-GS
10-anti-VEGF-A Fab with certain mutations and B. anti-VEGF-A light
chain.
[0052] FIGS. 20A, B. Protein Sequence of A. PDGFR.beta.-anti-VEGF-A
heavy chain and B. anti-VEGF-A light chain (1a).
[0053] FIGS. 21A, B. Protein Sequence of A. PDGFR-.beta.
(D2-D3)-anti-VEGF-A heavy chain and B. anti-VEGF-A light chain
(1b).
[0054] FIGS. 22A, B. Protein Sequence of A. PDGFR-.beta.
(D1-D3)-anti-VEGF-A Fab and B. anti-VEGF-A light chain (2b).
[0055] FIGS. 23A, B. Protein Sequence of A.
PDGFR-.beta.(D2-D3)-6xGS-anti-VEGF-A Fab and B. anti-VEGF-A light
chain (2b').
[0056] FIGS. 24A, B. Protein sequence of A.
PDGFR-.beta.-6xGS-anti-VEGF-A Fab and B. anti-VEGF-A light
chain.
[0057] FIGS. 25A, B: Protein Sequence of A. anti-VEGF-A
Fab-6xGS-PDGFR-.beta. (D2-D3) and B. anti-VEGF-A light chain
(3).
[0058] FIG. 26 shows the chemical structure of OG 1448.
[0059] FIG. 27 shows Compound L.
[0060] FIG. 28 shows Compound K.
[0061] FIG. 29 shows the synthesis of OG1802 from R3707.
[0062] FIG. 30 shows OG1786.
[0063] FIG. 31 shows the synthesis of OG1546 from OG1550.
[0064] FIG. 32 shows the synthesis of OG1784 from OG1546 and
OG1563.
[0065] FIG. 33 shows the synthesis of OG1405 from OG1784.
[0066] FIG. 34 shows the synthesis of OG1785 from OG1405.
[0067] FIG. 35 shows the synthesis of OG1786 from OG1785.
[0068] FIG. 36 shows OG1802.
[0069] FIG. 37 shows a graph of percent Grade IV laser lesions.
[0070] FIG. 38 shows Compound E.
[0071] FIG. 39 depicts OG1448.
[0072] FIG. 40 shows relative angiogenesis using OG1448, Avastin,
and an anti-PDGF-BB antibody and various combinations thereof.
[0073] FIG. 41 shows the % grade IV lesions compared to day in the
CNV monkey model for the compounds indicated.
[0074] FIG. 42 shows OG1448 ocular pharmacokinetics versus
aflibercept and ranibizumab in the rabbit vitreous.
BRIEF DESCRIPTION OF SEQ ID NOS.
[0075] SEQ ID NO. 1 is the protein sequence of PDGFRb-GS
10-LightChain anti-VEGF-A (Bevacizumab).
[0076] SEQ ID NO. 2 is the anti-VEGF-A Bevacizumab heavy chain.
[0077] SEQ ID NO. 3 is protein sequence of PDGFRb-GG-Light Chain
anti-VEGF-A
[0078] (Bevacizumab).
[0079] SEQ ID NO. 4 is PDGFR.beta.-GS 10-Heavy Chain-anti-VEGF-A
(Bevacizumab).
[0080] SEQ ID NO. 5 is the anti-VEGF-A Bevacizumab light chain.
[0081] SEQ ID NO. 6 is PDGFR-GG-Heavy Chain-anti-VEGF-A
(Bevacizumab).
[0082] SEQ ID NO. 7 is anti-VEGF-A Heavy Chain
(Bevacizumab)-GS21-PDGFR.beta..
[0083] SEQ ID NO. 8 is the amino acid sequence of the heavy chain
trap extracellular segment of TAF347: PDGFR-trap-anti-VEGF-A heavy
chain (Q347C).
[0084] SEQ ID NO. 9 is the amino acid sequence of the heavy chain
trap extracellular segment of TAF443: PDGFR-.beta. trap-anti-VEGF-A
heavy chain (L443C) and SEQ ID NO:10 is the amino acid sequence of
the light chain of anti-VEGF-A.
[0085] SEQ ID NO. 11 is human PDGFR-.beta..
[0086] SEQ ID NO. 12 is the ranibizumab light chain.
[0087] SEQ ID NO. 13 is the ranibizumab heavy chain.
[0088] SEQ ID NO. 14 is human VEGFR-1.
[0089] SEQ ID NO. 15 is human VEGFR-2.
[0090] SEQ ID NO. 16 is human VEGFR-3.
[0091] SEQ ID NO. 17 is a human IgG1 constant region.
[0092] SEQ ID NO. 18 is a human kappa light constant region.
[0093] SEQ ID NO. 19 is FIG. 7. PDGFR-GS 10-anti-VEGF-A light
chain.
[0094] SEQ ID NO. 20 is FIG. 8. PDGFR-GG-anti-VEGF-A light
chain.
[0095] SEQ ID NO. 21 is a Bevacizumab Fab.
[0096] SEQ ID NO. 22 is a PDGFR-.beta.-GS 10-anti-VEGF-A Fab.
[0097] SEQ ID NO. 23 is a PDGFR-.beta.-GG-anti-VEGF-A Fab.
[0098] SEQ ID NO. 24 is an anti-VEGF-A Fab-GS21-PDGFR-.beta..
[0099] SEQ ID NO. 25 is a PDGFR-.beta.-GS 10-anti-VEGF-A Fab with
certain mutations.
[0100] SEQ ID NO. 26 is a protein sequence of
PDGFR.beta.-anti-VEGF-A heavy chain (1a).
[0101] SEQ ID NO. 27 is a protein sequence of
PDGFR-.beta.-(D2-D3)-anti-VEGF-A heavy chain (1b).
[0102] SEQ ID NO. 28 is a protein sequence of PDGFR-.beta.
(D2-D3)-anti-VEGF-A Fab (2b).
[0103] SEQ ID NO. 29 is a protein sequence of PDGFR-.beta.
(D2-D3)-6xGS-anti-VEGF-A
[0104] SEQ ID NO. 30 is a protein sequence of anti-VEGF-A
Fab-6xGS-PDGFR-.beta. (D2-
[0105] SEQ ID NO. 31 is a nucleic acid encoding a heavy chain
anti-VEGF-PDGFR fusion.
[0106] SEQ ID NO. 32 is a nucleic acid encoding a light chain
anti-VEGF.
[0107] GGGGS (SEQ ID NO. 37), GGGS (SEQ ID NO. 38), GGGES (SEQ ID
NO. 39), GGGGSGGGGS (SEQ ID NO. 40) and GGGGSGGGGSGGGGSGGGGSG) (SEQ
ID NO. 41).
[0108] Ranibizumab CDRs are: CDR.sub.H1: GYDFTHYGMN, CDR.sub.H2:
WINTYTGEPTYAADFKR, and CDR.sub.H3: YPYYYGTSHWYFDV (SEQ ID NOS.
42-44), CDR.sub.L1: SASQDISNYLN, CDR.sub.L2: FTSSLHS and
CDR.sub.L3: QQYSTVPWT (SEQ ID NOS. 45-47). Bevacizumab CDR.sub.H1
is GYTFTNYGMN (SEQ ID NO. 48) and CDR.sub.H3 is YPHYYGSSHWYFDV (SEQ
ID NO:49).
Definitions
[0109] A "neovascular disorder" is a disorder or disease state
characterized by altered, dysregulated or unregulated angiogenesis.
Examples of neovascular disorders include neoplastic transformation
(e.g. cancer) and ocular neovascular disorders including diabetic
retinopathy and age-related macular degeneration.
[0110] An "ocular neovascular" disorder is a disorder characterized
by altered, dysregulated or unregulated angiogenesis in the eye of
a patient. Such disorders include optic disc neovascularization,
iris neovascularization, retinal neovascularization, choroidal
neovascularization, corneal neovascularization, vitreal
neovascularization, glaucoma, pannus, pterygium, macular edema,
diabetic retinopathy, diabetic macular edema, vascular retinopathy,
retinal degeneration, uveitis, inflammatory diseases of the retina,
and proliferative vitreoretinopathy.
[0111] A "polypeptide linker" is a polypeptide comprising two or
more amino acid residues joined by peptide bonds that are used to
link two polypeptides (e.g., a VH and VL domain or a VH domain and
an extracellular trap segment). Examples of such linker
polypeptides are well known in the art (see, e.g., Bolliger P,
Prospero T, Winter G. 1993. PNAS USA. 90:6444-6448; Poljak R J.
1994. Production and Structure of Diabodies. Structure 2:
1121-1123). Exemplary linkers include G, GG, GGGGS, GGGS, and
GGGES, and oligomers of such linkers (e.g., GGGGSGGGGS and
GGGGSGGGGSGGGGSGGGGSG).
[0112] Dual antagonists or other biologics described herein are
typically provided in isolated form. This means that an antagonist
is typically at least 50% w/w pure of interfering proteins and
other contaminants arising from its production or purification but
does not exclude the possibility that the antagonist is combined
with an excess of pharmaceutical acceptable excipient intended to
facilitate its use. Sometimes antagonists are at least 60, 70, 80,
90, 95 or 99% w/w pure of interfering proteins and contaminants
from production or purification. Often an antagonist is the
predominant macromolecular species remaining after its
purification.
[0113] The term antibody includes intact antibodies and binding
fragments thereof. A binding fragment refers to a molecule other
than an intact antibody that comprises a portion of an intact
antibody that binds the antigen to which the intact antibody binds.
Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2;
diabodies; linear antibodies; single-chain antibody molecules (e.g.
scFv); and multispecific antibodies formed from antibody fragments.
scFv antibodies are described in Houston J S. 1991. Methods in
Enzymol. 203:46-96. In addition, antibody fragments comprise single
chain polypeptides having the characteristics of a VH domain,
namely being able to assemble together with a VL domain, or of a VL
domain, namely being able to assemble together with a VH domain to
a functional antigen binding site and thereby providing the antigen
binding property of full length antibodies.
[0114] Specific binding of an antibody, extracellular trap segment
or dual antagonist to its target antigen(s) means an affinity of at
least 10.sub.6, 10.sub.7, 10.sub.8, 10.sub.9, or 10.sub.10
M.sup.-.sub.1. Specific binding is detectably higher in magnitude
and distinguishable from non-specific binding occurring to at least
one unrelated target. Specific binding can be the result of
formation of bonds between particular functional groups or
particular spatial fit (e.g., lock and key type) whereas
nonspecific binding is usually the result of van der Waals forces.
Specific binding does not however necessarily imply that an
antibody or fusion protein binds one and only one target.
[0115] A basic antibody structural unit is a tetramer of subunits.
Each tetramer includes two identical pairs of polypeptide chains,
each pair having one "light" (about 25 kDa) and one "heavy" chain
(about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition.
[0116] This variable region is initially expressed linked to a
cleavable signal peptide. The variable region without the signal
peptide is sometimes referred to as a mature variable region. Thus,
for example, a light chain mature variable region means a light
chain variable region without the light chain signal peptide.
However, reference to a variable region does not mean that a signal
sequence is necessarily present; and in fact signal sequences are
cleaved once the antibodies or fusion proteins of the invention
have been expressed and secreted. A pair of heavy and light chain
variable regions defines a binding region of an antibody. The
carboxy-terminal portion of the light and heavy chains respectively
defines light and heavy chain constant regions. The heavy chain
constant region is primarily responsible for effector function. In
IgG antibodies, the heavy chain constant region is divided into
CHI, hinge, CH2, and CH3 regions. The CHI region binds to the light
chain constant region by disulfide and noncovalent bonding. The
hinge region provides flexibility between the binding and effector
regions of an antibody and also provides sites for intermolecular
disulfide bonding between the two heavy chain constant regions in a
tetramer subunit. The CH2 and CH3 regions are the primary site of
effector functions and FcR binding.
[0117] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" segment of about 12 or more
amino acids, with the heavy chain also including a "D" segment of
about 10 or more amino acids. (See generally, Fundamental
Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7)
(incorporated by reference in its entirety for all purposes).
[0118] The mature variable regions of each light/heavy chain pair
form the antibody binding site. Thus, an intact antibody has two
binding sites, i.e., is divalent. In natural antibodies, the
binding sites are the same. However, bispecific antibodies can be
made in which the two binding sites are different (see, e.g.,
Songsivilai S, Lachmann P C. 1990. Bispecific antibody: a tool for
diagnosis and treatment of disease. Clin Exp Immunol. 79:315-321;
Kostelny S A, Cole M S, Tso J Y. 1992. Formation of bispecific
antibody by the use of leucine zippers. J Immunol. 148: 1547-1553).
The variable regions all exhibit the same general structure of
relatively conserved framework regions (FR) joined by three
hypervariable regions, also called complementarity determining
regions or CDRs. The CDRs from the two chains of each pair are
aligned by the framework regions, enabling binding to a specific
epitope. From N-terminal to C-terminal, both light and heavy chains
comprise the domains FRI, CDR1, FR2, CDR2, FR3, CDR3 and FR4. For
convenience, the variable heavy CDRs can be referred to as
CDR.sub.H1, CDR.sub.H2 and CDR.sub.H3; the variable light chain
CDRs can be referred to as CDR.sub.L1, CDR.sub.L2 and CDR.sub.L3.
The assignment of amino acids to each domain is in accordance with
the definitions of Kabat E A, et al. 1987 and 1991. Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md.) or Chothia C, Lesk A M. 1987. Canonical Structures
for the Hypervariable Regions of Immunoglobulins. J Mol Biol
196:901-917; Chothia C, et al. 1989. Conformations of
Immunoglobulin Hypervariable Regions. Nature 342:877-883. Kabat
also provides a widely used numbering convention (Kabat numbering)
in which corresponding residues between different heavy chain
variable regions or between different light chain variable regions
are assigned the same number. Although Kabat numbering can be used
for antibody constant regions, EU numbering is more commonly used,
as is the case in this application. Although specific sequences are
provided for exemplary dual antagonists, it will be appreciated
that after expression of protein chains one to several amino acids
at the amino or carboxy terminus of the light and/or heavy chain,
particularly a heavy chain C-terminal lysine residue, may be
missing or derivatized in a proportion or all of the molecules.
[0119] The term "epitope" refers to a site on an antigen to which
an antibody or extracellular trap segment binds. An epitope on a
protein can be formed from contiguous amino acids or noncontiguous
amino acids juxtaposed by tertiary folding of one or more proteins.
Epitopes formed from contiguous amino acids (also known as linear
epitopes) are typically retained on exposure to denaturing solvents
whereas epitopes formed by tertiary folding (also known as
conformational epitopes) are typically lost on treatment with
denaturing solvents. An epitope typically includes at least 3, and
more usually, at least 5 or 8-10 amino acids in a unique spatial
conformation. Methods of determining spatial conformation of
epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed. (1996).
[0120] Antibodies that recognize the same or overlapping epitopes
can be identified in a simple immunoassay showing the ability of
one antibody to compete with the binding of another antibody to a
target antigen. The epitope of an antibody can also be defined by
X-ray crystallography of the antibody (or Fab fragment) bound to
its antigen to identify contact residues.
[0121] Alternatively, two antibodies have the same epitope if all
amino acid mutations in the antigen that reduce or eliminate
binding of one antibody reduce or eliminate binding of the other.
Two antibodies have overlapping epitopes if some amino acid
mutations that reduce or eliminate binding of one antibody reduce
or eliminate binding of the other.
[0122] Competition between antibodies is determined by an assay in
which an antibody under test inhibits specific binding of a
reference antibody to a common antigen (see, e.g., Junghans et al.,
Cancer Res. 50: 1495, 1990). A test antibody competes with a
reference antibody if an excess of a test antibody (e.g., at least
2.times., 5.times., 10.times., 20.times. or 100.times.) inhibits
binding of the reference antibody by at least 50% but preferably
75%, 90% or 99% as measured in a competitive binding assay.
Antibodies identified by competition assay (competing antibodies)
include antibodies binding to the same epitope as the reference
antibody and antibodies binding to an adjacent epitope sufficiently
proximal to the epitope bound by the reference antibody for steric
hindrance to occur.
[0123] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0124] For purposes of classifying amino acids substitutions as
conservative or nonconservative, amino acids are grouped as
follows: Group I (hydrophobic side chains): met, ala, val, leu,
ile; Group II (neutral hydrophilic side chains): cys, ser, thr;
Group III (acidic side chains): asp, glu; Group IV (basic side
chains): asn, gin, his, lys, arg; Group V (residues influencing
chain orientation): gly, pro; and Group VI (aromatic side chains):
trp, tyr, phe. Conservative substitutions involve substitutions
between amino acids in the same class. Non-conservative
substitutions constitute exchanging a member of one of these
classes for a member of another.
[0125] Percentage sequence identities are determined with antibody
sequences maximally aligned by the Kabat numbering convention for a
variable region or EU numbering for a constant region. After
alignment, if a subject antibody region (e.g., the entire mature
variable region of a heavy or light chain) is being compared with
the same region of a reference antibody, the percentage sequence
identity between the subject and reference antibody regions is the
number of positions occupied by the same amino acid in both the
subject and reference antibody region divided by the total number
of aligned positions of the two regions, with gaps not counted,
multiplied by 100 to convert to percentage. Sequence identities of
other sequences can be determined by aligning sequences using
algorithms, such as BESTFIT, PASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis., using default gap parameters, or by
inspection, and the best alignment (i.e., resulting in the highest
percentage of sequence similarity over a comparison window).
[0126] Percentage of sequence identity is calculated by comparing
two optimally aligned sequences over a window of comparison,
determining the number of positions at which the identical residues
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0127] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises antibody may contain the
antibody alone or in combination with other ingredients.
[0128] The term "antibody-dependent cellular cytotoxicity", or
ADCC, is a mechanism for inducing cell death that depends upon the
interaction of antibody-coated target cells (i.e., cells with bound
antibody) with immune cells possessing lytic activity (also
referred to as effector cells). Such effector cells include natural
killer cells, monocytes/macrophages and neutrophils. ADCC is
triggered by interactions between the Fe region of an antibody
bound to a cell and Fey receptors, particularly Fc.gamma.RI and
Fc.gamma.RIII, on immune effector cells such as neutrophils,
macrophages and natural killer cells. The target cell is eliminated
by phagocytosis or lysis, depending on the type of mediating
effector cell. Death of the antibody-coated target cell occurs as a
result of effector cell activity.
[0129] The term opsonization also known as "antibody-dependent
cellular phagocytosis", or ADCP, refers to the process by which
antibody-coated cells are internalized, either in whole or in part,
by phagocytic immune cells (e.g., macrophages, neutrophils and
dendritic cells) that bind to an immunoglobulin Fe region.
[0130] The term "complement-dependent cytotoxicity" or CDC refers
to a mechanism for inducing cell death in which an Fe effector
domain(s) of a target-bound antibody activates a series of
enzymatic reactions culminating in the formation of holes in the
target cell membrane. Typically, antigen-antibody complexes such as
those on antibody-coated target cells bind and activate complement
component C1q which in turn activates the complement cascade
leading to target cell death. Activation of complement may also
result in deposition of complement components on the target cell
surface that facilitate ADCC by binding complement receptors (e.g.,
CR3) on leukocytes.
[0131] A humanized antibody is a genetically engineered antibody in
which the CDRs from a non-human "donor" antibody are grafted into
human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat.
Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539,
Carter, U.S. Pat. No. 6,407,213, Adair, U.S. Pat. No. 5,859,205
6,881,557, Foote, U.S. Pat. No. 6,881,557). The acceptor antibody
sequences can be, for example, a mature human antibody sequence, a
composite of such sequences, a consensus sequence of human antibody
sequences, or a germline region sequence. Thus, a humanized
antibody is an antibody having some or all CDRs entirely or
substantially from a donor antibody and variable region framework
sequences and constant regions, if present, entirely or
substantially from human antibody sequences. Similarly a humanized
heavy chain has at least one, two and usually all three CDRs
entirely or substantially from a donor antibody heavy chain, and a
heavy chain variable region framework sequence and heavy chain
constant region, if present, substantially from human heavy chain
variable region framework and constant region sequences. Similarly
a humanized light chain has at least one, two and usually all three
CDRs entirely or substantially from a donor antibody light chain,
and a light chain variable region framework sequence and light
chain constant region, if present, substantially from human light
chain variable region framework and constant region sequences.
Other than nanobodies and dAbs, a humanized antibody comprises a
humanized heavy chain and a humanized light chain. A CDR in a
humanized antibody is substantially from a corresponding CDR in a
non-human antibody when at least 85%, 90%, 95% or 100% of
corresponding residues (as defined by Kabat) are identical between
the respective CDRs. The variable region framework sequences of an
antibody chain or the constant region of an antibody chain are
substantially from a human variable region framework sequence or
human constant region respectively when at least 85, 90, 95 or 100%
of corresponding residues defined by Kabat are identical.
[0132] Although humanized antibodies often incorporate all six CDRs
(preferably as defined by Kabat) from a mouse antibody, they can
also be made with less than all CDRs (e.g., at least 3, 4, or 5
CDRs from a mouse antibody) (e.g., De Pascalis R, Iwahashi M,
Tamura M, et al. 2002. Grafting "Abbreviated"
Complementary-Determining Regions Containing
Specificity-Determining Residues Essential for Ligand Contact to
Engineer a Less Immunogenic Humanized Monoclonal Antibody. J
Immunol. 169:3076-3084; Vajdos F F, Adams C W, Breece T N, Presta L
G, de Vos A M, Sidhu, S S. 2002. Comprehensive functional maps of
the antigen-binding site of an anti-ErbB2 antibody obtained with
shotgun scanning mutagenesis. J Mol Biol. 320: 415-428; Iwahashi M,
Milenic D E, Padlan E A, et al. 1999. CDR substitutions of a
humanized monoclonal antibody (CC49): Contributions of individual
CDRs to antigen binding and immunogenicity. Mol Immunol.
36:1079-1091; Tamura M, Milenic D E, Iwahashi M, et al. 2000.
Structural correlates of an anticarcinoma antibody: Identification
of specificity-determining regions (SDRs) and development of a
minimally immunogenic antibody variant by retention of SDRs only. J
Immunol. 164:1432-1441).
[0133] A chimeric antibody is an antibody in which the mature
variable regions of light and heavy chains of a non-human antibody
(e.g., a mouse) are combined with human light and heavy chain
constant regions. Such antibodies substantially or entirely retain
the binding specificity of the mouse antibody, and are about
two-thirds human sequence.
[0134] A veneered antibody is a type of humanized antibody that
retains some and usually all of the CDRs and some of the non-human
variable region framework residues of a non-human antibody but
replaces other variable region framework residues that may
contribute to B- or T-cell epitopes, for example exposed residues
(Padlan E A. 1991. A possible procedure for reducing the
immunogenicity of antibody variable domains while preserving their
ligand-binding properties. Mol Immunol. 28:489-98) with residues
from the corresponding positions of a human antibody sequence. The
result is an antibody in which the CDRs are entirely or
substantially from a non-human antibody and the variable region
frameworks of the non-human antibody are made more human-like by
the substitutions. A human antibody can be isolated from a human,
or otherwise result from expression of human immunoglobulin genes
(e.g., in a transgenic mouse, in vitro or by phage display).
Methods for producing human antibodies include the trioma method of
Ostberg L, Pursch E. 1983. Human x (mouse x human) hybridomas
stably producing human antibodies. Hybridoma 2:361-367; Ostberg,
U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No.
4,634,666, use of transgenic mice including human immunoglobulin
genes (see, e.g., Lonberg et al., WO93/12227 (1993); U.S. Pat. Nos.
5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016,
5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553
(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO
91/10741 (1991) and phage display methods (see, e.g. Dower et al.,
WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. Nos.
5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and
5,565,332.
[0135] "Polymer" refers to a series of monomer groups linked
together. A polymer is composed of multiple units of a single
monomer (a homopolymer) or different monomers (a heteropolymer).
High MW polymers are prepared from monomers that include, but are
not limited to, acrylates, methacrylates, acrylamides,
methacrylamides, styrenes, vinyl-pyridine, vinyl-pyrrolidone and
vinyl esters such as vinyl acetate. Additional monomers are useful
in the high MW polymers of the present invention. When two
different monomers are used, the two monomers are called
"comonomers," meaning that the different monomers are copolymerized
to form a single polymer. The polymer can be linear or branched.
When the polymer is branched, each polymer chain is referred to as
a "polymer arm." The end of the polymer arm linked to the initiator
moiety is the proximal end, and the growing-chain end of the
polymer arm is the distal end. On the growing chain-end of the
polymer arm, the polymer arm end group can be the radical
scavenger, or another group.
[0136] "Initiator" refers to a compound capable of initiating a
polymerization using the monomers or comonomers of the present
invention. The polymerization can be a conventional free radical
polymerization or preferably a controlled/"living" radical
polymerization, such as Atom Transfer Radical Polymerization
(ATRP), Reversible Addition-Fragmentation-Termination (RAFT)
polymerization or nitroxide mediated polymerization (NMP). The
polymerization can be a "pseudo" controlled polymerization, such as
degenerative transfer. When the initiator is suitable for ATRP, it
contains a labile bond which can be homolytically cleaved to form
an initiator fragment, I, being a radical capable of initiating a
radical polymerization, and a radical scavenger, I', which reacts
with the radical of the growing polymer chain to reversibly
terminate the polymerization. The radical scavenger I' is typically
a halogen, but can also be an organic moiety, such as a nitrile. In
some embodiments of the present invention, the initiator contains
one of more 2-bromoisobutyrate groups as sites for polymerization
via ATRP.
[0137] A "chemical linker" refers to a chemical moiety that links
two groups together, such as a half-life extending moiety and a
protein. The linker can be cleavable or non-cleavable. Cleavable
linkers can be hydrolyzable, enzymatically cleavable, pH sensitive,
photolabile, or disulfide linkers, among others. Other linkers
include homobifunctional and heterobifunctional linkers. A "linking
group" is a functional group capable of forming a covalent linkage
consisting of one or more bonds to a bioactive agent. Non-limiting
examples include those illustrated in Table 1 of WO2013059137
(incorporated by reference).
[0138] The term "reactive group" refers to a group that is capable
of reacting with another chemical group to form a covalent bond,
i.e. is covalently reactive under suitable reaction conditions, and
generally represents a point of attachment for another substance.
The reactive group is a moiety, such as maleimide or succinimidyl
ester, is capable of chemically reacting with a functional group on
a different moiety to form a covalent linkage. Reactive groups
generally include nucleophiles, electrophiles and photoactivatable
groups.
[0139] "Phosphorylcholine," also denoted as "PC," refers to the
following:
##STR00001##
[0140] where * denotes the point of attachment. The
phosphorylcholine is a zwitterionic group and includes salts (such
as inner salts), and protonated and deprotonated forms thereof.
[0141] "Phosphorylcholine containing polymer" is a polymer that
contains phosphorylcholine. "Zwitterion containing polymer" refers
to a polymer that contains a zwitterion.
[0142] Poly(acryloyloxyethyl phosphorylcholine) containing polymer
refers to a polymer containing
2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC
shown below in Example 51) as monomer.
[0143] Poly(methacryloyloxyethyl phosphorylcholine) containing
polymer refers to a polymer containing
2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate
(HEMA-PC) as monomer.
[0144] "Molecular weight" in the context of the polymer can be
expressed as either a number average molecular weight, or a weight
average molecular weight or a peak molecular weight. Unless
otherwise indicated, all references to molecular weight herein
refer to the peak molecular weight. These molecular weight
determinations, number average (Mn), weight average (Mw) and peak
(Mp), can be measured using size exclusion chromatography or other
liquid chromatography techniques. Other methods for measuring
molecular weight values can also be used, such as the use of
end-group analysis or the measurement of colligative properties
(e.g., freezing-point depression, boiling-point elevation, or
osmotic pressure) to determine number average molecular weight, or
the use of light scattering techniques, ultracentrifugation or
viscometry to determine weight average molecular weight. In a
preferred embodiment of the present invention, the molecular weight
is measured by SEC-MALS (size exclusion chromatography-multi angle
light scattering). The polymeric reagents of the invention are
typically polydisperse (i.e., number average molecular weight and
weight average molecular weight of the polymers are not equal),
preferably possessing low polydispersity values of, for example,
less than about 1.5, as judged, for example, by the PDI value
derived from the SEC-MALS measurement. In other embodiments, the
polydispersities (PDI) are more preferably in the range of about
1.4 to about 1.2, still more preferably less than about 1.15, and
still more preferably less than about 1.10, yet still more
preferably less than about 1.05, and most preferably less than
about 1.03.
[0145] The phrase "a" or "an" entity refers to one or more of that
entity; for example, a compound refers to one or more compounds or
at least one compound. As such, the terms "a" (or "an"), "one or
more", and "at least one" can be used interchangeably herein.
[0146] "About" means variation one might see in measurements taken
among different instruments, samples, and sample preparations.
[0147] "Protected," "protected form," "protecting group" and
"protective group" refer to the presence of a group (i.e., the
protecting group) that prevents or blocks reaction of a particular
chemically reactive functional group in a molecule under certain
reaction conditions. Protecting groups vary depending upon the type
of chemically reactive group being protected as well as the
reaction conditions to be employed and the presence of additional
reactive or protecting groups in the molecule, if any. Suitable
protecting groups include those such as found in the treatise by
Greene et al., "Protective Groups In Organic Synthesis," 3rd
Edition, John Wiley and Sons, Inc., New York, 1999.
[0148] "Alkyl" refers to a straight or branched, saturated,
aliphatic radical having the number of carbon atoms indicated. For
example, C1-C6 alkyl includes, but is not limited to, methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are
not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl can include
any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,
1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The
alkyl group is typically monovalent, but can be divalent, such as
when the alkyl group links two moieties together.
[0149] The term "lower" referred to above and hereinafter in
connection with organic radicals or compounds respectively defines
a compound or radical which can be branched or unbranched with up
to and including 7, preferably up to and including 4 and (as
unbranched) one or two carbon atoms.
[0150] "Alkylene" refers to an alkyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkylene can be linked to
the same atom or different atoms of the alkylene. For instance, a
straight chain alkylene can be the bivalent radical of
--(CH2).sub.n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups
include, but are not limited to, methylene, ethylene, propylene,
isopropylene, butylene, isobutylene, sec-butylene, pentylene and
hexylene.
[0151] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a
variety of groups selected from: --OR', .dbd.O, .dbd.NR',
.dbd.N--OR'--NR'R'''--SR''-halogen'-SiR'R''R''''--OC(O)R''--C(O)R''--CO.s-
ub.2R''--CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.N H, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(OhR', --S(O).sub.2NR'R'', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'' and R'''each independently
refer to hydrogen, unsubstituted (C.sub.1-C.sub.8)alkyl and
heteroalkyl, unsubstituted aryl, aryl substituted with 1-3
halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or
aryl-(C.sub.1-C.sub.4) alkyl groups. When R' and R'' are attached
to the same nitrogen atom, they can be combined with the nitrogen
atom to form a 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "alkyl"
is include groups such as haloalkyl (e.g., --CF.sub.3 and
--CH.sub.2CF.sub.3) and acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like). Preferably, the substituted
alkyl and heteroalkyl groups have from 1 to 4 substituents, more
preferably 1, 2 or 3 substituents. Exceptions are those perhalo
alkyl groups (e.g., pentafluoroethyl and the like) which are also
preferred and contemplated by the present invention.
[0152] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR',
.dbd.N--OR''--NR'R'''--SR''-halogen'-SiR'R''R'''--OC(O)R''--C(O)R''--CO.s-
ub.2R''--CONR'R'''--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(OhR', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0153] "Alkoxy" refers to alkyl group having an oxygen atom that
either connects the alkoxy group to the point of attachment or is
linked to two carbons of the alkoxy group. Alkoxy groups include,
for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,
2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy,
etc. The alkoxy groups can be further substituted with a variety of
substituents described within. For example, the alkoxy groups can
be substituted with halogens to form a "halo-alkoxy" group.
[0154] "Carboxyalkyl" means an alkyl group (as defined herein)
substituted with a carboxy group. The term "carboxycycloalkyl"
means a cycloalkyl group (as defined herein) substituted with a
carboxy group. The term alkoxyalkyl means an alkyl group (as
defined herein) substituted with an alkoxy group. The term
"carboxy" employed herein refers to carboxylic acids and their
esters.
[0155] "Haloalkyl" refers to alkyl as defined above where some or
all of the hydrogen atoms are substituted with halogen atoms.
Halogen (halo) preferably represents chloro or fluoro, but may also
be bromo or iodo. For example, haloalkyl includes trifluoromethyl,
fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term
"perfluoro" defines a compound or radical which has all available
hydrogens that are replaced with fluorine. For example,
perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl,
perfluoromethyl refers to 1,1,1-trifluoromethyl, and
perfluoromethoxy refers to 1,1,1-trifluoromethoxy.
[0156] "Fluoro-substituted alkyl" refers to an alkyl group where
one, some, or all hydrogen atoms have been replaced by
fluorine.
[0157] "Cytokine" in the context of this invention is a member of a
group of protein signaling molecules that may participate in
cell-cell communication in immune and inflammatory responses.
Cytokines are typically small, water-soluble glycoproteins that
have a mass of about 8-35 kDa.
[0158] "Cycloalkyl" refers to a cyclic hydrocarbon group that
contains from about 3 to 12, from 3 to 10, or from 3 to 7
endocyclic carbon atoms. Cycloalkyl groups include fused, bridged
and spiro ring structures.
[0159] "Endocyclic" refers to an atom or group of atoms which
comprise part of a cyclic ring structure.
[0160] "Exocyclic" refers to an atom or group of atoms which are
attached but do not define the cyclic ring structure.
[0161] "Cyclic alkyl ether" refers to a 4 or 5 member cyclic alkyl
group having 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen
or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran,
tetrahydrothiophene); or a 6 to 7 member cyclic alkyl group having
1 or 2 endocyclic oxygen or sulfur atoms (e.g., tetrahydropyran,
1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane,
1,4-dithiane, 1,4-oxathiane).
[0162] "Alkenyl" refers to either a straight chain or branched
hydrocarbon of 2 to 6 carbon atoms, having at least one double
bond. Examples of alkenyl groups include, but are not limited to,
vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,
butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,
1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,
1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or
1,3,5-hexatrienyl. Alkenyl groups can also have from 2 to 3, 2 to
4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6
carbons. The alkenyl group is typically monovalent, but can be
divalent, such as when the alkenyl group links two moieties
together.
[0163] "Alkenylene" refers to an alkenyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkenylene can be linked to
the same atom or different atoms of the alkenylene. Alkenylene
groups include, but are not limited to, ethenylene, propenylene,
isopropenylene, butenylene, isobutenylene, sec-butenylene,
pentenylene and hexenylene.
[0164] "Alkynyl" refers to either a straight chain or branched
hydrocarbon of 2 to 6 carbon atoms, having at least one triple
bond. Examples of alkynyl groups include, but are not limited to,
acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl,
sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl,
1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,
1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or
1,3,5-hexatriynyl. Alkynyl groups can also have from 2 to 3, 2 to
4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6
carbons. The alkynyl group is typically monovalent, but can be
divalent, such as when the alkynyl group links two moieties
together.
[0165] "Alkynylene" refers to an alkynyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkynylene can be linked to
the same atom or different atoms of the alkynylene. Alkynylene
groups include, but are not limited to, ethynylene, propynylene,
butynylene, sec-butynylene, pentynylene and hexynylene.
[0166] "Cycloalkyl" refers to a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly
containing from 3 to 12 ring atoms, or the number of atoms
indicated. Monocyclic rings include, for example, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and
polycyclic rings include, for example, norbomane,
decahydronaphthalene and adamantane. For example, C.sub.3-8
cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclooctyl, and norbornane.
[0167] "Cycloalkylene" refers to a cycloalkyl group, as defined
above, linking at least two other groups, i.e., a divalent
hydrocarbon radical. The two moieties linked to the cycloalkylene
can be linked to the same atom or different atoms of the
cycloalkylene. Cycloalkylene groups include, but are not limited
to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,
and cyclooctylene.
[0168] "Heterocycloalkyl" refers to a ring system having from 3
ring members to about 20 ring members and from 1 to about 5
heteroatoms such as N, O and S. Additional heteroatoms can also be
useful, including, but not limited to, B, Al, Si and P. The
heteroatoms can also be oxidized, such as, but not limited to,
--S(O)-- and --S(Oh-. For example, heterocycle includes, but is not
limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino,
pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,
pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl,
quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.
[0169] "Heterocycloalkylene" refers to a heterocyclalkyl group, as
defined above, linking at least two other groups. The two moieties
linked to the heterocycloalkylene can be linked to the same atom or
different atoms of the heterocycloalkylene.
[0170] "Aryl" refers to a monocyclic or fused bicyclic, tricyclic
or greater, aromatic ring assembly containing 6 to 16 ring carbon
atoms. For example, aryl may be phenyl, benzyl or naphthyl,
preferably phenyl. "Arylene" means a divalent radical derived from
an aryl group. Aryl groups can be mono-, di- or tri-substituted by
one, two or three radicals selected from alkyl, alkoxy, aryl,
hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl,
alkylenedioxy and oxy-C.sub.2C.sub.3-alkylene; all of which are
optionally further substituted, for instance as hereinbefore
defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl.
Alkylenedioxy is a divalent substitute attached to two adjacent
carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.
Oxy-C.sub.2C.sub.3-alkylene is also a divalent substituent attached
to two adjacent carbon atoms of phenyl, e.g. oxyethylene or
oxypropylene. An example for oxy-C.sub.2C.sub.3-alkylene-phenyl is
2,3-dihydrobenzofuran-5-yl.
[0171] Preferred as aryl is naphthyl, phenyl or phenyl mono- or
disubstituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl,
especially phenyl or phenyl-mono- or disubstituted by alkoxy,
halogen or trifluoromethyl, and in particular phenyl.
[0172] Examples of substituted phenyl groups as R are, e.g.
4-chlorophen-1-yl, 3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl,
4-methylphen-1-yl, 4-aminomethylphen-1-yl,
4-methoxyethylaminomethylphen-1-yl,
4-hydroxyethylaminomethylphen-1-yl,
4-hydroxyethyl-(methyl)-aminomethylphen-1-yl,
3-aminomethylphen-1-yl, 4-N-acetylaminomethylphen-1-yl,
4-aminophen-1-yl, 3-aminophen-1-yl, 2-aminophen-1-yl,
4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phenyl,
4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,
4-(morpholin-1-ylmethyl)-phen-1-yl,
4-(2-methoxyethylaminomethyl)-phen-1-yl and
4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,
4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl,
and 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally
substituted in the heterocyclic ring.
[0173] "Arylene" refers to an aryl group, as defined above, linking
at least two other groups. The two moieties linked to the arylene
are linked to different atoms of the arylene. Arylene groups
include, but are not limited to, phenylene.
[0174] "Arylene-oxy" refers to an arylene group, as defined above,
where one of the moieties linked to the arylene is linked through
an oxygen atom. Arylene-oxy groups include, but are not limited to,
phenylene-oxy.
[0175] Similarly, substituents for the aryl and heteroaryl groups
are varied and are selected from: -halogen, --OR', --OC(O)R',
--NR'R'', --SR', --R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R'',
--C(O)R', --OC(O)NR'R'', --NR''C(O)R', --NR''C(OhR',
--NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --N.sub.3, --CH(Phh,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1C.sub.4) alkyl.
[0176] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2 or a single bond, and q is
an integer of from 0 to 2. Alternatively, two of the substituents
on adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CH.sub.2).sub.8--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted (C.sub.1-C.sub.6)alkyl.
[0177] "Heteroaryl" refers to a monocyclic or fused bicyclic or
tricyclic aromatic ring assembly containing 5 to 16 ring atoms,
where from 1 to 4 of the ring atoms are a heteroatom each N, O or
S. For example, heteroaryl includes pyridyl, indolyl, indazolyl,
quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl,
benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl,
oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,
thienyl, or any other radicals substituted, especially mono- or
di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents
2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl
represents 2- or 3-thienyl. Quinolinyl represents preferably 2-, 3-
or 4-quinolinyl. Isoquinolinyl represents preferably 1-, 3- or
4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents
preferably benzopyranyl or 3-benzothiopyranyl, respectively.
Thiazolyl represents preferably 2- or thiazolyl, and most preferred
4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl).
Tetrazolyl is preferably 5-tetrazolyl. Preferably, heteroaryl is
pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl,
triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl,
benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl,
oxazolyl, indazolyl, or any of the radicals substituted, especially
mono- or di-substituted.
[0178] The term "heteroalkyl" refers to an alkyl group having from
1 to 3 heteroatoms such as N, O and S. Additional heteroatoms can
also be useful, including, but not limited to, B, Al, Si and P. The
heteroatoms can also be oxidized, such as, but not limited to,
--S(O)-- and --S(O).sub.2--. For example, heteroalkyl can include
ethers, thioethers, alkyl-amines and alkyl-thiols.
[0179] The term "heteroalkylene" refers to a heteroalkyl group, as
defined above, linking at least two other groups. The two moieties
linked to the heteroalkylene can be linked to the same atom or
different atoms of the heteroalkylene.
[0180] "Electrophile" refers to an ion or atom or collection of
atoms, which may be ionic, having an electrophilic center, i.e., a
center that is electron seeking, capable of reacting with a
nucleophile. An electrophile (or electrophilic reagent) is a
reagent that forms a bond to its reaction partner (the nucleophile)
by accepting both bonding electrons from that reaction partner.
[0181] "Nucleophile" refers to an ion or atom or collection of
atoms, which may be ionic, having a nucleophilic center, i.e., a
center that is seeking an electrophilic center or capable of
reacting with an electrophile. A nucleophile (or nucleophilic
reagent) is a reagent that forms a bond to its reaction partner
(the electrophile) by donating both bonding electrons. A
"nucleophilic group" refers to a nucleophile after it has reacted
with a reactive group. Non limiting examples include amino,
hydroxyl, alkoxy, haloalkoxy and the like.
[0182] "Maleimido" refers to a pyrrole-2,5-dione-1l-yl group having
the structure:
##STR00002##
which upon reaction with a sulfhydryl (e.g., a thio alkyl) forms an
--S-maleimido group having the structure
##STR00003##
[0183] where " " indicates the point of attachment for the
maleimido group and indicates the point of attachment of the sulfur
atom the thiol to the remainder of the original sulfhydryl bearing
group.
[0184] For the purpose of this disclosure, "naturally occurring
amino acids" found in proteins and polypeptides are L-alanine,
L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine,
L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, and or L-valine.
"Non-naturally occurring amino acids" found in proteins are any
amino acid other than those recited as naturally occurring amino
acids. Non-naturally occurring amino acids include, without
limitation, the D isomers of the naturally occurring amino acids,
and mixtures of D and L isomers of the naturally occurring amino
acids. Other amino acids, such as N-alpha-methyl amino acids (e.g.
sarcosine), 4-hydroxyproline, desmosine, isodesmosine,
hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although
found in naturally occurring proteins, are considered to be
non-naturally occurring amino acids found in proteins for the
purpose of this disclosure as they are generally introduced by
means other than ribosomal translation of mRNA.
[0185] "Linear" in reference to the geometry, architecture or
overall structure of a polymer, refers to polymer having a single
polymer arm.
[0186] "Branched," in reference to the geometry, architecture or
overall structure of a polymer, refers to a polymer having 2 or
more polymer "arms" extending from a core structure contained
within an initiator. The initiator may be employed in an atom
transfer radical polymerization (ATRP) reaction. A branched polymer
may possess 2 polymer chains (arms), 3 polymer arms, 4 polymer
arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer
arms, 9 polymer arms or more. Each polymer arm extends from a
polymer initiation site. Each polymer initiation site is capable of
being a site for the growth of a polymer chain by the addition of
monomers. For example and not by way of limitation, using ATRP, the
site of polymer initiation on an initiator is typically an organic
halide undergoing a reversible redox process catalyzed by a
transition metal compound such as cuprous halide. Preferably, the
halide is a bromine.
[0187] "Pharmaceutically acceptable excipient" refers to an
excipient that can be included in the compositions of the invention
and that causes no significant adverse toxicological effect on the
patient and is approved or approvable by the FDA for therapeutic
use, particularly in humans. Non-limiting examples of
pharmaceutically acceptable excipients include water, NaCl, normal
saline solutions, lactated Ringer's, normal sucrose, normal glucose
and the like.
[0188] Dual antagonists are administered in an effective regime
meaning a dosage, route of administration and frequency of
administration that delays the onset, reduces the severity,
inhibits further deterioration, and/or ameliorates at least one
sign or symptom of a disorder. If a patient is already suffering
from a disorder, the regime can be referred to as a therapeutically
effective regime. If the patient is at elevated risk of the
disorder relative to the general population but is not yet
experiencing symptoms, the regime can be referred to as a
prophylactically effective regime. In some instances, therapeutic
or prophylactic efficacy can be observed in an individual patient
relative to historical controls or past experience in the same
patient. In other instances, therapeutic or prophylactic efficacy
can be demonstrated in a preclinical or clinical trial in a
population of treated patients relative to a control population of
untreated patients.
[0189] The "biological half-life" of a substance is a
pharmacokinetic parameter which specifies the time required for one
half of the substance to be removed from a tissue or an organism
following introduction of the substance.
[0190] "HEMA-PC" is
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate.
[0191] "TAP" means a PDGFR.beta.-GS 10-anti-VEGF-A heavy
chain/anti-VEGF-A light chain wherein amino acids 1-282 of the
heavy chain correspond to amino acids 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1), fused as a single
open reading frame via a glycine-serine linker (GGGGSGGGGS) linked
to the N terminus of a bevacizumab heavy chain sequence having the
following mutations in the variable region: T28D, N31H, H97Y,
S100aT (Ferrara N, Damico L, Shams N, et al. 2006. Development of
Ranibizumab, an anti-vascular endothelial growth factor antigen
binding fragment, as therapy for neovascular age-related macular
degeneration. Retina 26(8):859-870); and the following in the Fe
region: L234A, L235A, and G237A (EU numbering) (Strohl W R. 2009.
Optimization of Fe-mediated effector functions of monoclonal
antibodies. Curr Opin in Biotech. 20: 685-691). The light chain is
the bevacizumab light chain having an M4L mutation. TAP normally
exists as a dimer having two heavy chains and two light chains. TAP
may or may not have carbohydrate or other post-translational
modifications after being expressed from cells. TAP is also
sometimes called TAFwt or TAFWT, which indicates that the molecule
in question does not have either the Q347C or L443C mutations in
the heavy chain (Fe region) as do TAF347 or TAF443, defined
infra.
[0192] "TAF347" is the same as TAP except that it has the Q347C
mutation.
[0193] "TAF443" is the same as TAP except that it has the L443C
mutation. TAF443 is sometimes referred to herein as OG 1321.
[0194] "OG 1786" is a 9-arm initiator used for polymer synthesis
with the structure shown in FIG. 35, which depicts that salt form
of OG 1786 with trifluororacetic acid. OG 1786 may be used in
accordance with the present invention as other salts or as the free
base.
[0195] "OG 1801" is an approximately (+/-15%) 750 kDa polymer
(either by Mn or Mp) made using OG 1786 as an intiator for ATRP
synthesis using the monomer HEMA-PC.
[0196] "OG 1802" is OG 1801 with a maleimide functionality added
and is shown in FIG. 36 wherein each of n.sub.1, n.sub.2, n.sub.3,
n.sub.4, n.sub.5, n.sub.6, n.sub.7, n.sub.8, and n.sub.9 is an
integer (positive) (from 0 up to about 3000) such that the total
molecular weight of the polymer is (Mw) 750,000.+-.15% daltons.
[0197] "OG 1448" is TAF443 conjugated to the OG 1802
biopolymer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
General
[0198] The present invention provides a dual VEGF/PDGF antagonist
comprising a VEGF antagonist linked to a PDGF antagonist. The VEGF
antagonist is an antibody to a VEGF or VEGFR or is a VEGFR
extracellular trap segment (i.e., a segment from the extracellular
region of one or more VEGFR receptors that inhibits binding of at
least one VEGFR to at least one VEGF). The PDGF antagonist is an
antibody to a PDGF or PDGFR or is a PDGFR extracellular trap
segment (i.e., segment from the extracellular region of one or more
PDGFRs, which inhibits binding of at least one PDGFR and at least
one PDGF). At least one of the antagonists is not an antibody, or
put another way, at least one of the antagonists is an
extracellular trap segment. Preferably, the dual antagonist
includes an antibody antagonist and one extracellular trap segment
antagonist. In such a dual antagonist the extracellular trap
segment is preferably fused, optionally via a linker to the
N-terminus of the antibody heavy chain. The antibody light chain is
complexed with the antibody heavy chain in similar manner to that
in a natural antibody. Such dual antagonists are preferably
provided in the form of conjugates with a half-life extending
moiety conjugated to the dual antagonist. Preferably, a cysteine
residue is used for conjugation which has been introduced into the
antagonist. More preferably, the cysteine residue is at positions
347 or 443 of an IgG 1 heavy chain. It is preferred that the
half-life extending moiety is a zwitterionic polymer. Most
preferably the zwitterionic polymer is a phosphorylcholine
containing polymer.
[0199] Angiogenesis is the process by which new blood vessels are
created and plays a crucial role in development (going from embryo
to adult) and in wound healing (restoring blood flow to damaged or
injured tissue). However, when angiogenesis is dysregulated, it
contributes to the pathologies of many disorders, including cancer,
psoriasis, arthritis and blindness. Carmeliet P. 2003. Angiogenesis
in health and disease. Nature Med 9(6):653-660.
[0200] Abnormal angiogenesis is associated with wet age related
macular degeneration (a leading cause of blindness in the elderly)
and with cancer. Angiogenesis is characterized by an increase in
proliferating endothelial and stromal cells and vasculature with
altered morphology. See, generally, Folkman J. 2007. Angiogenesis:
an organizing principle for drug discovery?. Nat Rev Drug 6:273-286
and Baluk P, Hashizume H, McDonald D M. 2005. Cellular
abnormalities of blood vessels as targets in cancer. Curr Opin
Genet Dev. 15:102-111.
[0201] As mentioned above, neovascularization (NV) is a normal
process occurring both in development and in wound healing but can
become pathological when angiogenesis is dysregulated and occurs in
tissues associated with tumors (cancer), avascular cornea or the
subretinal space (wet AMD). The proliferation, invastion and
migration of NV vessels is controlled by a complex interplay
between growth factors, vascular endothelial cells, extracellular
matrix molecules, chemokines and cell signaling molecules.
[0202] NV tissue is composed of endothelial cells (EC), pericytes
and inflammatory cells (e.g. macrophages). Pericytes are derived
via differentiation from mast cells. The process of
neovascularization first involves the formation of angiogenic
sprouts composed of EC from existing capillaries into the avascular
space. VEGF signaling is understood to be the master switch for
this NV process. In this regard, VEGF has been localized in the tip
cell fiopodia which leads the angiogenic sprout.
[0203] Following sprout formation, the newly formed vessels are
coated by pericytes, leading to maturation of the NV. Pericyte
coating of NV leads to stabilization and support of NV both
physically and through signaling, including pericyte production of
VEGF. Armulik A, Abramsson A, Betsholtz C. 2005.
Endothelial/Pericyte Interactions. Circ Res. 97:512-523.
[0204] Approved wet AMD therapies are all directed at the
suppression of VEGF signaling. These therapies include pegaptanib
(Macugen.RTM.), approved in 2004, Genentech's bevacizumab
(Avastin.RTM.), approved in 2004 for cancer, used off label for
AMD, Genentech's ranibizumab (Lucentis.RTM.), approved in 2006, and
Regeneron's aflibercept (Eylea.RTM.) approved in 2011. Pegaptanib
is an aptamer based therapeutic, but with a limited market compared
with protein based therapeutics likely due to the limited gains in
visual acuity for patients. Bevacizumab is an anti-VEGFA IgG 1
antibody approved for cancer treatment, but is widely used off
label for treatment of AMD. Ranibizumab is a Fab which was affinity
matured from bevacizumab and is approved for AMD. However, the
market for Ranibizumab is substantially undercut by use of the much
cheaper bevacizumab. Finally, aflibercept is a VEGF trap, employing
a soluble receptor fragment decoy.
[0205] Anti-VEGF monotherapy has not lead to disease-modifying
regression of pathological NV. Brown D M, Kaiser P K, Michels M, et
al. 2006. ANCHOR Study Group. Ranibizumab versus verteporfin for
neovascular age-related macular degeneration. N Engl J Med
355(14):1432-1444; Rosenfeld P J, Brown D M, Heier J S, et al.
2006. MARINA study group. Ranibizumab for neovascular age-related
macular degeneration. N Engl J Med 355(14):1419-1431; Regillo C D,
Brown D M, Abraham P, et al. 2008. Randomized, double-masked,
sham0controlloed trial of ranibizumab for neovacular age-related
macular degeneration: PIER study year 1. Am J Ophthalmol.
145:239-248. Instead the majority of the efficacy or therapeutic
benefit of anti-VEGF therapies is due to their anti-permeability
property. Zebrowski B K, Yano S, Liu W, et al. 1999. Vascular
endothelial growth factor levels and induction of permeability in
malignant pleural effusions. Clin Cancer Res 5:3364-3368.
[0206] Because conventional anti-VEGF therapies do not cause
regression of pathological NV, visual acuity gains for many
patients have been quite limited. Moreover, neovasculature can also
lead to subretinal fibrosis which is a cause of blindness in wet
AMD patients.
[0207] Subretinal scarring develops in nearly half of treated eyes
within two years of anti-VEGF therapy. Daniel E, Toth C A, Grunwald
J E. 2014. Risk of scar in the comparison of age-related macular
degeneration in clinical settings. Retina 32: 1480-1485. Subretinal
fibrosis formation can cause permanent dysfunction of the macular
system; it causes destruction of photoreceptors, retinal pigment
epithelium and choroidal vessels. Ishikawa K, Ram K, Hinton D R.
2015. Molecular mechanisms of subretinal fibrosis in age-related
macular degeneration. Eye Res. Mar. 13, 2015 Epub 1-7. Although
anti-VEGF therapy generally stabilizes or improves visual acuity,
scar formation has been identified as one of the causes of loss of
visual acuity after treatment. Cohen S Y, Oubraham H, Uzzan J, et
al. 2012. Causes of unsuccessful ranibizumab treatment in exudative
age-related macular degeneration in clinical settings. Retina 32:
1480-1485.
[0208] Proangiogenic factors are generally upregulated in
pathological angiogenesis, including two members of the vascular
endothelial growth factor (VEGF) family: VEGF-A and placental
growth factor (PGF). VEGF-A and PGF activate quiescent endothelial
cells, promote cell proliferation and vascular permeability. VEGF-A
has been identified as a major factor in vascular leak in wet AMD.
Dvorak H F, Nagy J A, Feng D, Brown L F, Dvorak A M. 1999. Vascular
permeability factor/vascular endothelial growth factor and the
significance of microvascular hyperpermeability in angiogenesis.
Curr Top Microbial Immunol. 237:97-132.
[0209] Platelet derived growth factor "PDGF" signaling plays an
important role in NV maturation and in particular to the coating of
NV by pericytes. The coating of NV endothelial cells by pericytes
begins with EC expression of the paracrine platelet-derived growth
factor B, which forms the homodimer PDGF-BB. PDGF-BB is highly
retained in the tip cells of the angiogenic sprouts by heparin
sulfate proteoglycan. This PDGF-BB is then recognized by the
pericyte bound receptor PDGFR-.beta., which initiates the
proliferation and migration of pericytes along the growing
neovascularization.
[0210] PDGF-DD had also been discovered to play a central role in
pathological angiogenesis. Kumar A, Hou X, Chunsik L, et al. 2010.
Platelet-derived Growth Factor-DD Targeting Arrests Pathological
Angiogenesis by Modulating Glycogen Synthase Kinase-3
Phosphorylation. J Biol Chem 285(20):15500-15510. PDGF-DD
overexpression induces blood vessel maturation during angiogenesis.
Kong D, Wang Z, Sarkar F H, et al. 2008. Platelet-Derived Growth
Factor-D Overexpression Contributes to Epithelial-Mesenchymal
Transition of PC3 Prostate Cancer Cells. Stem Cells 26:1425-1435.
PDGF-DD is highly expressed in the eye. Ray S, Gao C, Wyatt K, et
al. 2005. Platelet-derived Frowth Factor D, Tissue-specific
Expression in the Eye, and a Key Role in Control of Lens Epithelial
Cell Proliferation. J Biol Chem. 280:8494-8502. Kumar et al. (2010)
found that PDGF-DD expression was upregulated during pathological
angiogenesis and that inhibition of PDGF-DD signaling decreased
choroidal and retinal neovascularization.
[0211] The term "PDGF" as used herein means any member of the class
of growth factors that (i) bind to a PDGF receptor such as
PDGFR-.beta., or PDGFR-.alpha.; (ii) activates a tyrosine kinase
activity associated with the PDGF receptor; and (iii) thereby
affects angiogenesis or an angiogenic process. The term "PDGF"
generally refers to those members of the class of growth factors
that induce DNA synthesis and mitogenesis through the binding and
activation of a platelet-derived growth factor cell surface
receptor (i.e., PDGFR) on a responsive cell type. PDGFs effect
specific biological effects including, for example: directed cell
migration (chemotaxis) and cell activation; phospholipase
activation; increased phosphatidylinositol turnover and
prostaglandin metabolism; stimulation of both collagen and
collagenase synthesis by responsive cells; alteration of cellular
metabolic activities, including matrix synthesis, cytokine
production, and lipoprotein uptake; induction, indirectly, of a
proliferative response in cells lacking PDGF receptors; fibrosis
and potent vasoconstrictor activity. The term "PDGF" is meant to
include both a "PDGF" polypeptide and its corresponding "PDGF"
encoding gene or nucleic acid.
[0212] The PDGF family consists of disulfide bonded homo-ffdimers
of PDGF-A (Swiss Protein P04085), -B (P01127), -C (Q9NRA1) and -D
(Q9GZPO) and the hetero dimer PDGF-AB. The various PDGF isoforms
exert their effect by binding to .alpha. and .beta.-tyrosine kinase
receptors (PDGFR-.alpha. (P16234) and PDGFR-.beta. (P09619)
respectively). See generally U.S. Pat. No. 5,872,218 which is
incorporated herein by reference for all purposes. The .alpha. and
.beta. receptors are structurally similar: both have extracellular
domains with five immunoglobulin (lg) like domains and
intracellular domains with a kinase function. PDGF binding occurs
mainly through domains 2 and 3 of the receptors and causes
dimerization of the receptors. lg like domain 4 is involved in
receptor dimerization. Receptor dimerization is a key component of
PDGF signaling: receptor dimerization leads to receptor
auto-phosphorylation. Auto-phosphorylation in turns causes a
conformational change in the receptor and activates the receptor
kinase. PDGF-A, --B, --C and -D bind to the two different receptors
with different affinities and effects. PDGF-AA, -AB, -BB and -CC
induce aa receptor homodimers, PDGF-BB and -DD induced .beta..beta.
homodimers and PDGF-AB, -BB, -CC and -DD produce .alpha..beta.
receptor heterodimers.
[0213] In terms of function, PDGFR-.alpha. and PDGFR-.beta. appear
to have substantially different roles. PDGFR-.alpha. signaling is
involved in gastrulation and in development of the cranial and
cardiac neural crest, gonads, lung, intestine, skin, CNS and
skeleton. PDGFR-.beta. signaling is involved in blood vessel
formation and early hematopoiesis. Andrae J, Radiosa G, Betsholtz
C. 2008. Role of platelet-derived growth factors in physiology and
medicine. Genes Develop 22: 1276-1312. In terms of interaction of
the various PDGF ligands with the receptors, PDGF-AA and PDGF-CC
exclusively bind to and interact with PDGFR-.alpha.. PDGF-BB and
PDGF-AB bind with .alpha. and .beta. receptors. PDGF-DD exclusively
interacts with PDGFR-.beta.. Raica M, Cimpean A M. 2010.
Platelet-Derived Growth Factor (PDGF)/PDGF Receptors (PDGFR) Axis
as Target for Antitumor and Antiangiogenic Therapy. Pharmaceut.
3:572-599.
[0214] Unless otherwise apparent from the context reference to a
PDGF means any of PDGF-A, -B, -C and -D in any of the natural
isoforms or natural variants or induced variants having at least
90, 95, 98 or 99% sequence identity to a natural form. Preferably,
such PDGFs are human PDGFs. Likewise reference to a PDGFR means
PDGFR-A (P16234) or PDGFR-B including any natural isoform or
natural variant, or an induced variant having at least 90, 95, 98
or 99% or 100% sequence identity to a natural sequences.
[0215] The amino acid sequence of human
PDGFR-(UniProtKB/Swiss-Prot: P09619.1) is set forth in FIG. 1,
which shows a full-length human PDGFR-.beta. (including the leader
sequence), a 1106 amino acid protein. Amino acids 1-32 are part of
the leader peptide which is cleaved off in the mature protein.
PDGFR-.beta. has five extracellular lg-like domains D 1-D5.
Williams A F, Barclay A N. 1988. The immunoglobulin
superfamily-domains for cell surface recognition. Annu Rev Immunol.
6:381-405. The full-length extracellular region runs from about
amino acid 33 to 532, the transmembrane domain from about residue
533 to 553 and the cytoplasmic domain from about residue 554 to
1106. The extracellular region includes five immunoglobulin-like
domains, D1-D5. The D1 domain is typically considered to be from
about amino acid 33 (Leu) to about 123 (Pro). In accordance with
the present invention, D1 may also be from 33 to 122 (Val). D2 is
typically considered to be from about 124 (Thr) to about 213 (Ser).
In accordance with the present invention, D2 may be 129 (Pro) to
210 (Gln). D3 is typically considered to be from about amino acid
214 (Ile) to 314 (Gly). In accordance with the present invention,
D3 may be from 214 (Ile) to 309 (Thr). D4 is typically considered
to be from about amino acid 315 (Tyr) to 416 (Pro). D5 is typically
considered to be from about amino acid 417 (Val) to 531 (Lys).
[0216] The exact boundaries of the D1-D5 domains can vary depending
on how the analysis is done. Preferably, the boundaries vary by 9
amino acids or less. Typically they vary by 7 amino acids of less,
more typically by 5 amino acids or less. Usually, boundary variance
is 3 amino acids or less. Most typically the boundaries vary by
only an amino acid. The essential characteristic of each domain is
its ability to bind to its cognate ligands.
[0217] A "PDGF antagonist" or a molecule that "antagonizes PDGF" is
an agent that reduces, or inhibits, either partially or fully, at
least one activity of a PDGF including its ability to specifically
bind to a PDGFR, and consequent cellular responses, such as
proliferation. PDGF antagonists include antibodies that
specifically bind to a PDGF or PDGFR and extracellular trap
segments from a PDGFR.
[0218] One or more portions of a PDGFR-.beta. extracellular
receptor sequence can be used as an antagonist for
PDGF-PDGFR-.beta. signaling. The term extracellular trap segment
refers to a full length extracellular region or any portion
thereof, or combination of portions from different PDGF receptors
that can antagonize PDGF-PDGFR-beta signaling. Such portions are
typically used free of the transmembrane and intracellular sequence
of the PDGFR and are consequently referred to as being soluble. The
portions antagonize by acting as a trap or decoy for a cognate
PDGF. PDGF binds to the soluble PDGFR-.beta. segment trap and is
unable to bind to the corresponding membrane bound receptor.
Preferably, such traps include one of more of PDGFR-.beta. domains
D1-D5. Preferably, the trap contains at least one of D2 and D3.
More preferably, the trap contains D1, D2 and D3. More preferably
the trap is a contiguous segment corresponding to amino acids 33 to
314 of FIG. 8 which contains D1-D3. PDGFR-alpha likewise includes
domains D1 through D5 and extracellular trap segments incorporating
corresponding domains of PDGFR-alpha can likewise be used instead
of PDGFR-beta.
[0219] Antibodies can also be used as antagonists of PDGFR-.beta.,
including antibodies which bind to the receptor (e.g., 2A1E2 [U.S.
Pat. No. 7,060,271]; HuM4 Ts.22 [U.S. Pat. No. 5,882,644]; or 1B3
or 2C5 [U.S. Pat. No. 7,740,850]), and anti-PDGF antibodies such as
anti-PDGF BB, anti-PDGF-DD, anti-PDGF-BB and anti-PDGF-AB.
[0220] "VEGF" or "vascular endothelial growth factor" is a human
vascular endothelial growth factor that affects angiogenesis or an
angiogenic process. In particular, the term VEGF means any member
of the class of growth factors that (i) bind to a VEGF receptor
such as VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), or VEGFR-3 (FLT-4);
(ii) activates a tyrosine kinase activity associated with the VEGF
receptor; and (iii) thereby affects angiogenesis or an angeogenic
process.
[0221] The VEGF family of factors is made up of five related
glycoproteins: VEGF-A (also known as VPE), --B, --C, -D and PGF
(placental growth factor). Of these, VEGF-A is the most well
studied and is the target of anti-angiogenic therapy. Ferrara et
al, (2003) Nat. Med. 9:669-676. VEGF-A exists as a number of
different isotypes which are generated both by alternative splicing
and proteolysis: VEGF-A.sub.206, VEGF-A.sub.189, VEGF-A.sub.165,
and VEGF-A121. The isoforms differ in their ability to bind heparin
and non-signaling binding proteins called neuropilins. The isoforms
are all biologically active as dimers.
[0222] The various effects of VEGF are mediated by the binding of a
VEGF, e.g., VEGF-A (P15692), -B (P49766), -C (P49767) and -D
(Q43915), to receptor tyrosine kinases (RTKs). The VEGF family
receptors belong to class V RTKs and each carry seven lg-like
domains in the extracellular domain (ECD). In humans, VEGF binds to
three types of RTKs: VEGFR-1 (Flt-1) (P17948), VEGFR-2 (KDR, Flk-1)
(P935968) and VEGFR-3 (Flt-4) (P35916). A sequence of VEGFR-1 is
shown in FIG. 2. Unless otherwise apparent from the context
reference to a VEGF means any of VEGF-A, -B, -C, -D, and PGF, in
any of the natural isoforms or natural variants or induced variants
having at least 90, 95, 98 or 99% or 100% sequence identity to a
natural form. Preferably, such VEGFs are human VEGFs. Likewise
reference to a VEGFR means any of VEGR-1, R-2 or R-3, including any
natural isoform or natural variant, or an induced variant having at
least 90, 95, 98 or 99% or 100% sequence identity to a natural
sequences.
[0223] The extracellular region runs from about amino acid 27-758,
the transmembrane domain from about amino acid 759 to 780 and the
intracellular region from about 781-1338. The extracellular region
includes seven immunoglobulin-like domains, D1-D7. Domain 1 of
VEGFR-1 is from 32 (P) to 128 (I), Domain 2 from 134 (P) to 125
(Q), Domain 3 from 232 (V) to 331 (K), Domain 4 from 333 (F) to 428
(P), Domain 5 is from 431 (Y) to 553 (T), Domain 6 from 558 (G) to
656 (R) and Domain 7 from 662 (Y) to 751 (T). See generally U.S.
Pat. No. 8,273,353, incorporated herein by reference for all
purposes. The exact boundaries of the domains D1-D7 of VEGFR-1 can
vary depending on how the analysis is done. Preferably, the
boundaries vary by 9 amino acids or less. Typically they vary by 7
amino acids of less, more typically by 5 amino acids or less.
Usually, boundary variance is 3 amino acids or less. Most typically
the boundaries vary by only an amino acid.
[0224] The protein sequence of VEGFR-2 is shown below in FIG.
3.
[0225] The extracellular region runs from about residues 20-764,
the transmembrane domain from about residues 765-785 and the
intracellular domain from about residues 786 to 1356. The
extracellular region includes seven immunoglobulin-like domains,
D1-D7. Domain 1 of VEGFR-2 is from 32 (P) to 118 (V), Domain 2 is
from 124 (P) to 220 (G), Domain 3 is from 226 (V) to 327 (K),
Domain 4 is from 329 (F) to 421 (P), Domain 5 is from 424 (G) to
548 (T), Domain 6 is from 553 (I) to 662 (L), and Domain 7 is from
668 (T) to 757 (A). See generally U.S. Pat. No. 8,273,353,
incorporated herein by reference for all purposes. The exact
boundaries of the domains D1-D7 of VEGFR-2 can vary depending on
how the analysis is done. Preferably, the boundaries vary by 9
amino acids or less. Typically they vary by 7 amino acids of less,
more typically by 5 amino acids or less. Usually, boundary variance
be by 3 amino acids or less. Most typically the boundaries 1 vary
by only an amino acid.
[0226] The protein sequence of VEGFR-3 is shown below in FIG. 4.
The extracellular region runs from about residues 25-775, the
transmembrane domain from about residues 776-796 and the
intracellular domain from about residues 797-1363. The
extracellular domain includes seven immunoglobulin-like domains
D1-D7. Domain 1 of VEGFR-3 is from 30 (P) to 132 (V), Domain 2 is
from 138 (P) to 226 (G), Domain 3 is from 232 (I) to 330 (N),
Domain 4 is from 332 (F) to 422 (P), Domain 5 is from 425 (H) to
552 (T), Domain 6 is from 557 (G) to 673 (Q), and Domain 7 is from
679 (R) to 768 (S). See generally U.S. Pat. No. 8,273,353,
incorporated herein by reference for all purposes. The exact
boundaries of the domains D1-D7 of VEGFR-3 can vary depending on
how the analysis is done. Preferably, the boundaries vary by 9
amino acids or less. Typically they vary by 7 amino acids of less,
more typically by 5 amino acids or less. Usually, boundary variance
is 3 amino acids or less. Most typically the boundaries vary by
only an amino acid.
[0227] VEGFR-2 is expressed predominately on vascular endothelial
cells. VEGFR-1 is also expressed on the vascular endothelium, but
in addition is also expressed by a number of other cell types:
neutrophils, monocytes, macrophages, mural cells and endothelial
progenitor cells. VEGFR-1 has a higher affinity for VEGF-A than
does VEGFR-2. However, when VEGFR-1 is bound to VEGF-A in
endothelial cells, VEGFR-1 exhibits only very weak tyrosine
phosphorylation. Hence, it is believed that the effects of VEGF-A
(including its various isoforms) on the vascular endothelium are
mediated by the binding of VEGF-A to VEGFR-2.
[0228] PGF and VEGF-B bind only to VEGFR-1. PGF and VEGF-B have
been implicated in pathogenic vascular remodeling. Carmeliet P,
Moons L, Lutten A, et al. 2001. Synergism between vascular
endothelial growth factor and placental growth factor contributes
to angiogenesis and plasma extravasation in pathological
conditions. Nat Med. 7(5); 575-583. VEGF-C and -D bind with high
affinity to VEGFR-3, which is primarily found on lymphatic
endothelial cells in the adult. VEGF-C and -D are thought to play a
role in regard to Lymphangio genesis.
[0229] A "VEGF antagonist" or a molecule that "antagonizes VEGF" is
an agent that reduces, or inhibits, either partially or fully, an
activity of a VEGF including its ability to specifically bind to
its receptor a VEGFR and consequent cellular responses, such as
angiogenesis and cellular proliferation. VEGF antagonists include
antibodies specifically binding to a VEGF or a VEGFR or a VEGFR
extracellular trap segment.
[0230] The term extracellular trap segment refers to a full length
extracellular region or any portion thereof, or combination of
portions from different VEGFR receptors that can antagonize
signaling between at least one VEGF and VEGFR. Preferably, the
extracellular trap segment includes at least one domain from one of
VEGFR-1, -2 or -3 defined above, and more preferably at least two
contiguous domains, such as D2 and D3. Optionally, an extracellular
domain includes at least one domain as defined above from at least
two different VEGFRs. A preferred extracellular domain comprises or
consists essentially of D2 of VEGFR-1 and D3 of VEGFR-2.
[0231] VEGF antagonist therapies have been approved for the
treatment of certain cancers and wet AMD. Bevacizumab
(AVASTIN.RTM., Genentech/Roche) is a humanized mouse monoclonal
antibody that binds to and neutralizes human VEGF, in particular to
all isoforms of VEGF-A and to bioactive proteolytic fragments of
VEGF-A. See, e.g., Ferrara N, Hillan K J, Gerber H P, Novotny W.
2004. Discovery and development of bevacizumab, an anti-VEGF
antibody for treating cancer. Nat Rev Drug Discov. 3(5):391-400.
Bevacizumab has been approved for the treatment of certain cancers.
The protein sequence of the heavy and light chains of bevacizumab
(DrugBank DB00112) is shown below in FIG. 5 with CDRs underlined
(see also SEQ ID NOs. 2 and 5).
[0232] Bevacizumab variable light chain CDRs are CDR.sub.L1:
SASQDISNYLN, CDR.sub.L2: FTSSLHS and CDR.sub.L3: QQYSTVPWT.
Bevacizumab variable heavy chain CDRs are CDR.sub.H1: GYTFTNYGMN,
CDR.sub.H2: WINTYTGEPTY AADFKR, and CDR.sub.H3: YPHYYGSSHWYFDV.
CDRs are defined by Kabat except CDR.sub.H1 is the composite
Kabat/Chothia definition.
[0233] Another anti-VEGF molecule, derived from the same mouse
monoclonal antibody as bevacizumab has been approved as a treatment
for wet AMD: ranibizumab (LUCENTIS.RTM., Genentech/Roche).
Ranibizumab is an antibody fragment or Fab. Ranibizumab was
produced by affinity maturation of the variable heavy and light
chains of bevacizumab. The sequence of the heavy and light chains
of ranibizumab is shown below (as published by Novartis) in FIG. 6
(see also SEQ ID NOs. 12 and 13):
[0234] Ranibizumab variable light chain CDRs are CDR.sub.L1:
SASQDISNYLN, CDR.sub.L2: FTSSLHS and CDR.sub.L3: QQYSTVPWT.
Ranibizumab variable heavy chain CDRs are CDR.sub.H1: GYDFTHYGMN,
CDR.sub.H2: WINTYTGEPTYAADFKR, and CDR.sub.H3: YPYYYGTSHWYFDV.
[0235] Antibodies competing with bevacizumab for binding to VEGF-A
or binding to the same epitope on VEGF-A as bevacizumab can also be
used.
[0236] Another anti-VEGF therapy is a VEGF Trap. For example,
aflibercept (Eylea.RTM., Regeneron), consists of the second lg like
domain of VEGFR-1 and the third lg like domain of VEGFR-2 expressed
as an in line fusion with the constant region (Fe) of human IgG 1.
Papadopoulos N, et al. 2012. Binding and neutralization of vascular
endothelial growth factor (VEGF) and related ligands by VEGF Trap,
ranibizumab and bevacizumab. Angiogenesis 15:171-185. In theory,
aflibercept binds not only VEGF-A, but also VEGF-B and PGF thereby
antagonizing their interaction with VEGFR-1.
[0237] In accordance with the present invention, a dual VEGF/PDGF
antagonist is provided comprising a VEGF antagonist linked to a
PDGF antagonist. The linkage preferably includes a fusion of
protein chains to form a hybrid chain formed from components of
both antagonists. Alternatively, the components can be joined by
chemical cross linking. As an example, of linkage by fusion, if the
dual antagonist is formed from an antibody and an extracellular
trap segment, then a heavy or light chain of the antibody can be
fused to the extracellular trap segment. Preferably, the
extracellular trap segment is fused directly or indirectly via a
linker to the N-terminus of the antibody heavy or light chain.
Whichever chain is not fused to the extracellular trap segment can
associate with the chain that is in similar fashion to heavy light
chain association in a natural antibody. For example, an exemplary
format has an extracellular trap segment fused to the N-terminus of
an antibody heavy chain via a linker and the antibody light chain
complexed with the antibody heavy chain. The antibody in such a
dual antagonist can be an intact antibody or any of the binding
fragments described above, such as a Fab fragment. Preferably, in
such dual antagonists, the VEGF antagonist is an antibody to
VEGF-A, such as bevacizumab or ranibizumab, and the PDGF antagonist
is an extracellular trap segment from PDGR-1.
[0238] In an alternative format, the VEGF antagonist and PDGF
antagonist are both extracellular trap segments. The two segments
can be fused in either orientation with respect to one another,
directly or via a linker. That is the VEGFR extracellular trap
region can be joined to the N-terminus or the C-terminus of the
PDGFR extracellular trap region. The C-terminus of such a fusion
protein can be linked to an Fe region of an antibody forming an Fe
fusion proteins. In preferred embodiments, the PDGFR is
PDGFR-.beta. and the extracellular trap segment comprises one or
more of domains D1-D5 of PDGFR-.beta.. More preferably, the
extracellular trap segment comprises domains D1-D3 of PDGFR-.beta..
Still more preferably, the extracellular trap segment comprises or
consists of amino acids 33 to 314 of SEQ ID NO. 11. In preferred
embodiments, the VEGF antagonist is an anti-VEGF antibody,
preferably an anti-VEGF-A antibody.
[0239] In dual antagonists having antibody and extracellular trap
components fused to one another, the respective components,
typically the antibody heavy chain and the extracellular trap
segment are separated by a linker sequence. The linker is
preferably GGGGSGGGGS, GG, or GGGGSGGGGSGGGGSGGGGSG or an oligomers
of any of these. More preferably, the linker is GGGGSGGGGS.
[0240] In accordance with an aspect of the present invention, the
anti-VEGF-A antibody heavy chain has at least the following CDR
sequences: CDR.sub.H1: GYDFTHYGMN, CDR.sub.H2: WINTYTGEPTYAADFKR,
and CDR.sub.H3: YPYYYGTSHWYFDV. Preferably, the anti-VEGF-A light
chain has at least the following CDRs: CDR.sub.L2: SASQDISNYLN,
CDR.sub.L2: FTSSLHS and CDR.sub.L2: QQYSTVPWT. In the case of the
anti-VEGF-A antibody heavy chain, it is preferred that its isotype
is IgG 1 and has a CH.sub.1, hinge, CH.sub.2 and CH.sub.3 domains.
It is also preferred that the light chain isotype is kappa. The
constant region of the preferred IgG 1 sequence is set forth in SEQ
ID NO. 17. The sequence of the light chain constant region is
preferably set forth in SEQ ID NO. 18.
[0241] The IgG 1 domain of the anti-VEGF-A antibody preferably has
one or more mutations to reduce or lower effector function.
Preferred amino acids to use for effector function reducing
mutations include (EU numbering) E233P, L234V, L235, G236, G237,
delG236, D270A, K322A, A327G, P329A, A330, A330S, P331S, and P331A,
in which the second mentioned amino acid is the mutation.
Preferably, the mutations include one or more of the following:
E233P, L234V, L234A, L235A, G237A, A327G, A330S and P331S (EU
numbering). More preferably, the anti-VEGF-A heavy chain has the
following mutations: L234A, L235A and G237A. The number of such
mutations relative to a natural human IgG 1 sequence is no more
than 10, and preferably no more than 5, 4, 3, 2 or 1.
[0242] Alternatively, the IgG domain can be IgG2, IgG3 or IgG4,
preferably, human IgG2, IgG3 or IgG4, or a composite in which a
constant regions is formed from more than one of these isotypes
(e.g., CH1 region from IgG2 or IgG4, hinge, CH2 and CH3 regions
from IgG1). Such domains can contain mutations to reduce effector
function at one or more of the EU position mentioned for IgG 1.
Human IgG2 and IgG4 have reduced effector functions relative to
human IgG 1 and IgG3.
[0243] The anti-VEGF-A heavy chain can also contain a cysteine
residue added as a mutation by recombinant DNA technology which can
be used to conjugate a half-life extending moiety. Preferably, the
mutation is (EU numbering) Q347C and/or L443C. More preferably, the
mutation is L443C. Preferably, the stoichiometry of dual antagonist
to polymer is 1:1; in other words, a conjugate consists essentially
of molecules each comprising one molecule of dual antagonist
conjugated to one molecule of polymer.
[0244] A preferred dual antagonist including an antibody to VEGF-A
and a PDGFR extracellular trap segment comprises a fusion protein
of the antibody heavy chain and the PDGFR extracellular trap
segment having the amino acid sequence of SEQ ID NO. 9 and the
antibody light chain having the amino acid sequence of SEQ ID NO.
10, or variants thereof including sequences differing each of from
SEQ ID NO: 9 and 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
amino acids.
[0245] In another aspect of the present invention, a dual VEGF/PDGF
antagonist is presented having a PDGF antagonist constituting one
or more segments of a PDGFR as described above and a VEGF
antagonist constituting an anti-VEGF Fab fragment. For this aspect
of the present invention, the PDGFR extracellular trap comprises
one or more of domains D1-D5 of PDGFR-.beta.. More preferably, the
PDGFR trap constitutes domains D1-D3 of PDGFR--More preferably, the
PDGFR trap is amino acids 33 to 314 of SEQ ID NO. 11.
[0246] The PDGFR trap is preferably located C-terminal of the Fab
heavy or light chain. The PDGFR trap is also preferentially located
N-terminal of the Fab heavy or light chain. Preferably, the dual
antagonist includes an anti-VEGF-A Fab fragment heavy chain fused
via a linker to a PDGFR extracellular trap segment and an
anti-VEGF-A light chain.
[0247] In another aspect of the invention, a dual VEGF/PDGF
antagonist is presented wherein the extracellular trap segment
binds to one or more of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and
PDGF-DD. Preferably, the extracellular trap binds PDGF-AB, PDGF-BB
and PDGF-DD. Still more preferably, the extracellular trap inhibits
PDGF-AB, PDGF-BB and PDGF-DD from binding to any one of
PDGFR-.alpha..alpha., PDGFR-.alpha..beta., and PDGFR-.beta..beta.
receptors.
[0248] A linker is preferably located between the PDGFR trap and
the anti-VEGF Fab fragment heavy chain. Preferably, the linker is
selected from group consisting of GGGGSGGGGS, GG, and
GGGGSGGGGSGGGGSGGGGSG, and oligomers of any of these. More
preferably, the linker is GGGGSGGGGS.
[0249] The anti-VEGF Fab fragment heavy chain preferably has at
least the following CDRs: CDR.sub.H1: GYDFTHYGMN, CDR.sub.H2:
WINTYTGEPTYAADFKR, and CDR.sub.H3:
[0250] YPYYYGTSHWYFDV. The anti-VEGF-A light chain preferably has
at least the following CDRs: CDR.sub.L2: SASQDISNYLN, CDR.sub.L2:
FTSSLHS and CDR.sub.L3: QQYSTVPWT.
[0251] A preferred anti-VEGF Fab fragment heavy chain isotype is
IgG 1 and comprises a CH.sub.1 domain and the light chain isotype
is kappa.
[0252] The dual VEGF/PDGF antagonist can have a half-life extending
moiety attached. Preferably the half-life extending moiety is a
zwitterionic polymer but PEG or other half-life extenders discussed
below can alternatively be used. More preferably, the zwitterionic
polymer is formed of monomers having a phosphorylcholine group.
Preferably the monomer is
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate. More
preferably, the monomer is
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate
(HEMA-PC).
[0253] A polymer conjugated to a dual antagonist preferably has at
least 2 and more preferably 3 or more arms. Some polymers have 2,
3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms. Still more preferably the
polymer has 3, 6 or 9 arms. Most preferably, the polymer has 9
arms. Preferably, the polymer peak molecular weight is between
300,000 and 1,750,000 Da. More preferably, the polymer has a peak
molecular weight between 500,000 and 1,000,000 Da. Still more
preferably, the polymer has a peak molecular weight between 600,000
to 800,000 Da.
[0254] The polymer can be covalently bonded to the dual antagonist
via conjugation. Preferably, the polymer is conjugated to the dual
VEGF/PDGF antagonist via a group such as an amino group, a hydroxyl
group, a sulfbydryl group or a carboxyl group. The sulfbydryl group
can be from a naturally occurring cysteine residue. The sulfbydryl
group can also be from a cysteine residue added by recombinant DNA
technology.
[0255] In a preferred aspect of the present invention, the polymer
is conjugated to the cysteine residue at position 731 of SEQ ID NO.
9, or aligned position of any variants of SEQ ID NO: 9 disclosed
herein.
[0256] In another aspect of the present invention, a dual VEGF/PDGF
antagonist having a VEGFR trap containing one or more extracellular
segments of a VEGFR, such as VEGFR-1, VEGFR-2 or VEGFR-3, fused to
an anti-PDGF antibody or Fab fragment heavy or light chain and an
anti-PDGF antibody or Fab fragment heavy or light chain not
included in fusion.
[0257] In accordance with an aspect of the present invention, the
extracellular segment of VEGFR is preferably one or more of domains
D1-D7. More preferably, the extracellular segment comprises D2 from
VEGFR-1 and D3 from VEGFR-2. Still more preferably, the D2 is
N-terminal to the D3 and further comprises a linker between the
domains.
[0258] In preferred embodiments of this aspect of the present
invention, the PDGF antagonist is an antibody. More preferably, the
antibody is selected from the group consisting of humanized 2A 1E2,
HuM4 Ts.22, humanized 1B3, humanized 2C5, anti-PDGF-BB,
anti-PDGF-DD, anti-PDGF-BB and anti-PDGF-AB. The PDGF antagonist is
also preferably a Fab fragment.
[0259] In accordance with this aspect of the present invention, the
antibody heavy chain is preferably IgG 1, more preferably human IgG
1 and the light chain is preferably kappa, human kappa. The heavy
chain can have a cysteine added via recombinant DNA technology.
Preferably, the cysteine is selected from the group consisting of
Q347C and L443C. Preferably, there is a half-life extending moiety
conjugated to the cysteine.
[0260] Preferably, the half-life extending moiety is a zwitterionic
polymer having one or more monomer units and wherein at least one
monomer unit has a zwitterionic group. Preferably, the zwitterionic
group is phosphorylcholine. The monomer is preferably
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate. More
preferably, the monomer is
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate
(HEMA-PC).
[0261] In accordance with this aspect of the present invention, the
polymer preferably has at least 2 and more preferably 3 or more
arms. Some polymers have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms.
Still more preferably the polymer has 3, 6 or 9 arms. Most
preferably, the polymer has 9 arms. In accordance with an aspect of
the present invention, the polymer peak molecular weight of between
300,000 and 1,750,000 Da. More preferably, the polymer has a peak
molecular weight between 500,000 and 1,000,000 Da. Still more
preferably, the polymer has a peak molecular weight between 600,000
to 800,000 Da.
[0262] In accordance with an aspect of the present invention, the
polymer is covalently bound to the polymer via conjugation.
Preferably, the polymer is conjugated to the dual VEGF/PDGF
antagonist via a group selected from the group consisting of an
amino group, a hydroxyl group, a sulfhydryl group and a carboxyl
group. Preferably, the sulfhydryl group is from a naturally
occurring cysteine residue. In other preferred embodiments, the
sulfhydryl group is from a cysteine residue added by recombinant
DNA technology.
[0263] In preferred aspects of the present invention, the PDGF
trap-VEGF trap is conjugated to a half-life extending moiety as
discussed with other dual antagonists.
[0264] Preferably, the half-life extending moiety is a zwitterionic
polymer having one or more monomer units and wherein at least one
monomer unit has a zwitterionic group. Preferably, the zwitterionic
group is phosphorylcholine. The monomer is preferably
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate. More
preferably, the monomer is
2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate
(HEMA-PC).
[0265] In accordance with this aspect of the present invention, the
polymer preferably has at least 2 and more preferably 3 or more
arms. Some polymers have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms.
Still more preferably the polymer has 3, 6 or 9 arms. Most
preferably, the polymer has 9 arms. In accordance with an aspect of
the present invention, the polymer peak molecular weight of between
300,000 and 1,750,000 Da. More preferably, the polymer has a peak
molecular weight between 500,000 and 1,000,000 Da. Still more
preferably, the polymer has a peak molecular weight between 600,000
to 800,000 Da.
[0266] In accordance with an aspect of the present invention, the
polymer is covalently bound to the polymer via conjugation.
Preferably, the polymer is conjugated to the dual VEGF/PDGF
antagonist via a group such as an amino group, a hydroxyl group, a
sulfbydryl group or a carboxyl group. In some conjugates, the
sulfbydryl group is from a naturally occurring cysteine residue. In
some conjugates, the sulfbydryl group is from a cysteine residue
added by recombinant DNA technology.
[0267] Dual PDGF/VEGF antagonists can be produced by recombinant
expression including the production of recombinant DNA by genetic
engineering, (ii) introducing recombinant DNA into prokaryotic or
eukaryotic cells by, for example and without limitation,
transfection, electroporation or microinjection, (iii) cultivating
the transformed cells, (iv) expressing dual antagonists, e.g.
constitutively or on induction, and (v) isolating the dual
antagonist, e.g. from the culture medium or by harvesting the
transformed cells, in order to (vi) obtain purified dual
antagonist.
[0268] Dual antagonists can be produced by expression in a suitable
prokaryotic or eukaryotic host system characterized by producing a
pharmacologically acceptable dual antagonist molecule. Examples of
eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293,
BHK, SK-Hip, and HepG2. Other suitable expression systems are
prokaryotic (e.g., coli with pET/BL21 expression system), yeast
(Saccharomyces cerevisiae and/or Pichia pastoris systems), and
insect cells.
[0269] A wide variety of vectors can be used for the preparation of
the dual antagonist and are selected from eukaryotic and
prokaryotic expression vectors. Examples of vectors for prokaryotic
expression include plasmids such as, and without limitation,
preset, pet, and pad, wherein the promoters used in prokaryotic
expression vectors include one or more of, and without limitation,
lac, trc, trp, recA, or araBAD. Examples of vectors for eukaryotic
expression include: (i) for expression in yeast, vectors such as,
and without limitation, pAO, pPIC, pYES, or pMET, using promoters
such as, and without limitation, AOX 1, GAP, GALl, or AUG 1; (ii)
for expression in insect cells, vectors such as and without
limitation, pMT, pAc5, pB, pMIB, or pBAC, using promoters such as
and without limitation PH, p 10, MT, Ac5, OpIE2, gp64, or polh, and
(iii) for expression in mammalian cells, vectors such as, and
without limitation, pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and
vectors derived from, in one aspect, viral systems such as and
without limitation vaccinia virus, adeno-associated viruses, herpes
viruses, or retroviruses, using promoters such as and without
limitation CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and beta-actin.
[0270] The half-life of dual antagonists can be extended by
attachment of a "half-life extending moieties" or "half-life
extending groups," which terms are herein used interchangeably to
refer to one or more chemical groups attached to one or more amino
acid site chain functionalities such as --SH, --OH, --COOH,
--CONH2, --NH2, or one or more N- and/or 0-glycan structures and
that can increase in vivo circulatory half-life of
proteins/peptides when conjugated to these proteins/peptides.
Examples of half-life extending moieties include polymers described
herein, particularly those of zwitterionic monomers, such as
HEMA-phosphorylcholine, PEG, biocompatible fatty acids and
derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g. Hydroxy Ethyl
Starch (HES), Poly Ethylene Glycol (PEG), Poly (Glyx-Sery) (HAP),
Hyaluronic acid (HA), Heparosan polymers (HEP), Fleximers, Dextran,
Poly-sialic acids (PSA), Fe domains, Transferrin, 25 Albumin,
Elastin like (ELP) peptides, XTEN polymers, PAS polymers, PA
polymers, Albumin binding peptides, CTP peptides, FcRn binding
peptides and any combination thereof.
[0271] In one embodiment a half-life extending moiety can be
conjugated to a dual antagonist via free amino groups of the
protein using N-hydroxysuccinimide (NHS) esters. Reagents targeting
conjugation to amine groups can randomly react to E-amine group of
lysines, a-amine group of N-terminal amino acids, and 8-amine group
of histidines.
[0272] However, dual antagonists of the present have many amine
groups available for polymer conjugation. Conjugation of polymers
to free amino groups, thus, might negatively impact the ability of
the dual antagonist proteins to bind to VEGF and/or PDGF.
[0273] In another embodiment, a half-life extending moiety is
coupled to one or more free SH groups using any appropriate
thiol-reactive chemistry including, without limitation, maleimide
chemistry, or the coupling of polymer hydrazides or polymer amines
to carbohydrate moieties of the dual antagonist after prior
oxidation. The use of maleimide coupling is a particularly
preferred embodiment of the present invention. Coupling preferably
occurs at cysteines naturally present or introduced via genetic
engineering.
[0274] Polymers are preferably covalently attached to cysteine
residues introduced into dual antagonist by site directed
mutagenesis. It is particularly preferred to employ cysteine
residues in the Fe portion of the dual antagonist. For preferred
sites to introduce cysteine residues into an Fe region see WO
2013/093809, U.S. Pat. No. 7,521,541, WO 2008/020827, U.S. Pat.
Nos. 8,008,453, 8,455,622 and US2012/0213705, incorporated herein
by reference for all purposes. Particularly preferred cysteine
mutations are Q347C and L443C referring to the human IgG heavy
chain by EU numbering.
[0275] The invention provides conjugates of dual antagonist and
high MW polymers serving as half-life extenders. A preferred
conjugate comprises a dual antagonist is coupled to a zwitterionic
polymer wherein the polymer is formed from one or more monomer
units and wherein at least one monomer unit has a zwitterionic
group. Preferably, the zwitterionic group is phosphorylcholine.
[0276] Preferably, one of the monomer units is
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate or
2-(methacryloyloxyethyl)-2' (trimethylammoniumethyl) phosphate
(HEMA-PC). In other preferred embodiments, polymer is synthesized
from a single monomer which is preferably
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate or
2-(methacryloyloxyethyl)-2' (trimethylammoniumethyl) phosphate.
[0277] Some dual antagonist conjugates have 2 or more preferably 3
or more polymer arms wherein the monomer is HEMA-PC. Preferably,
the conjugates have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer
arms wherein the monomer is HEMA-PC. More preferably, the
conjugates have 3, 6 or 9 arms. Most preferably, the conjugate has
9 arms.
[0278] Polymer-dual antagonist conjugates preferably have a polymer
portion with a molecular weight of between 100,000 and 1,500,000
Da. More preferably the conjugate has a polymer portion with a
molecular weight between 500,000 and 1,000,000 Da. Still more
preferably the conjugate has a polymer portion with a molecular
weight between 600,000 to 800,000 Da. Most preferably the conjugate
has a polymer portion with a molecular weight between 600,000 and
850,000 Da and has 9 arms. When a molecular weight is given for a
dual VEGF/PDGF antagonist conjugated to a polymer, the molecular
weight will be the addition of the molecular weight of the protein,
including any carbohydrate moieties associated therewith, and the
molecular weight of the polymer.
[0279] In accordance with an aspect of the present invention, a
dual VEGF/PDGF antagonist having a HEMA-PC polymer which has a
molecular weight measured by Mw of between about 100 kDa and 1500
kDa. More preferably, the molecular weight of the polymer as
measured by Mw is between about 500 kDa and 1000 kDa. Still more
preferably, the molecular weight of the polymer as measured by Mw
is between about 600 kDa to about 900 kDa. Most preferably, the
polymer molecular weight as measured by Mw is 750 kDa plus or minus
15%.
[0280] In this aspect of the present invention, the polymer is
preferably made from an initiator suitable for ATRP having one or
more polymer initiation sites. Preferably, the polymer initiation
site has a 2-bromoisobutyrate site. Preferably, the initiator has 3
or more polymer initiation sites. More preferably, the initiator
has 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polymer initiation sites.
More preferably, the initiator has 3, 6 or 9 polymer initiation
sites. Still more preferably, the initiator has 9 polymer
initiation sites. Most preferably, the initiator is OG 1786.
[0281] The invention provides methods for synthesizing a
zwitterionic polymer-dual antagonist conjugate, the conjugate
having one or more functional agents and one or more polymer arms
wherein each of the polymer arms has one or more monomer units
wherein at least one of the units has a zwitterion. The method can
have the steps of [0282] a. providing an initiator having one or
more sites for monomer polymerization and a first linker having an
amine group wherein the initiator is a trifluoro acetic acid salt;
[0283] b. providing one or more monomers suitable for
polymerization wherein at least one of the monomers is
zwitterionic; [0284] c. reacting the monomers with the initiator to
form one or more polymer arms each corresponding to the sites for
monomer polymerization to provide an initiator-polymer conjugate
having the first linker with the amine group; [0285] d. providing a
second linker having at least second and third reactive groups;
[0286] e. coupling one of the second and third reactive groups of
the second linker to the amine group of the first linker of the
initiator-polymer conjugate to provide a linker-initiator-polymer
conjugate having one or more reactive groups that were not used in
the coupling step; and [0287] f. coupling one or more functional
agents to one or more of the unreacted reactive groups of the
linker-initiator-polymer moiety to provide the polymer-functional
agent conjugate.
[0288] Prior to the instant invention, the initiator molecule or
entity had to contain a deprotectable functional group that would
allow coupling of the functional agent. An example of such an
initiator having a protected maleimide is shown below:
##STR00004##
[0289] After polymer synthesis, the protected maleimide is
deprotected with heat to allow for generation of maleimide which
could be used to couple functional agent. If one wanted to vary the
nature of the chemical entity in between the maleimide and the
polymer initiation site, one would have to synthesize an entire new
initiator.
[0290] Each time the initiator is changed or altered in any way, a
new scaled up synthesis procedure would have to be developed. Each
change in the nature of the initiator molecule can have a wide
range of effects on polymer synthesis. However, in accordance with
the present invention, a method is presented where the conjugation
group (e.g. maleimide) is added after polymer synthesis. This is
sometimes referred to as a "snap-on strategy" or "universal polymer
strategy. A single initiator moiety can be used for large scale
polymer and bioconjugate discovery and development. Thus,
conditions can be developed for scaled up optimal polymer
synthesis. Such polymer can then be adapted to various types of
functional agents by "snapping-on" various types of linkers and
functional conjugation chemistries.
[0291] For example, if it is desired to conjugate a larger
functional agent to a polymer of the instant invention such as an
antibody of even a Fab fragment, a longer linker sequence can be
snapped on to the polymer. In contrast, smaller functional agents
may call for relatively shorter linker sequences.
[0292] In preferred embodiments of the methods, the initiator has
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 sites for polymer
initiation. Preferably, the initiator has 3, 6 or 9 sites for
polymer initiation.
[0293] In accordance with an aspect of the present invention, a
second linker has second, third, fourth, fifth, and sixth reactive
groups. More preferably, a second linker has just second and third
reactive groups.
[0294] In accordance with an aspect of the present invention, each
polymer arm has from about 20 to about 2000 monomer units.
Preferably, each arm has from about 100 to 500 monomer units or
from about 500 to 1000 monomer units or from about 1000 to 1500
monomer units or from about 1500 to 2000 monomer units.
[0295] In accordance with an aspect of the present invention, the
peak molecular weight of the polymer-functional agent conjugate is
about 100,000 to 1,500,000 Da. Preferably, the peak molecular
weight of the polymer-functional agent conjugate is about 200,000
to about 300,000 Da, about 400,000 to about 600,000 Da or about
650,000 to about 850,000 Da.
[0296] In accordance with another aspect of the present invention,
the first linker is preferably alkyl, substituted alkyl, alkylene,
alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether,
alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene,
heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylene-oxy,
heteroaryl, amino, amido or any combination thereof. More
preferably, the first linker has the formula:
##STR00005##
wherein m is 1 to 10. More preferably, the first linker has the
above formula and m is 4.
[0297] In still other aspects of the present invention, the
initiator preferably includes a structure selected from group
consisting of
##STR00006##
[0298] In preferred embodiments of the present invention, the
monomer is selected from the group consisting of
##STR00007##
wherein R7 is H or C.sub.1-6 alkyl and t is 1 to 6.
[0299] More preferably, the monomer is selected from the group
consisting of 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)
phosphate (HEMA-PC) and
2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate.
[0300] Most preferably, the monomer is 2-(methacryloyloxyethyl)-2'
(trimethylammoniumethyl) phosphate.
[0301] The second linker moiety preferably comprises an activated
ester having the structure
##STR00008##
wherein R8 is selected from the group consisting of
##STR00009##
and R9 is
##STR00010##
[0302] wherein p is 1 to 12.
[0303] In more preferred embodiments of the present invention, the
polymer has 9 arms, m of R2 is 2-4, R9 is
##STR00011##
and p is 4 to 15. Still more preferably, m is 4 and p is 12.
[0304] When a polymer is to be conjugated via a cysteine (or other
specified residue), the polymer can be linked directly or
indirectly to the residue (e.g., with an intervening initiator, and
or spacer or the like).
[0305] Dual antagonists can be incorporated into a pharmaceutical
composition with a pharmaceutically acceptable excipient.
Pharmaceutical compositions adapted for oral administration may be
presented as discrete units such as capsules, as solutions, syrups
or suspensions (in aqueous or non-aqueous liquids; or as edible
foams or whips; or as emulsions). Suitable excipients for tablets
or hard gelatine capsules include lactose, maize starch or
derivatives thereof, stearic acid or salts thereof. Suitable
excipients for use with soft gelatine capsules include for example
vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For
the preparation of solutions and syrups, excipients which may be
used include for example water, polyols and sugars. For the
preparation of suspensions oils (e.g. vegetable oils) may be used
to provide oil-in-water or water in oil suspensions.
[0306] Pharmaceutical compositions can be adapted for nasal
administration wherein the excipient is a solid include a coarse
powder having a particle size for example in the range 20 to 500
microns which is administered in the manner in which snuff is
taken, i.e. by rapid inhalation through the nasal passage from a
container of the powder held close up to the nose. Suitable
compositions wherein the excipient is a liquid, for administration
as a nasal spray or as nasal drops, include aqueous or oil
solutions of the active ingredient. Pharmaceutical compositions
adapted for administration by inhalation include fine particle
dusts or mists which may be generated by means of various types of
metered dose pressurized aerosols, nebulizers or insufflators.
[0307] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solution which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation substantially isotonic
with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents. Excipients which may be used for injectable
solutions include water, alcohols, polyols, glycerine and vegetable
oils, for example. The compositions may be presented in unit-dose
or multi-dose containers, for example sealed ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carried, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets. Pharmaceutical
compositions can be substantially isotonic, implying an osmolality
of about 250-400 mOsm/kg water.
[0308] The pharmaceutical compositions may contain preserving
agents, solubilizing agents, stabilizing agents, wetting agents,
emulsifiers, sweeteners, colorants, odorants, salts (substances of
the present invention may themselves be provided in the form of a
pharmaceutically acceptable salt), buffers, coating agents or
antioxidants. They may also contain therapeutically active agents
in addition to the substance of the present invention. The
pharmaceutical compositions of the invention may be employed in
combination with one or more pharmaceutically acceptable
excipients. Such excipients may include, but are not limited to,
saline, buffered saline (such as phosphate buffered saline),
dextrose, liposomes, water, glycerol, ethanol and combinations
thereof.
[0309] The dual antagonists and pharmaceutical compositions
containing them may be administered in an effective regime for
treating or prophylaxis of a patient's disease including, for
instance, administration by oral, intravitreal, intravenous,
subcutaneous, intramuscular, intraosseous, intranasal, topical,
intraperitoneal, and intralesional administration. Parenteral
infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration or routes among
others. In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic or
substantially isotonic.
[0310] For administration to mammals, and particularly humans, it
is expected that the dosage of the active agent is from 0.01 mg/kg
body weight, typically around 1 mg/kg. The physician can determine
the actual dosage most suitable for an individual which depends on
factors including the age, weight, sex and response of the
individual, the disease or disorder being treated and the age and
condition of the individual being treated. The above dosages are
exemplary of the average case. There can, of course, be instances
where higher or lower dosages are merited.
[0311] This dosage may be repeated as often as appropriate (e.g.,
weekly, fortnightly, monthly, quarterly). If side effects develop
the amount and/or frequency of the dosage can be reduced, in
accordance with normal clinical practice. In one embodiment, the
pharmaceutical composition may be administered once every one to
thirty days.
[0312] The dual antagonists and pharmaceutical compositions of the
invention can be employed alone or in conjunction with other
compounds, such as therapeutic compounds or molecules, e.g.
anti-inflammatory drugs, analgesics or antibiotics. Such
administration with other compounds may be simultaneous, separate
or sequential. The components may be prepared in the form of a kit
which may comprise instructions as appropriate.
[0313] The dual antagonists and pharmaceutical compositions
disclosed herein can be used for treatment or prophylaxis of
disease, particularly the ocular diseases or conditions described
herein. Although both antagonist modalities within the dual
antagonist are believed to contribute to efficacy as discussed
above and shown in Example 40 an understanding of mechanism is not
required for practice of the invention. Preferably, a dual
antagonist is more effective than an equimolar concentration of
each antagonist administered alone, or a 1:1 combination of the
antagonists administered as separate molecules.
[0314] So used, the conjugates are typically formulated for and
administered by ocular, intraocular, and/or intravitreal injection,
and/or juxtascleral injection, and/or subretinal injection and/or
subtenon injection, and/or superchoroidal injection and/or topical
administration in the form of eye drops and/or ointment. Such dual
antagonists and compositions can be delivered by a variety of
methods, e.g. intravitreally as a device and/or a depot that allows
for slow release of the compound into the vitreous, including those
described in references such as Intraocular Drug Delivery, Jaffe,
Ashton, and Pearson, editors, Taylor & Francis (March 2006). In
one example, a device may be in the form of a minipump and/or a
matrix and/or a passive diffusion system and/or encapsulated cells
that release the compound for a prolonged period of time
(Intraocular Drug Delivery, Jaffe, Ashton, and Pearson, editors,
Taylor & Francis (March 2006).
[0315] Formulations for ocular, intraocular or intravitreal
administration can be prepared by methods and using ingredients
known in the art. A main requirement for efficient treatment is
proper penetration through the eye. Unlike diseases of the front of
the eye, where drugs can be delivered topically, retinal diseases
require a more site-specific approach. Eye drops and ointments
rarely penetrate the back of the eye, and the blood-ocular barrier
hinders penetration of systemically administered drugs into ocular
tissue. Accordingly, usually the method of choice for drug delivery
to treat retinal disease, such as AMD and CNV, is direct
intravitreal injection. Intravitrial injections are usually
repeated at intervals which depend on the patient's condition, and
the properties and half-life of the drug delivered.
[0316] Therapeutic dual agonists and related conjugates according
to the present invention generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle. Such compositions may also be supplied in the form of
pre-filled syringes.
[0317] A "stable" formulation is one in which the protein or
protein conjugated to a polymer of other half-life extending moiety
therein essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. By "stable" is
also meant a formulation which exhibits little or no signs of
instability, including aggregation and/or deamidation. For example,
in accordance with an aspect of the present invention, the
formulations provided by the present invention may remain stable
for at least two year, when stored as indicated at a temperature of
5-8.degree. C.
[0318] Various analytical techniques for measuring protein
stability are available in the art and are reviewed in Peptide and
Protein Drug Delivery, 247-301 (Vincent Lee ed., New York, N.Y.,
1991) and Jones, 1993 Adv. Drug Delivery Rev. 10: 29-90, for
examples. Stability can be measured at a selected temperature for a
selected time period. Storage of stable formulations is preferably
for at least 6 months, more preferably 12 months, more preferably
12-18 months, and more preferably for 2 or more years.
[0319] A protein, such as an antibody or fragment thereof, "retains
its physical stability" in a pharmaceutical formulation if it shows
no signs of aggregation, precipitation, deamidation and/or
denaturation upon visual examination of color and/or clarity, or as
measured by UV light scattering or by size exclusion
chromatography.
[0320] A protein "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the protein is considered to still retain its
biological activity. Chemical stability can be assessed by
detecting and quantifying chemically altered forms of the protein.
Chemical alteration may involve size modification (e.g., clipping),
which can be evaluated using size exclusion chromatography,
SDS-PAGE and/or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for
examples. Other types of chemical alteration include charge
alteration (e.g., occurring as a result of deamidation), which can
be evaluated by ion-exchange chromatography, for example. An
antibody "retains its biological activity" in a pharmaceutical
formulation, if the biological activity of the antibody at a given
time is within about 10% (within the errors of the assay) of the
biological activity exhibited at the time the pharmaceutical
formulation was prepared as determined in an antigen binding assay,
for example.
[0321] A protein-polymer conjugate "retains its chemical stability"
the chemical bond between the protein and the polymer is maintained
intact, e.g., it is not hydrolyzed or otherwise disrupted. The
protein part of the conjugate retains its chemical stability as
described above.
[0322] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood or the
vitreous for intravitreal injections. Isotonic formulations will
generally have an osmotic pressure from about 250 to 400 mOsm.
Isotonicity can be measured using a vapor pressure or ice-freezing
type osmometer, for example.
[0323] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention has a pH in the range of
preferably from about 3.0 to about 8.0; for example from about 4.5
to 8; or about pH 6 to about 7.5; or about 6.0 to about 7.0, or
about 6.5-7.0, or about pH 7.0 to about 7.5; or about 7.1 to about
7.4. A pH of any point in between the above ranges is also
contemplated.
[0324] "PBS" phosphate buffered saline, Tris based buffers and
histidine based buffers are particularly preferred buffers for the
instantly invented dual antagonists. In the case of OG 1448, PBS is
particularly preferred. More preferably, in the case of OG 1448,
the PBS buffer has a pH of 7-8 and the concentration of OG 1448 is
from about 10 mg/ml to about 100 mg/ml. Still more preferably, the
OG 1448 is from about 25 to about 65 mg/ml and the pH is about 7.4.
In the most preferred embodiments of the present invention, the
concentration of OG 1448 is 50 mg/ml to 60 mg/ml.
[0325] In preferred embodiments of the present invention, the PBS
buffer is made up of at least Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4
and NaCl adjusted so as to provide the appropriate pH. In
particularly preferred embodiments of the present invention, the
buffer may contain other pharmaceutical excipients such as KCl and
other salts, detergents and/or preservatives so as to provide a
stable storage solution.
[0326] A "preservative" is a compound which can be included in the
formulation to essentially reduce bacterial action therein, thus
facilitating the production of a multi-use formulation, for
example. Examples of potential preservatives include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a mixture of alkylbenzyldimethylammonium
chlorides in which the alkyl groups are long-chain compounds), and
benzethonium chloride. Other types of preservatives include
aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl
parabens such as methyl or propyl paraben, catechol, resorcinol,
cyclohexanol, 3-pentanol, and m-cresol.
[0327] In accordance with an aspect of the present invention,
formulations of dual PDGF/VEGF antagonists according to the present
invention to be safe for human use or for animal testing must have
sufficiently low levels of endotoxin. "Endotoxin" is
lipopolysaccharide (LPS) derived from the cell membrane of
Gram-negative bacteria. Endotoxin is composed of a hydrophilic
polysaccharide moiety covalently linked to a hydrophobic lipid
moiety (lipid A). Raetz C R, Ulevitch R J, Wright S D, Sibley C H,
Ding A, Nathan C F. 1991. Gram-negative endotoxin: an extraordinary
lipid with profound effects on eukaryotic signal transduction.
FASEB J. 5(12):2652-2660. Lipid A is responsible for most of the
biological activities of endotoxin, i.e., its toxicity. Endotoxins
are shed in large amount upon bacterial cell death as well as
during growth and division. They are highly heat-stable and are not
destroyed under regular sterilizing conditions. Extreme treatments
with heat or pH, e.g., 180-250.degree. C. and over 0.1 M of acid or
base must be used (Petsch D, Anspach F. 2000. Endotoxin removal
from protein solutions. J Biotechnol. 76: 97-119). Such conditions
of course would be highly detrimental to biological drugs.
[0328] In the biotech and pharmaceutical industries, it is possible
to find endotoxin during both production processes and in final
products. As bacteria can grow in nutrient poor media, including
water, saline and buffers, endotoxins are prevalent unless
precautions are taken. Endotoxin injection into an animal or human
causes a wide variety of pathophysiological effects, including
endotoxin shock, tissue injury and even death. Ogikubo Y, Ogikubo
Y, Narimatsu M, Noda K, Takahashi J, Inotsume M, Tsuchiya M, Tamura
Y. 2004. Evaluation of the bacterial endotoxin test for
quantifications of endotoxin contamination of porcine vaccines.
Biologics 32:88-93.
[0329] Pyrogenic reactions and shock are induced in mammals upon
intravenous injection of endotoxin at low concentrations (1 ng/mL)
(Fiske J M, Ross A, VanDerMeid RK, McMichael J C, Arumugham. 2001.
Method for reducing endotoxin in Moraxella catarrhalis UspA2
protein preparations. J Chrom B. 753:269-278). The maximum level of
endotoxin for intravenous applications of pharmaceutical and
biologic product is set to 5 endotoxin units (EU) per kg of body
weight per hour by all pharmacopoeias (Daneshiam M, Guenther A,
Wendel A, Hartung T, Von Aulock S. 2006. In vitro pyrogen test for
toxic or immunomodulatory drugs. J Immunol Method 313: 169-175). EU
is a measurement of the biological activity of an endotoxin. For
example, 100 pg of the standard endotoxin EC-5 and 120 pg of
endotoxin from Escherichia coli 0111:B4 have activity of 1 EU
(Hirayama C, Sakata M. 2002. Chromatographic removal of endotoxin
from protein solutions by polymer particles. J Chrom B
781:419-432). Meeting this threshold level has always been a
challenge in biological research and pharmaceutical industry
(Berthold W, Walter J. 1994. Protein Purification: Aspects of
Processes for Pharmaceutical Products. Biologicals 22: 135-150;
Petsch D, Anspach F B. 2000. Endotoxin removal from protein
solutions. J Biotech 76:97-119).
[0330] The presence of endotoxin in drugs to be delivered via
intravitreal injection is of particular concern. Intravitreal
injection of drug (penicillin) was first performed in 1945 by
Rycroft. Rycroft B W. 1945. Penicillin and the control of deep
intraocular infection. British J Ophthalmol 29 (2): 57-87. The
vitreous is a chamber where high level of drug can be introduced
and maintained for relatively long periods of time. The
concentration of drug that can be achieved via intravitreal
injection far exceeds what can be generated by topical
administration or by systemic administration (e.g.
intravenous).
[0331] One of the most dangerous complications potentially arising
from intravitreal injections is endophthalmitis. Endophthalmitis
falls into two classes: infectious and sterile. Infectious
endophthalmitis is generally cause by bacteria, fungi or parasites.
The symptoms of infectious endophthalmitis include severe pain,
loss of vision, and redness of the conjunctiva and the underlying
episclera. Infectious endophthalmitis requires urgent diagnosis and
treatment. Possible treatments include intravitreal injection of
antibiotics and pars plana vitrectomy in some cases. Enucleation
may be called for to remove a blind and painful eye. See, e.g.,
Christy N E, Sommer A. 1979. Antibiotic prophylaxis of
postoperative endophthalmitis. Ann Ophthalmol 11 (8):
1261-1265.
[0332] Sterile endophthalmitis in contrast does not involve an
infectious agent and can be defined as the acute intraocular
inflammation of the vitreous cavity that resolves without the need
of intravitreal antibiotics and/or vitreoretinal surgery. If a
vitreous microbiological study has been done, it needs to be
negative culture proven to sustain a diagnosis of sterile
endophthalmitis. Marticorena J, Romano V, Gomez-Ulla F. 2012
"Sterile Endophthalmitis after Intravitreal Injections" Med Inflam.
928123.
[0333] It has been observed that intravitreal injection of
biological drugs contaminated with endotoxin can result in sterile
endophthalmitis. Marticorena, et al. Bevacizumab (Avastin) is
approved by the Food and Drug Administration for the treatment of
glioblastoma and of metastatic colorectal cancer, advanced
nonsquamous non-small-cell lung cancer and metastatic kidney
cancer. Bevacizumab is also widely used off label as a treatment
for wet AMD. Bevacizumab comes from the manufacturer as a 100 mg/4
ml. This solution cannot be directly used for intravitreal
injection and must be compounded by a pharmacist. Clusters of
sterile endophthalmitis have been observed and are theorized to be
cause by inadvertent contamination of bevacizumab by endotoxin by
the compounding pharmacist.
[0334] Given the dire clinical results of intravitreal injection of
endotoxin, the total amount of endotoxin that can be given to a
patient via intravitreal dosing is highly limited. In accordance
with an aspect of the present invention, a solution having a dual
VEGF/PDGF antagonist according to the present invention is provided
having an endotoxin level that does not exceed 5.0 EU/ml. More
preferably, the endotoxin level does not exceed 1.0 EU/ml. Still
more preferably, the endotoxin level does not exceed 0.5 EU/ml.
Still more preferably, the endotoxin level does not exceed 0.2
EU/ml. In still more preferred embodiments, the endotoxin level
does not exceed 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02
or 0.01 EU/ml.
[0335] Two commonly used FDA-approved tests for the presence of
endotoxin are the rabbit pyrogen test and Limulus Amoebodyte Lysate
(LAL) assay (Hoffman S, et al. 2005. International validation of
novel pyrogen tests based on human monocytoid cells J. Immunol.
Methods 298: 161-173; Ding J L, Ho B A. 2001. New era in pyrogen
testing. Biotech. 19:277-281). The rabbit pyrogen test was
developed in the 1920s and involves monitoring the temperature rise
in a rabbit injected with a test solution. However, use of the
rabbit pyrogen test has greatly diminished over the years due to
expense and long turnaround time. Much more common is the LAL test.
LAL is derived from the blood of a horseshoe crab and clots upon
exposure to endotoxin.
[0336] One of the simplest LAL assays is the LAL gel-clot assay.
Essentially, the LAL clotting assay is combined with a serial
dilution of the sample in question. Formation of the gel is
proportional to the amount of endotoxin in the sample. Serial
dilutions are prepared from the sample and each dilution assayed
for its ability to form LAL gel. At some point a negative reaction
is contained. The amount of endotoxin in the original sample can be
estimated from the dilution assay.
[0337] Other LAL tests have also been developed, including the
turbidimetric LAL assay (Ong K G, Lelan J M, Zeng K F, Barrett G,
Aourob M, Grimes C A. 2006. A rapid highly-sensitive endotoxin
detection system. Biosensors and Bioelectronics 21:2270-2274) and
the chromogenic LAL assay (Haishima Y, Hasegawa C, Yagami T,
Tsuchiya T, Matsuda R, Hayashi Y. 2003. Estimation of uncertainty
in kinetic-colorimetric assay of bacterial endotoxins. J Pharm
Biomed Analysis. 32:495-503). The turbidimetric and chromogenic
assays are much more sensitive and quantitative than the simple
gel-clot dilution assay.
[0338] The present invention provides a method of reducing the
amount of endotoxin in a composition having a dual VEGF/PDGF
antagonist, the method having the steps of contacting the
composition with an affinity chromatography resin that binds to the
dual VEGF/PDGF antagonist; eluting the dual VEGF/PDGF antagonist
from the affinity chromatography resin to form an affinity
chromatography eluent having the antagonist; contacting the
affinity chromatography eluent with an ion-exchange resin that
binds the dual VEGF/PDGF antagonist; and eluting the dual VEGF/PDGF
antagonist from the ion-exchange resin, wherein the dual VEGF/PDGF
antagonist eluted from the ion-exchange resin is substantially free
from endotoxin.
[0339] The above method for reducing the amount of endotoxin, or
other method or process recited herein, can be performed in the
order described in the steps above or it can optionally be
performed by varying the order of the steps or even repeating one
or more of the steps. In one embodiment, the method of reducing the
amount of endotoxin in a composition is performed in the order of
the described steps. In some embodiments, the affinity
chromatography resin contacting, washing and eluting steps are
repeated in the same order more than one time before contacting the
affinity chromatography eluent with the ion exchange resin. The
method can also include a filtering step using, for example, a 0.1
micron, 0.22 micron, or 0.44 micron filter, that can be performed
on either one or more of the eluents removed after each resin
binding step.
[0340] In certain instances, the steps of contacting the
composition with affinity chromatography resin, washing and eluting
the antibody from the affinity chromatography resin can be repeated
more than one time before contacting the first eluent with an
ion-exchange resin. In one embodiment, the affinity chromatography
resin comprises a recombinant Protein A ("rProteinA") resin. One
example of a suitable recombinant Protein A resin is rProteinA
Sepharose FF.RTM. resin (Amersham, Piscataway, N.J.). In another
embodiment, a suitable affinity chromatography resin would comprise
a protein G chromatography resin. In other embodiments, a suitable
affinity chromatography resin comprises a mixed Protein A/Protein G
resin. In other embodiments, a suitable affinity chromatography
resin comprises a hydrophobic charge induction resin that comprises
a 4-mercaptoethylpyridine ligand such as a MEP HyperCel.RTM. resin
(BioSepra, Cergy, Saint Christophe, France).
[0341] In some embodiments, it is preferred that the ion exchange
resin comprises an anion-exchange resin. As will be known by the
person skilled in the art, ion exchangers may be based on various
materials with respect to the matrix as well as to the attached
charged groups. For example, the following matrices may be used, in
which the materials mentioned may be more or less cross-linked:
MacroCap Q (GE Healthcare Biosciences, Piscataway, N.J.), agarose
based (such as Sepharose CL-6B.RTM., Sepharose Fast Flow.RTM. and
Sepharose High Performance.RTM.), cellulose based (such as DEAE
Sephacel.RTM.), dextran based (such as Sephadex.RTM.), silica based
and synthetic polymer based. For the anion exchange resin, the
charged groups, which are covalently attached to the matrix, may,
for example, be diethylaminoethyl, quaternary aminoethyl, and/or
quaternary ammonium. It is preferred that the anion-exchange resin
comprises a quaternary amine group. An exemplarily anion-exchange
resin that has a quaternary amine group for binding the anti-M-CSF
antibody is a Q Sepharose.RTM. resin (Amersham, Piscataway,
N.J.).
[0342] In other aspects, if the endotoxin levels are higher than
desired after subjecting the composition to the aforementioned
anion-exchange chromatography step, the composition may in the
alternative be subjected to a cation exchange resin. In accordance
with this aspect of the present invention, any endotoxin in the
composition should have a differential binding to the ion-exchange
resin than the protein in question to allow purification of the
protein from the endotoxin. In this regard, endotoxin is negatively
charged and will generally bind to an anion exchange resin. If both
the protein and the endotoxin bind to the anion exchange resin,
purification of one from the other may be effectuated by using a
salt gradient to elute the two into different fractions. The
relative binding of the protein to a particular resin may also be
effected by changing the pH of the buffer relative to the pl of the
protein. In a preferred aspect of the present invention,
cation-exchange chromatography is the sole ion-exchange
chromatography employed.
[0343] In accordance with another aspect of the present invention,
if the endotoxin levels are too high after the anion exchange
resin, the composition may be further subjected to a second
ion-exchange step, for example, by contacting the compositions with
a cation exchange resin and followed by a wash step, then elution
from the ion-exchange resin. In preferred embodiments, the cation
exchange resin comprises a sulfonic group for binding. Exemplary
cation exchange resins are SP Sepharose.RTM. resin FF (Amersham,
Piscataway, N.J.) Porns XS (CEX) (Life Technology, Grand Island,
N.Y.).
[0344] In accordance with an aspect of the invention, after the
solution of dual PDGF/VEGF antagonist protein is produced having
the specified level of endotoxin, there are a number of steps prior
to final formulation of the protein. In some embodiments of the
present invention, a half-life extending moiety is conjugated to
the protein. The conjugate is then formulated into a final drug
formulation which is injected into the patients. In some
embodiments, the conjugate is again purified on an ion-exchange
resin which can preferably be a cation-exchange resin. In other
embodiments, the protein is formulated. In all cases, normal
laboratory procedures must be employed to prevent the introduction
of endotoxin contaminants into the protein sample or into the
protein-polymer conjugate.
EXAMPLES
Example 1. Protein Sequence of PDGFR-GS 10-Anti-VEGF-A Light
Chain/Anti-VEGF-A Heavy Chain (Wild Type Fe)
[0345] A PDGFR-.beta. trap-anti-VEGF-A light chain/anti-VEGF-A
heavy chain was constructed having the sequence set forth below in
FIGS. 7A, B. PDGFR-GS 10-anti-VEGF-A light chain amino acids 1-282
correspond to 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1), followed by the linker sequence
GGGGSGGGGS and the bevacizumab light chain sequence. Optionally, no
linker need be used between the PDGFR-.beta. segment and the
anti-VEGF segment. Alternatively, the linker may be the GGGGS motif
x1, x2 (as noted above), x3, x4, etc. such that the activity of the
two proteins is optimized. Other linker motifs known to those of
skill in the art may also be used in accordance with the present
invention, including G, GG, GGGS and GGGES x1, x2, x3, x4, etc. The
linker may be combinations of the above. The sequence of FIG. 7A is
set forth in SEQ ID NO. 19. FIG. 7B shows the bevacizumab heavy
chain sequence (SEQ ID NO. 2). The bevacizumab light chain
optionally has an M4L mutation (Kabat numbering). The bevacizumab
heavy chain optionally has one or more of the following mutations:
T28D, N31H, H97Y, S100aT (Kabat numbering), L234A, L235A, G237A,
Q347C and L443C EU numbering).
Example 2. Protein Sequence of PDGFR.beta.-GG-Anti-VEGF-A Light
Chain/Anti-VEGF-A Heavy Chain (Wild Type Fe)
[0346] Another PDGFR-.beta. trap-anti-VEGF-A light
chain/anti-VEGF-A heavy chain was constructed having the sequence
set forth below in FIGS. 8A, B. FIG. 8A amino acids 1-282
correspond to 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1), followed by the linker sequence
GG and the bevacizumab light chain sequence. Alternatively, the
linker may be the GGGGS motif x1, x2, x3, x4, etc. such that the
activity of the two proteins is optimized. Other linker motifs may
also be used in accordance with the present invention, including G,
GG (as noted above), GGGS and GGGES x1, x2, x3, x4, etc. The linker
may be combinations of the above. The protein sequence of FIG. 8A
is set forth in SEQ ID NO. 3. FIG. 8B shows bevacizumab heavy chain
sequence (SEQ ID NO. 2). The bevacizumab light chain of FIG. 8A
optionally has an M4L mutation. The bevacizumab heavy chain
optionally has one or more of the following mutations: T28D, N31H,
H97Y, S 100aT (Kabat numbering), L234A, L235A, G237A, Q347C and
L443C (EU numbering).
Example 3. Protein Sequence of PDGFR.beta.-GS 10-Anti-VEGF-A Heavy
Chain (Wild Type Fc)/Anti-VEGF-A Light Chain
[0347] Another PDGFR-.beta. trap-anti-VEGF-A heavy chain (wild type
Fc)/anti-VEGF-A light chain was constructed having the sequence set
forth in FIGS. 9A, B. FIG. 9A amino acids 1-282 correspond to 33 to
314 of human PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1),
followed by the linker sequence GGGGSGGGGS and the bevacizumab
heavy chain sequence, optionally having Q347C or L443C (EU
numbering). Alternatively, the linker may be the GGGGS motif x1, x2
(as noted above), x3, x4, etc. such that the activity of the two
proteins is optimized. Other linker motifs may also be used in
accordance with the present invention, including G, GG, GGGS and
GGGES x1, x2, x3, x4, etc. The linker may be combinations of the
above. The protein sequence of FIG. 9A is set forth in SEQ ID NO.
4. The protein of FIG. 9B is the bevacizumab light chain sequence
(SEQ ID NO. 5). The bevacizumab light chain optionally has an M4L
mutation. The bevacizumab heavy chain optionally has one or more of
the following mutations: T28D, N31H, H97Y, S1OOaT (Kabat
numbering), L234A, L235A, G237A, Q347C and L443C (EU
numbering).
Example 4. Protein Sequence of PDGFR-GG-Anti-VEGF-A Heavy Chain
(Wild Type Fc)/Anti-VEGF-A Light Chain
[0348] Another PDGFR-.beta. trap-anti-VEGF-A heavy chain (wild type
Fc)/anti-VEGF-A light chain was constructed having the sequence set
forth below in FIGS. 10A, 10B. FIG. 10A amino acids 1-282
correspond to 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1), followed by the linker sequence
GG and the bevacizumab heavy chain sequence, optionally having
Q347C or L443C. Alternatively, the linker may be the GGGGS motif
x1, x2, x3, x4, etc. such that the activity of the two proteins is
optimized. Other linker motifs may also be used in accordance with
the present invention, including G, GG (as noted above), GGGS and
GGGES x1, x2, x3, x4, etc. The linker may be combinations of the
above. The protein sequence of FIG. 10A is set forth in SEQ ID NO.
6. The protein of FIG. 10B is the bevacizumab light chain sequence
(SEQ ID NO. 5). The bevacizumab light chain optionally has an M4L
mutation. The bevacizumab heavy chain optionally has one or more of
the following mutations: T28D, N31H, H97Y, S1OOaT (Kabat
numbering), L234A, L235A, G237A, Q347C and L443C (EU
numbering).
Example 5. Protein Sequence of Anti-VEGF-A Heavy Chain (Wild Type
Fc)-GS21-PDGFR/Anti-VEGF-A Light Chain
[0349] A PDGFR-.beta. trap-anti-VEGF-A antibody construct was
constructed with the anti-VEGF-A heavy chain being upstream or
N-terminal to the PDGFR-.beta. trap having the sequence set forth
below in FIGS. 11A, B. FIG. 11A amino acids 1-451 correspond to the
bevacizumab heavy chain sequence, optionally having Q347C or L443C,
followed by linker sequence GGGGSGGGGSGGGGSGGGGSG. Alternatively,
the linker may be the GGGGS motif x1, x2 (as noted above), x3, x4,
etc. such that the activity of the two proteins is optimized. Other
linker motifs also be used in accordance with the present
invention, including G, GG, GGGS and GGGES x1, x2, x3, x4, etc. The
linker may be combinations of the above. The linker is followed by
amino acid sequences 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1). The protein sequence of FIG. 11A
is set forth in SEQ ID NO. 7. FIG. 11B shows the bevacizumab light
chain sequence (SEQ ID NO. 5). The bevacizumab light chain
optionally has an M4L mutation. The bevacizumab heavy chain
optionally has one or more of the following mutations: T28D, N31H,
H97Y, S1OOaT (Kabat numbering), L234A, L235A, G237A, Q347C and
L443C (EU numbering).
Example 6. Protein Sequence of PDGFR-GS 10-Anti-VEGF-A Heavy Chain
(Q347C)/Anti-VEGF-A Light Chain (TAF347)
[0350] Another PDGFR-.beta. trap-anti-VEGF-A heavy chain
(Q347C)/anti-VEGF-A light chain was constructed having the sequence
set forth below in FIGS. 12A, B. FIG. 12A amino acids 1-282
correspond to 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1). Immediately following the PDGFR
sequence is a 10 amino acid linker GGGGSGGGGS. Optionally, no
linker need be used between the PDGFR-.beta. segment and the
anti-VEGF segment. The linker may be combinations of the above.
Joined to the carboxyl terminus of the serine residue of the linker
is the bevacizumab heavy chain with the following amino acid: T28D,
N31H, H97Y, S100aT (Kabat numbering), L234A, L235A, G237A and Q347C
(EU numbering). The protein sequence of FIG. 12A is set forth in
SEQ ID NO. 8. The protein of FIG. 12B is ranibizumab light chain
(bevacizumab w/M4L) (SEQ ID NO. 12).
Example 7. Protein Sequence of PDGFR-GS 10-Anti-VEGF-A Heavy Chain
(L443C)/Anti-VEGF-A Light Chain
[0351] Another PDGFR-.beta. trap-anti-VEGF-A heavy chain
(L443C))/anti-VEGF-A light chain was constructed having the
sequence set forth below in FIGS. 13A, B. FIG. 13A amino acids
1-282 correspond to 33 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1). Immediately following the PDGFR
sequence is a 10 amino acid linker GGGGSGGGGS. Joined to the
carboxyl terminus of the serine residue of the linker is the
bevacizumab heavy chain with the following amino acid: T28D, N31H,
H97Y, S100aT (Kabat numbering), L234A, L235A, G237A and L443C (EU
numbering). The TAF443 light chain is the same as bevacizumab
except for a M4L change (Kabat numbering). The protein sequence of
FIG. 13A is set forth in SEQ ID NO. 9. FIG. 13B shows the
ranibizumab light chain (bevacizumab w/M4L) (SEQ ID NO. 12).
Example 8. Protein Sequence of PDGFR.beta.-GS 10-Anti-VEGF-A Light
Chain/Anti-VEGF-A Fab
[0352] A PDGFR-.beta. trap-anti-VEGF-A light chain/anti-VEGF-A Fab
was constructed having the sequence set forth below in FIGS. 14A,
B. FIG. 14A amino acids 1-282 correspond to 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1), followed by the
linker sequence GGGGSGGGGS and the bevacizumab light chain
sequence. Optionally, no linker need be used between the
PDGFR-.beta. segment and the anti-VEGF segment. Alternatively, the
linker may be the GGGGS motif x1, x2 (as noted above), x3, x4, etc.
such that the activity of the two proteins is optimized. Other
linker motifs may also be used in accordance with the present
invention, including G, GG, GGGS and GGGES x1, x2, x3, x4, etc. The
linker may be combinations of the above. The protein sequence of
FIG. 14A is set forth in SEQ ID NO. 1. The protein of FIG. 14B is
the bevacizumab Fab (SEQ ID NO. 21). The bevacizumab light chain of
FIG. 14A optionally has an M4L mutation. The bevacizumab Fab of the
second protein optionally has one or more of the following
mutations: T28D, N31H, H97Y, and S1OOaT. The bevacizumab Fab of the
second chain optionally has a cysteine moiety added to the
C-terminus for conjugating a half-life extending moiety.
Preferably, the cysteine moiety is added via SGGGC or CAA.
Alternatively, SGGGC or CAA may be added to the C-terminus of the
light chain.
Example 9. Protein Sequence of PDGFR.beta.-GG-Anti-VEGF-A Light
Chain/Anti-VEGF-A Fab
[0353] A PDGFR-.beta. trap-anti-VEGF-A light chain/anti-VEGF-A Fab
was constructed having the sequence set forth below in FIGS. 15A,
B. FIG. 15A amino acids 1-282 correspond to 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619. 1), followed by the
linker sequence GG and the bevacizumab light chain sequence.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2, x3, x4, etc. such that the activity of the two
proteins is optimized. Other linker motifs may also be used in
accordance with the present invention, including G, GG (as above),
GGGS and GGGES x1, x2, x3, x4, etc. The linker may be combinations
of the above. The protein sequence of FIG. 15A is set forth in SEQ
ID NO. 3. FIG. 15B shows the heavy chain of bevacizumab Fab (SEQ ID
NO. 21). The bevacizumab light chain of FIG. 15A optionally has an
M4L mutation (Kabat numbering). The bevacizumab Fab of FIG. 15B
optionally has one or more of the following mutations: T28D, N31H,
H97Y, and S1OOaT (Kabat numbering). The bevacizumab Fab of FIG. 15B
optionally has a cysteine moiety added to the C-terminus for
conjugating a half-life extending moiety. Preferably, the cysteine
moiety is added via SGGGC or CAA. Alternatively, SGGGC or CAA may
be added to the C-terminus of the light chain.
Example 10. Protein Sequence of PDGFR.beta.-GS 10-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain
[0354] Another PDGFR-.beta. trap-anti-VEGF-A Fab/anti-VEGF-A light
chain was constructed having the sequence set forth below in FIGS.
16A, B. FIG. 16A amino acids 1-282 correspond to 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1), followed by the
linker sequence GGGGSGGGGS and the bevacizumab Fab sequence.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2 (as above), x3, x4, etc. such that the activity
of the two proteins is optimized. Other linker motifs may also be
used in accordance with the present invention, including G, GG,
GGGS and GGGES x1, x2, x3, x4, etc. The linker may be combinations
of the above. The protein sequence of FIG. 16A is set forth in SEQ
ID NO. 22. FIG. 16B shows the bevacizumab light chain sequence (SEQ
ID NO. 5). The bevacizumab light chain optionally has an M4L
mutation. The bevacizumab heavy chain optionally has one or more of
the following mutations: T28D, N31H, H97Y, and S 100aT (Kabat
numbering). The heavy chain optionally has a cysteine moiety added
to the C-terminus for conjugating a half-life extending moiety.
Preferably, the cysteine moiety is added via SGGGC or CAA.
Alternatively, SGGGC or CAA may be added to the C-terminus of the
light chain.
Example 11. Protein Sequence of PDGFR.beta.-GG-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain
[0355] Another PDGFR-.beta. trap-anti-VEGF-A Fab/anti-VEGF-A light
chain was constructed having the sequence set forth below in FIGS.
17A, B. FIG. 17A amino acids 1-282 correspond to 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1), followed by the
linker sequence GGGGSGGGGS and the bevacizumab Fab sequence.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2 (as above), x3, x4, etc. such that the activity
of the two proteins is optimized. Other linker motifs may also be
used in accordance with the present invention, including G, GG,
GGGS and GGGES x1, x2, x3, x4, etc. The linker may be combinations
of the above. The protein sequence of FIG. 17A is set forth in SEQ
ID NO. 23. FIG. 17B shows the bevacizumab light chain sequence (SEQ
ID NO. 5). The bevacizumab light chain optionally has an M4L
mutation. The bevacizumab heavy chain optionally has one or more of
the following mutations: T28D, N31H, H97Y, and S100aT (Kabat
numbering). The bevacizumab Fab heavy chain optionally has a
cysteine moiety added to the C-terminus for conjugating a half-life
extending moiety. Preferably, the cysteine moiety is added via
SGGGC or CAA. Alternatively, SGGGC or CAA may be added to the
C-terminus of the light chain.
Example 12. Protein Sequence of Anti-VEGF-A
Fab-GS21-PDGFR.beta./Anti-VEGF-A Light Chain
[0356] A PDGFR-.beta. trap-anti-VEGF-A antibody construct was
constructed with the anti-VEGF-A heavy chain being upstream or
N-terminal to the PDGFR-.beta. trap having the sequence set forth
below in FIGS. 18A, B. FIG. 18A amino acids 1-231 correspond to the
bevacizumab Fab followed by linker sequence GGGGSGGGGSGGGGSGGGGSG.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2, x3, x4, etc. such that the activity of the two
proteins is optimized. Other linker motifs may also be used in
accordance with the present invention, including G, GG, GGGS and
GGGES x1, x2, x3, x4, etc. The linker may be combinations of the
above. The linker is followed by amino acid sequences 33 to 314 of
human PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1). The protein
sequence of FIG. 18A is set forth in SEQ ID NO. 24. FIG. 18B shows
the bevacizumab light chain sequence (SEQ ID NO. 5). The
bevacizumab light chain optionally has an M4L mutation. The
bevacizumab heavy chain optionally has one or more of the following
mutations: T28D, N31H, H97Y, and S 100aT. The protein of FIG. 18A
optionally has a cysteine moiety added to the C-terminus for
conjugating a half-life extending moiety. Preferably, the cysteine
moiety is added via SGGGC or CAA. Alternatively, SGGGC or CAA may
be added to the C-terminus of the light chain of FIG. 18B.
Example 13. Protein Sequence of PDGFR.beta.-GS 10-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain
[0357] Another PDGFR-.beta. trap-anti-VEGF-A Fab/anti-VEGF-A light
chain was constructed having the sequence set forth below in FIGS.
19A, B. FIG. 19A amino acids 1-282 correspond to 33 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1). Immediately
following the PDGFR sequence is a 10 amino acid linker GGGGSGGGGS.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2 (as above), x3, x4, etc. such that the activity
of the two proteins is optimized. Other linker motifs may also be
used in accordance with the present invention, including G, GG,
GGGS and GGGES x1, x2, x3, x4, etc. The linker may be combinations
of the above. Joined to the carboxyl terminus of the serine residue
of the linker is the bevacizumab Fab having the mutations T28D,
N31H, H97Y, and S1OOaT (Kabat numbering). The protein sequence of
FIG. 19A is set forth in SEQ ID NO. 25. The protein of FIG. 19B is
the ranibizumab light chain (bevacizumab w/M4L) (SEQ ID NO. 12).
The protein of FIG. 19A optionally has a cysteine moiety added to
the C-terminus for conjugating a half-life extending moiety.
Preferably, the cysteine moiety is added via SGGGC or CAA.
Alternatively, SGGGC or CAA may be added to the C-terminus of the
light chain of FIG. 19B.
Example 14. Protein Sequence of PDGFR.beta.-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain (1a)
[0358] Another PDGFR-.beta. trap-anti-VEGF-A Fab/anti-VEGF-A light
chain was constructed having the sequence set forth in FIGS. 20A,
B. FIG. 20A amino acids 1-283 correspond to 32 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1), followed by the
bevacizumab heavy chain. The protein sequence of FIG. 20A is set
forth in SEQ ID NO. 26. The protein of FIG. 20B is the bevacizumab
light chain sequence (SEQ ID NO. 5). As set forth in this example,
no linker need be used between the PDGFR-.beta. segment and the
anti-VEGF segment. Alternatively, the linker may be the GGGGS motif
x1, x2, x3, x4, etc. such that the activity of the two proteins is
optimized. Other linker motifs may also be used in accordance with
the present invention, including G, GG, GGGS and GGGES x1, x2, x3,
x4, etc. The linker may be combinations of the above. The
bevacizumab light chain optionally has an M4L mutation. The
bevacizumab heavy chain optionally has one or more of the following
mutations: T28D, N31H, H97Y, S1OOaT (Kabat numbering), Q347C and
L443C (EU numbering).
Example 15. Protein Sequence of PDGFR-.beta. (D2-D3)-Anti-VEGF-A
Heavy Chain/Anti-VEGF-A Light Chain (1b)
[0359] Another PDGFR-.beta. trap (D2-D3)-anti-VEGF-A heavy
chain/anti-VEGF-A light chain was constructed having the sequence
set forth below in FIG. 21A, B. FIG. 21A amino acids 1-190
correspond to 125 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1), followed by the bevacizumab heavy
chain. The protein sequence of FIG. 21A is set forth in SEQ ID NO.
27. As set forth in this example, no linker need be used between
the PDGFR-.beta. segment and the anti-VEGF segment. Alternatively,
the linker may be the GGGGS motif x1, x2, x3, x4, etc. such that
the activity of the two proteins is optimized. Other linker motifs
may also be used in accordance with the present invention,
including G, GG, GGGS and GGGES x1, x2, x3, x4, etc. The linker may
be combinations of the above. FIG. 21B shows the bevacizumab light
chain sequence (SEQ ID NO. 5). The bevacizumab light chain
optionally has an M4L mutation. The bevacizumab heavy chain
optionally has one or more of the following mutations: T28D, N31H,
H97Y, S1OOaT (Kabat numbering), Q347C and L443C (Eu numbering).
Example 16. Protein Sequence of PDGFR-.beta. (D2-D3)-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain (2b)
[0360] Another PDGFR-.beta. trap (D2-D3)-anti-VEGF-A
Fab/anti-VEGF-A light chain was constructed having the sequence set
forth in FIGS. 22A, B. FIG. 22A amino acids 1-190 correspond to 125
to 314 of human PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1),
followed by the bevacizumab Fab. As set forth in this example, no
linker need be used between the PDGFR.beta.-segment and the
anti-VEGF segment. Alternatively, the linker may be the GGGGS motif
x1, x2, x3, x4, etc. such that the activity of the two proteins is
optimized. The GGGGSGGGGS linker is particularly preferred. Other
linker motifs may also be used in accordance with the present
invention, including G, GG, GGGS and GGGES x1, x2, x3, x4, etc. The
linker may be combinations of the above. The sequence of FIG. 22A
is set forth in SEQ ID NO. 28. FIG. 22B shows the bevacizumab light
chain sequence (SEQ ID NO. 5). The bevacizumab light chain
optionally has an M4L mutation. The bevacizumab heavy chain
optionally has one or more of the following mutations: T28D, N31H,
H97Y, and S100aT (Kabat numbering). The bevacizumab Fab of FIG. 22A
optionally has a cysteine moiety added to the C-terminus for
conjugating a half-life extending moiety. Preferably, the cysteine
moiety is added via SGGGC or CAA. Alternatively, SGGGC or CAA may
be added to the C-terminus of the light chain of FIG. 22B.
Example 17. Protein Sequence of PDGFR-.beta. (D2-D3)-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain (2b')
[0361] Another PDGFR-.beta. trap (D2-D3)-6xGS-anti-VEGF-A
Fab/anti-VEGF-A light chain was constructed having the sequence set
forth below in FIGS. 23A, B. FIG. 23A amino acids 1-190 correspond
to 125 to 314 of human PDGFR-.beta. (UniProtKB/Swiss-Prot:
P09619.1), followed by the linker GGGSGGGGSGGGGSGGGGSGGGGSGGGGS and
then by bevacizumab Fab. Optionally, no linker need be used between
the PDGFR-.beta. segment and the anti-VEGF segment. Alternatively,
the linker may be the GGGGS motif x1, x2, x3, x4, etc. such that
the activity of the two proteins is optimized. Other linker motifs
known to those of skill in the art may also be used in accordance
with the present invention, including G, GG, GGGS and GGGES x1, x2,
x3, x4, etc. The linker may be combinations of the above. The
protein sequence of FIG. 23A is set forth in SEQ ID NO. 29. FIG.
23B shows the bevacizumab light chain sequence (SEQ ID NO. 5). The
bevacizumab light chain optionally has an M4L mutation. The
bevacizumab heavy chain optionally has one or more of the following
mutations: T28D, N31H, H97Y, and S1OOaT (Kabat numbering). The
bevacizumab Fab heavy chain optionally has a cysteine moiety added
to the C-terminus for conjugating a half-life extending moiety.
Preferably, the cysteine moiety is added via SGGGC or CAA.
Alternatively, SGGGC or CAA may be added to the C-terminus of the
light chain.
Example 18. Protein Sequence of PDGFR-(D2-D3)-Anti-VEGF-A
Fab/Anti-VEGF-A Light Chain (2b')
[0362] Another anti-VEGF-A Fab-6xGS-PDGFR-.beta. trap
(D2-D3)/anti-VEGF-A light chain was constructed having the sequence
set forth below in FIGS. 24A, B. FIG. 24A amino acids 1-190
correspond to 125 to 314 of human PDGFR-.beta.
(UniProtKB/Swiss-Prot: P09619.1), followed by the linker
GGGSGGGGSGGGGSGGGGSGGGGSGGGGS and then by bevacizumab Fab.
Optionally, no linker need be used between the PDGFR-.beta. segment
and the anti-VEGF segment. Alternatively, the linker may be the
GGGGS motif x1, x2, x3, x4, etc. such that the activity of the two
proteins is optimized. Other linker motifs may also be used in
accordance with the present invention, including G, GG, GGGS and
GGGES x1, x2, x3, x4, etc. The linker may be combinations of the
above. The sequence of FIG. 24A is set forth in SEQ ID NO. 29. FIG.
24B shows the bevacizumab light chain sequence (SEQ ID NO. 5). The
bevacizumab light chain of FIG. 24B optionally has an M4L mutation.
The bevacizumab heavy chain of the first protein optionally has one
or more of the following mutations: T28D, N31H, H97Y, and S1OOaT
(Kabat numbering). The bevacizumab Fab of FIG. 24A optionally has a
cysteine moiety added to the C-terminus for conjugating a half-life
extending moiety. Preferably, the cysteine moiety is added via
SGGGC or CAA. Alternatively, SGGGC or CAA may be added to the
C-terminus of the light chain.
Example 19. Protein Sequence of Anti-VEGF-A Fab-6xGS-PDGFR-.beta.
(D2-D3)/Anti-VEGF-A Light Chain (3)
[0363] Another anti-VEGF-A Fab-6xGS-PDGFR-.beta.
(D2-D3)/anti-VEGF-A light chain was constructed having the sequence
set forth below in FIGS. 25A, B. FIG. 25A amino acids 1-231
correspond to bevacizumab Fab, followed by the linker
GGGSGGGGSGGGGSGGGGSGGGGSGGGGS and then 125 to 314 of human
PDGFR-.beta. (UniProtKB/Swiss-Prot: P09619.1). Optionally, no
linker need be used between the PDGFR-.beta. segment and the
anti-VEGF segment. Alternatively, the linker may be the GGGGS motif
x1, x2, x3, x4, etc. such that the activity of the two proteins is
optimized. Other linker motifs may also be used in accordance with
the present invention, including G, GG, GGGS and GGGES x1, x2, x3,
x4, etc. The linker may be combinations of the above. The sequence
of FIG. 25A is set forth in SEQ ID NO. 30. FIG. 25B shows the
bevacizumab light chain sequence (SEQ ID NO. 5). The bevacizumab
light chain optionally has an M4L mutation. The bevacizumab heavy
chain optionally has one or more of the following mutations: T28D,
N31H, H97Y, and S1OOaT (Kabat numbering). The PDGFR-.beta. of FIG.
25A optionally has a cysteine moiety added to the C-terminus for
conjugating a half-life extending moiety. Preferably, the cysteine
moiety is added via SGGGC or CAA. Alternatively, SGGGC or CAA may
be added to the C-terminus of the light chain.
Example 20. Production of Dual PDGFR/VEGF Antagonist Protein
[0364] The TAF443 heavy and light chains were cloned into
expression plasmids and transfected into CHO cells. Cells were
grown up in appropriate media and harvested. TAF443 was purified as
follows. 10 L culture medium from CHO cells expressing SEQ ID NOS.
31 and 32 were adjusted with 5% (v/v) 1.1 M HEPES, 0.22 M EDTA, pH
6.7 or 10% 0.55 M Hepes, 0.11M EDTA, 5.5% Triton X-100, pH 6.7, and
loaded onto a 167/400 ml Protein A column (2-run) packed with Mab
Select Sure resin equilibrated in 50 mM Tris, 150 mM NaCl, pH 7.5
(5-CV). The column was washed with 50 mM Tris, 150 mM NaCl, pH 7.5
(2-CV), 50 mM Tris, 0.5M CaCh, pH 7.5 (5-CV), and then 10 mM Tris,
10 mM NaCl, pH 7.5 (3-CV) before the protein was eluted using 150
mM Glycine, 40 mM NaCl, pH 3.5 (4-CV). Fractions were pooled,
adjusted to pH 3.5 using 2M Glycine, pH 2.7, and then neutralized
to pH 7 using 2M HEPES, pH 8.0. The Protein A pool was loaded onto
a 274 ml TMAE column equilibrated in 50 mM Hepes, 65 mM NaCl, pH
7.0 (5-CV). The column was washed with 50 mM Hepes, 65 mM NaCl, pH
7.0 (3-CV), and then eluted with 50 mM Tris, 200 mM NaCl, pH 7.5
(5-CV). The elution fractions were pooled and buffer exchanged in a
1150 mL Sephadex G-25 Coarse column equilibrated with PBS-CMF, pH
7.2. The pool was filtered, concentrated to >5 mg/ml via 30k
MWCO VivaFlow200. The concentrated protein was filtered through a
0.22 um filter, and then characterized by SDS-PAGE, analytical SEC,
O.D.280/320, end toxin LAL assay, Protein A ELISA, IEF, and
Freeze/Thaw Analysis.
[0365] The table below summarizes the properties of an example
batch of purified TAF443.
TABLE-US-00001 TAF443 Purified Lot Characteristics Concentration
(UV) 5.69 mg/ml Purity (SEC) 98.6% MW (SDS-PAGE) ~200 kDa (NR) pI
(IEF) 4.2-4.5 Endotoxin (LAL) 0.1 EU/mg Protein A (Elisa) <10
ng/ml Final Yield ~700 mg/L(CM)
Example 21. TAP Bi-Functional Molecule Stability at High
Concentration in Representative Formulations
[0366] The TAP bi-functionals were concentrated to 50-85 mg/ml in a
series of standard formulation buffers ranging from pH 4.5 to 7.5,
in the presence of excipients such as sucrose. Aliquots of these
samples were stored at room temperature (RT) and 4.degree. C. over
a period of 6 weeks, and sampled at time zero and after each
subsequent week to measure the percentage of aggregated material by
analytical SEC. The effect of pH on aggregation of TAP443 can be
seen in the following table.
TABLE-US-00002 % Aggregates Observed in TAF Solution at various pHs
over Time Tris pH 7.5 His pH 6.0 His pH 5.5 Lac pH 4.5 Time 0 <1
<1 <1 <2 Day 4 <1 <1 <1 ~3 Week 1 <1 <1
<1 ~4 Week 2 <1 <1 ~2 ~6 Week 4 <1 <1 ~3 ~10 Week 6
<1 <1 ~3 ~10
Example 22. Transfection of Constructs into CHO Cells
[0367] DNA constructs for TAPwt, TAP443 and TAP347 were transfected
into CHO-K 1 SV SSI: 3 pools/construct. The normal 3 weeks of
recovery was observed in most of the cell lines. However, TAPwt and
TAP347 cell lines lagged approximately 1 week behind the other cell
lines. Once the pools were established, day 4 for most and day 3
for TAPwt and TAP347, conditioned media samples were run on Octet.
3-day conditioned media for TAPwt and TAP347 showed about 7 mg/ml
by Octet. 4-day conditioned media showed about 21 mg/ml for TAP443.
Small differences were observed between pools and the pools were
used to make pools of pools which were carried forward for protein
generation.
Example 23. SEC-MALS of Proteins
[0368] The PDGPR segment of TAP has 7 putative glycosylation sites.
The protein appears to be heavily glycosylated from SEC-MALS
measurements:
TABLE-US-00003 Construct Protein (kDa) Sugar (kDa) Total (kDa)
TAFwt 184 63 247 TAF334 182 62 244 TAF443 187 63 250
[0369] The samples run on SEC-MALS were all greater than 98% pure.
The molecular weights measured were reasonable. Some high molecular
weight material was observed, probably a tri- to pentamer (data not
shown).
Example 24. Thermal Stability of TAP Proteins
[0370] Thermal stability profiles were run of TAFwt, TAF443 and
TAF347 in PBS, pH 7.2. Each protein had three peaks (data not
shown). The relative positions of the peaks are set forth in the
table below:
TABLE-US-00004 Sample T.sub.m1(.degree. C.) T.sub.m2(.degree. C.)
T.sub.m3(.degree. C.) TAFwt 58.1 .+-. 0.1 71.9 .+-. 0.1 83.2 .+-.
0.1 TAF347 58.2 .+-. 0.1 71.9 .+-. 0.1 81.7 .+-. 0.1 TAF443 58.2
.+-. 0.1 71.9 .+-. 0.1 84.4 .+-. 0.1
[0371] The stabilities of the proteins over the temperature range
are very similar. It is noted however that there are some small
changes in T.sub.m3. T.sub.m3 likely corresponds to the CH.sub.3
domain of the antibody domain of the three TAF proteins and the
changes reflect the Cys mutations. The low overall stability of the
TAF proteins is likely due to unfolding of the PDGFR segment of the
proteins.
Example 25. TAP Forced Aggregation
[0372] The percentage of aggregates in a solution of the three TAP
proteins as a function of heat was examined (data not shown).
Solutions of each of the proteins (TAFwt, TAF347 and TAF443)
started to show aggregates starting around 54.degree. C. The
percentage of aggregates for each of the proteins increased sharply
as the temperature was increased. At 64.degree. C., roughly 40% of
each of the TAF proteins constituted aggregates. It is noted that
the aggregation starts to occur at the lowest T.sub.m, seemingly
corresponding to the unfolding of the PDGFR portion of the
protein.
Example 26. TAF443 Thermal Stability as a Function of pH
[0373] The thermal stability of TAF443 was examined at various pHs
as set forth in the table below. In non PBS buffers, 4 thermal
denaturation peaks are seen:
TABLE-US-00005 Buffer T.sub.m1(.degree. C.) T.sub.m2(.degree. C.)
T.sub.m3(.degree. C.) T.sub.m4(.degree. C.) Tris pH 7.5 57 67 74
85.9 His pH 6.0 53.3 62.9 75.4 84.9 Succinate 55.9 66.7 74.9 85.8
Lucentis buffer, 53.9 61.3 75.1 82.9 pH 4.8 PBS pH 7.2 58.2 71.9
84.4
[0374] As can be seen, there is a weak pH dependence. Notably, the
T.sub.m2 and T.sub.m3 domain (presumably CH2, Pab) overlap in PBS,
but not in other buffers.
Example 27. Affinity of Dual PDGP/VEGP Antagonist Proteins and
Conjugates to Targets
[0375] Surface plasmon resonance (SPR) was used to characterize the
binding kinetics of recombinant human PDGP-BB (PeproTech, 100-14B)
to TAP-WT, TAP-347, TAP-443, TAP443-6A250K, and TAP443-3A250K dual
PDGP/VEGP antagonist variants. Initially, an anti-human IgG
antibody (GE Healthcare, BR-1008-39) was covalently amine coupled
onto all four flow cells of a CM5 carboxymethylated dextran coated
sensorchip to a density of about 10,000 resonance units (RUs)
following the manufacturer's protocol. Each PDGP/VEGP variant was
captured to a level of approximately 150 RUs. The running and
sample buffer for the PDGP analysis was HBS-EP+300 mM NaCl (10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4,
300 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.05%
(v/v) Tween-20). A 2-fold serial dilution series of PDGP-BB ranging
in concentration from 1 nM to 0.125 nM was injected at a flow rate
of 100 .mu.L/minute for a 110 second association with dissociations
that varied from 300 to 2700 seconds. The surface was then
regenerated with a 30 second pulse of 3M MgCh, a 30 second pulse of
an ionic regeneration buffer (0.46M KSCN, 1.83 M MgCl2, 0.92 M
urea, and 1.83 M guanidine-HCl pH7.4, Andersson et al., Analytical
Chemistry, 1999) and then equilibrated with a 30 second pulse of
HBS-EP+300 mM NaCl running buffer.
[0376] Similarly, SPR was used to determine the binding affinities
of recombinant human VEGP121 (PeproTech, 100-20A) against the
TAP-WT, TAP-347, and TAP-443 dual PDGP/VEGP antagonist variants.
The running and sample buffer for the VEGP analysis was HBS-EP+
with a final concentration of 150 mM NaCl. A 2-fold dilution series
of VEGP121 ranging in concentration from 100 nM to 12.5 nM was
injected at a flow rate of 50 uL/minute for about a 50 second
association with dissociations that varied from 300 to 3600
seconds. The surface was then regenerated with a 30 second pulse of
3M MgCh, a 30 second pulse of ionic regeneration buffer (0.46M
KSCN, 1.83 M MgCl2, 0.92 M urea, and 1.83 M guanidine-HCl pH7.4,
Andersson et al., Analytical Chemistry, 1999), and then
equilibrated with a 30 second pulse of HBS-EP+150 mM NaCl running
buffer.
[0377] All SPR assays were performed at 25.degree. C. with a data
collection rate of 1 Hz using a Biacore T200 instrument (GE
Healthcare). The resulting PDGP and VEGP sensorgrams were double
referenced using both a control surface and buffer injections. The
rate constants were determined by fitting the data to a 1:1
Langmuir model with Biacore T200 evaluation software v2.0 and the
equation K.sub.D=ka/k.sub.a.
TABLE-US-00006 Biacore Affinity to PDGF-BB ka kd t1/2 Rmax KD
Analyte Ligand (1/Ms) (1/2) (min) (Ru) Chi2/Rmax (pM)* PDGF-A*
TAF-WT 7.97E+07 8.01E-05 144.28 15.16 0.16% 1.01 PDGF-B* TAF-WT
8.01E+07 9.19E-05 125.68 15.15 0.20% 1.15 PDGF-C* TAF-WT 8.65E+07
1.03E-04 111.94 15.32 0.15% 1.19 AVG +/- STDEV 1.1 .+-. 0.1 PDGF-A*
TAF-347C 4.15E+07 8.41E-05 137.33 13.99 0.86% 2.03 PDGF-B* TAF-347C
5.82E+07 7.87E-05 146.79 13.08 1.05% 1.35 AVG +/- STDEV 1.7 .+-.
0.3 PDGF-A* TAF-443C 3.22E+07 4.96E-05 233.15 13.15 0.81% 1.54
PDGF-B* TAF-443C 5.62E+07 8.76E-05 131.89 12.19 0.96% 1.56 AVG +/-
STDEV 1.55 .+-. 0.01 PDGF-A* R3643-6A 7.60E+07 9.46E-05 122.09 8.11
0.41% 1.25 (TAF443- 6A250K) PDGF-B* R3643-6A 5.62E+07 5.85E-05
197.55 8.33 0.30% 1.04 (TAF443- 6A250K) PDGF-C* R3643-6A 3.48E+07
5.13E-05 225.41 8.45 0.80% 1.47 (TAF443- 6A250K) AVG +/- STDEV 1.3
.+-. 0.2 PDGF-A* R3644-3A 5.76E+07 5.92E-05 195.04 8.15 0.31% 1.03
(TAF443- 3A250K) PDGF-B* R3644-3A 2.86E+07 4.96E-05 233.05 8.52
0.60% 1.73 (TAF443- 3A250K) PDGF-C* R3644-3A 4.71E+07 7.52E-05
153.56 8.15 0.49% 1.60 (TAP443- 3A250K) AVG +/- STDEV 1.5 .+-. 0.4
*A, B and C refer to separate runs or measurements concerning the
same analyte PDGP-BB
TABLE-US-00007 Biacore Affinity to VEGP121 ka kd T1/2 Rmax KD
Analyte* Ligand (1/Ms) (1/s) (min) (RU) Chi2/Rmax (pM) VEGF-A
TAF-WT 1.14E+05 2.01E-05 573.89 29.4 0.17% 176.60 VEGF-B TAF-WT
6.85E+05 5.89E-05 196.00 13.90 0.16% 86.03 VEGF-C TAF-WT 1.40E+05
2.96E-05 390.68 27.36 0.17% 212.00 VEGF-D TAF-WT 1.55E+05 2.69E-05
429.78 24.92 0.23% 173.00 AVG/STD DEV 161.96 .+-. 53.46 VEGF-A
TAF347 1.37E+05 3.83E-05 301.55 26.57 0.87% 280.30 VEGF-B TAF347
1.42E+05 2.59E-05 446.21 24.56 0.35% 182.40 AVG 231.35 .+-. 48.95
VEGF-A TAF443 1.46E+05 3.20E-05 361.01 25.87 0.75% 219.30 VEGF-B
TAF443 1.35E+05 3.10E-05 372.18 25.47 0.31% 229.70 AVG 224.5 .+-.
5.2 *A, B, C, D, refer to different runs of the same analyte
(VEGF121).
Example 28. Decapping of TAF443 Prior to Maleimide Conjugation
[0378] The TAF443 Cysteine residue is typically "capped" or
oxidized by chemicals in the cell culture media and is not
available for conjugation. In this regard, purified TAF443 (OG
1321) is subjected to a decapping (i.e. reducing) procedure to
remove the cap and enable the free (i.e. those not involved in
Cys-Cys disulfide bonds) cysteine residue to be conjugated to the
maleimide functionality of a polymer. Decapping is done by mixing
TAP protein with a 30.times. molar excess for 1 hour at 25.degree.
C. of the reducing agent TCEP (3,3',3''-Phosphanetriyltripropanoic
acid). The reduction reaction with TCEP is monitored by SDS-PAGE.
Undenatured TAP runs as a single band at about 250 kDa (about 40
kDa of this weight is carbohydrate). When fully denatured the
single 250 kDa band is converted into bands corresponding to the
light and heavy chains. Following denaturation, the TAP protein was
washed by UFdF using a Pellion XL Ultrafiltration Cassette with 20
mM Tris pH7.5, 150 mM NaCl, 0.5 mM TCEP buffer to remove the cap.
The TCEP reagent was then removed in the same UFdF setup with 20 mM
Tris pH7.5, 150 mM NaCl. Reduced TAP was allowed to refold using
air (ambient Oxygen) which was again followed by SDS-PAGE as an
assay.
[0379] A detailed procedure for decapping is as follows:
[0380] 500 mg of OG 1321 was thawed from -80.degree. C. at
4.degree. C. overnight, and warmed up in the 25.degree. C. water
bath before mixing with TCEP at 30.times. molar excess. The
reaction was incubated in the 25.degree. C. water bath for 1 hour.
Samples were taken out at 15, 30, and 60 minutes to run on SDS-PAGE
in order to evaluate the reduction completeness. A UFdF cassette
with 10 kD MWCO was used to carry out buffer exchange. First buffer
exchange step was done with 20 mM Tris pH7.5, 100 mM NaCl, 0.5 mM
TCEP for -100.times. to thoroughly remove the cap. A second buffer
exchange step was done with 20 mM Tris pH7.5, 100 mM NaCl for
-1000.times. for TCEP remove prior to air refolding. The final TCEP
concentration in the sample was -0.5 .mu.M. Samples were taken out
from both buffer exchange steps for both SDS-PAGE and SEC analyses
to evaluate the protein reoxidation status and protein aggregation.
After the second buffer exchange step, the OG 1321 was concentrated
to -2 mg/ml, 0.22 .mu.m filtered, and allowed to re-oxidize with
air at 4.degree. C. Samples were taken out for SDS-PAGE and SEC
analyses at different time points to evaluate the re-oxidation
status. Re-oxidized OG 1321 was 0.22 .mu.m filtered and further
concentrated. Continued to concentrate the sample with VIVACELL 100
30k MWCO spin concentrators to 4-6 mg/ml and sterile filtered the
sample. Quantified by OD280.
Example 29. Conjugation of TAF443 to Biopolymer
[0381] TAF443 which is also called OG 1321 was conjugated to
polymer OG 1802 (see below) after decapping using a 15.times.
excess of polymer in pH 7.5 Tris buffer to produce OG 1448, shown
in FIG. 26, showing the chemical structure of OG 1448 which is
TAF443 conjugated to biopolymer OG 1802. TAF443 is on the extreme
right hand of the molecule shown in the figure, conjugated via the
cysteine 443 residue to the 5 member ring. Conjugation was
monitored by SDS-PAGE and driven to near completion. Conjugate was
purified via anion exchange chromatography and buffer exchanged
into the formulation buffer by UF/DF.
[0382] In general, there are three steps involved in the synthesis
of OG 1448 from components OG 1802 and OG 1321. Step A: OG 1321
much be reduced or decapped to free up the sulfhydryl groups at
cysteine position 443. Although the cysteine position at 443 of the
heavy chain of TAP is not believed to be involved in
cysteine-cysteine disulfide pairing, this cysteine is typically
capped by components of the media and absent reduction is not
available to react with maleimide. Step B: reduced TAP is then
conjugated to OG1802. Step C: conjugated TAP (OG 1448) is then
separated from unconjugated TAP and polymer via chromatography.
[0383] These three general steps are broken down into seven smaller
steps in the following table:
TABLE-US-00008 General Step Description IPC Assays Target Range A
Step 1: To reduce OG1321 using tris Non-reducing >95% reduction
(2-carboxyethyl) phosphine (TCEP). 30x SDS-PAGE molar of TCEP at
25.degree. C. for 1 hour. Step 2: To remove TCEP reducing agent
Non-reducing Band shift upon and cap groups using UF/DF. First,
wash SDS-PAGE. removal of the with 0.5 mM TCEP in Tris pH 7.5 for a
reducing agent. 100-fold volume exchange factor; followed by a
2.sup.nd wash with Tris buffer pH 7.5 for 1,000 fold volume
exchange factor to remove the TCEP, targeting final TCEP level
lower than 0.5 .mu.M. Step 3: To refold protein to ensure the
Non-reducing Band upshift native disulfide pairs are fully oxidized
SDS-PAGE upon oxidation of while the internal cysteine residues
remain the native reduced. disulfide pairs. UV/Vis for Final
protein protein concentration at 6-8 mg/ml. B Step 4: To conjugate
OG1321 protein to Non-reducing <20% full length OG1802
biopolymer. Conjugate by SDS-PAGE band remains. mixing the oxidized
OG1321 with Analytical AE- <20% unreacted OG1802. The process
requires 15x molar HPLC protein at OD 280 of biopolymer to decapped
protein and nm. constant mixing. Low temperature at 2-8.degree. C.
for 20 hours and overlay the reaction with nitrogen gas to minimize
oxidation. C Step 5: To separate OG1448 conjugate Analytical AE-
<5% unreacted from the unreacted OG1321 protein, HPLC for
protein at OD 180 unreacted OG1802 biopolymer, protein unreacted
nm: aggregates and other process contaminants. polymer and <15%
unreacted Purify OG1448 using MacroCap Q (AEX). unreacted protein
polymer at OD The chromatography is performed at pH 220 nm. 7.5 in
20 mM Tris buffer and eluted using a Non-reducing <5% unreacted
NaCl gradient. A pool is made by SDS PAGE protein combining
fractions. Step 6: To concentrate the OG1448 and to UV/Vis for
OG1448 at 50 exchange the chromatography buffers for protein mg/ml
the formulation buffers. The pooled concentration fractions from
the previous step are diafiltered and then concentrated by UF/DF to
achieve the target OG1448. Step 7: To remove bioburden from the
UV/Vis for OG1448 at 50 final product and to dispense into storage
protein mg/ml containers. The UF/DF final pool is 0.2 concentration
.mu.m filtered into sterile comakers, and pH pH Conductivity pH
7.2-7.5 and conductivity of the final filtrate is established. The
drug substance is stored at -20.degree. C.
Example 30. Purification of OG 1448 Via Anion Exchange (Macrocap
Q)
[0384] After conjugation of TAF443 to OG 1802 as described above,
OG 1448 was purified as follows: After conjugation of TAF443 to OG
1802 as described above, OG 1448 was purified as follows:
2.times.400 ml of Macrocap Q columns were packed according to -3:1
ratio of resin:conjugate. The columns were flushed with 5M of NaCl
and equilibrated with 20 mM Tris pH7.5, 20 mM NaCl (equilibration
buffer) by syphoning. The conjugation reaction mixture was diluted
with 20 mM Tris pH7 0.5 and loaded on the columns. The columns were
then chased with the equilibration buffer, and washed with 20 mM
Tris pH7.5, 50 mM NaCl (Wash 1) and then 20 mM Tris pH7.5, 100 mM
NaCl (Wash 2). Elution was done with 20 mM Tris pH7.5, with step
gradient of 150 mM, 200 mM, 220 mM, 250 mM, 300 mM, and 500 mM
NaCl. All the column flow-through, washes, and elution were
collected in clean bottles for SDS-PAGE and AEX analyses. Elution
fractions containing the conjugate were pooled and concentrated
using Pellicon XL TFF cassette with 30 kD MWCO and PES membrane.
The concentrated pool was then buffer exchanged against 1.times.PBS
pH7.4 buffer for -100.times. using the same TFF cassette and
transferred to the VIVACELL 100 spin concentrators to further
concentrate until the targeted concentration (-30 mg/ml) was
achieved. The final conjugate was filtered through a 0.2 .mu.m PES
syringe filter for lot release.
Example 31. Reduction of Bacterial Endotoxin
[0385] To reduce levels of endotoxin in the final protein (OG 1321)
or conjugate (OG 1448), purification procedures may be employed for
either protein or conjugate which utilize cation exchanges in place
of anion exchanges. For example, in the above procedure for
purifying OG 1321, the anion exchange TMAE resin is employed. In
place of TMAE resin, the cation exchange resin CEX may be used.
However, in order to use CEX residue the pH of the solution
containing the protein in question must be reduced to below the
protein's pl. For OG 1321, the pH of the protein solution after the
protein A column, is reduced to pH 3.5. The OG 1321 is bound to the
Porns XS column at pH5. Then, Porox XS (CEX) can be used to bind
and elute the OG1321.
Example 32. Route 1 Synthesis of OG 1802
[0386] A first route for the synthesis of OG 1802 is as follows.
First, TFA/amine salt initiator (Compound L) having the structure
shown in FIG. 27 was synthesized as follows.
[0387] First, Compound K, having the structure shown in FIG. 28 was
synthesized as follows. Into a 200 mL round bottom flask under
nitrogen was placed Compound J (OG 1563) (1.9 g, 2.67 mmol, 3.3
equiv)
##STR00012##
and Compound E (0.525 g, 0.81 mmol, 1.0 equiv) (see FIG. 38)
followed by dimethylformamide (10 mL) then diisopropylethylamine
(2.5 mL, 14.6 mmol, 18 equiv). The flask was cooled to 0.degree. C.
using an ice bath. To this was added propylphosphonic anhydride
solution (50 wt. % in ethyl acetate, 2.5 mL, 4.04 mmol, 5 equiv)
over -6 minutes.
[0388] The reaction was warmed to room temperature and stirred for
15 minutes. The reaction was quenched by adding water (20 mL),
saturated aqueous sodium bicarbonate (20 mL) and ethyl acetate (100
mL). The organic layer was separated and the aqueous layer
extracted with ethyl acetate (75 mL). The combined organic layers
were washed with saturated aqueous sodium bicarbonate (30 mL), 0.5
M aqueous citric acid (40 mL), water (25 mL), and saturated aqueous
sodium chloride (40 mL), then dried (sodium sulfate), filtered and
concentrated under vacuum. The residue which was used without
further purification resulted in 2.0 g (0.80 mmol, 99%) of Compound
K.
[0389] 1H NMR (400 MHz DMSO-d6): D D=1.36 (s, 9H, OCCH3), 1.90 (s,
54H, CC(CH3)2Br), 2.31 (t, J=7.2 Hz, 6H, CCH2CH2NH), 2.98 (d, J=5.6
Hz, 6H, CCH2NH), 3.04 (q, J=6.0 Hz, 2H, OCH2CH2NH), 3.18 (s, 2H,
OCH2C), 3.3-3.37 (m, 8H, CH2), 3.47-3.55 (m, 12H, CH2), 3.58 (s,
6H, OCH2C), 3.87 (s, 6H, O.dbd.CCH20), 4.27 (s, 18H, CCH2OC=0),
6.74 (br t, 1H, CH2NHC=0), 7.69 (t, J=6.8 Hz, 3H, CH2NHC=0), 7.84
(t, J=6.0 Hz, 3H, CH2NHC=0). LC-MS (ES, m/z): [(M+2H-boc)/2]+ Calcd
for (C84H136Br9N7033+2H-Boc)/2=1196.6; Found 1196.6.
[0390] Next Compound L (FIG. 27) was synthesized as follows: into a
100 mL round bottom under nitrogen was added Compound K (2.0 g, 0.8
mmol), dichloromethane (10 mL) followed by trifluoroacetic acid (5
mL). The reaction was stirred at room temperature for 30
minutes.
[0391] The reaction was concentrated under a vacuum. The reaction
was diluted using dichloromethane (10 mL) and concentrated under a
vacuum. The residue was dissolved using acetonitrile (10 mL),
filtered through a syringe filter (Acrodisc CR25, PN 4225T) and
loaded onto a preparatory HPLC column and eluted with 60%
acetonitrile in water (with 0.1% trifluoroacetic acid) up to 98%
acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing
product were pooled, concentrated under vacuum, frozen and placed
on a lyophilizer. This resulted in 990 mgs (0.4 mmol, 50% over 2
steps) Compound L as a white powder.
[0392] 1H NMR (400 MHz DMSO-d6): D D=1.90 (s, 54H, CC(CH3)2Br),
2.31 (t, J=7.2 Hz, 6H, CCH2CH2NH), 2.97-3.0 (m, 8H, CCH2NH and
OCH2CH2NH), 3.17 (s, 2H, OCH2C), 3.3 (q, 6H, CH2CH2NHC=0), 3.4-3.59
(m, 20H, CH2), 3.87 (s, 6H, 0=CCH20), 4.27 (s, 18H, CCH20C=0),
7.69-7.84 (m, 9H, both CH2NHC=0 and NH3+). LC-MS (ES, m/z):
[(M+2H)/2]+ Calcd for (C84H136Br9N7033+2H)/2=1196.6; Found
1197.4.
[0393] Next, compound L was used as an initiator to synthesize MPC
polymer. Initiator is typically prepared as a stock solution in DMF
of about 100 mg/mL. The initiator and the ligand (2,2'-bipyridyl)
were introduced into a Schlenk tube. The resultant solution was
cooled to -78.degree. C. using a dry ice/acetone mixture, and was
degassed under vacuum for 10 min. The tube was refilled under Argon
and the catalyst (CuBr unless otherwise indicated), kept under
Argon, was introduced into the Schlenck tube (the Molar ratio of
atom bromine on the initiator/catalyst (CuBr)/ligand was kept at
1/1/2). The solution became dark brown immediately. The Schlenk
tube was sealed and immediately purged by applying a short cycle
vacuum/Argon. A solution of HEMA-PC was prepared by mixing a
defined quantity of monomer, prepared in a glovebox kept under
nitrogen, with 200 proof degassed ethanol. The monomer solution was
added drop wise into the Schlenk tube (via cannula) (and
homogenized by light stirring), The temperature was maintained at
-78.degree. C. A thorough vacuum was applied to the reaction
mixture for at least 10 to 15 min. until bubbling from the solution
ceased. The tube was then refilled with Argon and warmed to room
temperature. The solution was stirred, and as the polymerization
proceeded, the solution became viscous. After 3 to 8 hours or just
left overnight, the reaction was quenched by direct exposure to air
in order to oxidize Cu (I) to Cu (II), the mixture became
blue-green in color, and was passed through a silica column in
order to remove the copper catalyst. The collected solution was
concentrated by rotary evaporation and the resulting mixture was
either precipitated with tetrahydrofuran or dialyzed against water
followed by freeze drying to yield a free-flowing white powder. The
table below sets forth polymer data for polymer employing compound
L as an initiator.
TABLE-US-00009 Theor. MW Polymer (kDa) ID No. Initiator Mn(kDa)
Mp(kDa) PDI 500 130 L 490 530 1.1 750 150 L 645 750 1.1
[0394] Next, the maleimide Mal-PEG4-PFP ester was snapped on (as
set forth in FIG. 29) to the 750 kDa polymer referred to above to
provide OG 1802. Into a 20 mL vial was placed Polymer R3707 (750
kDa polymer made using L as initiator, 515 mg) and dissolved using
ethanol (4.0 mL) after stirring for 40 minutes. To this was added a
1% solution of 4-methylmorpholine in acetonitrile (22 uL). In a
separate vial was dissolved Mal-PEG4-PFP (1.97 mg) in acetonitrile
(1.0 mL) and this solution was added to the polymer solution over
-2 minute at room temperature and the resulting solution was
stirred for overnight. The reaction was diluted with 0.1% aqueous
trifluoroacetic acid (2 mL) (pH -5) followed by water (-12 mL),
filtered through a syringe filter (Acrodisc Supor, PN 4612) and
placed evenly into 3 Amicon centrifuge membrane dialysis tubes
(30,000 mwco). The tubes were diluted and mixed with water (-5 mL
each), placed into centrifuge (rpm 3200) for 25 minutes. The
filtrate is removed for analysis while the retentate is diluted and
mixed with water (-10 mL/tube). The centrifuge procedure repeated 5
more times, after which the retentate is removed and placed into a
vial. The Amicon membrane tubes were rinsed with water (2.times.-2
mL each tube) and this combined with the retentate. The retentate
solution was filtered through a syringe filter (Acrodisc Supor, PN
4612), frozen and placed on a lyophilizer. This resulted in 485 mgs
as a white powder.
Example 33. Biacore Binding Studies of TAP (OG 1448 and OG
1321)
[0395] The binding affinity of OG 1448 (and OG 1321) to its
intended targets was evaluated via Biacore assay. Binding studies
were performed at 25.degree. C. and 37.degree. C. using BioRad
Protean XPR36 and Biacore 2000 optical biosensors equipped with GLM
(Protean) and CM4 (Biacore) sensor chips and equilibrated with
running buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween-20, 0.2
mg/ml BSA). OG 1448, OG 1321, bevacizumab, aflibercept and
anti-PDGF were immobilized to the sensor surface via
amine-coupling.
[0396] Binding of the coupled proteins to the ligands was
determined by standard methodology. For example, rhVEGFA-165 was
tested for binding in a three-fold dilution series starting at 52
nM. rhVEGFA-165 was injected across the surface for five minutes
and then the dissociation phase was monitored for >1000 seconds
as the surfaces were washed with buffer. The rhVEGFA-165/001448
complex appeared quite stable, as indicated by the apparently flat
response during the wash phase (>300 seconds) (data not shown).
The dissociation phase for the 52 nM rhVEGFA-165 was monitored for
more than 2 hours. No decrease in the binding response over time
was observed.
[0397] Similarly, rhPDGF-BB was tested for binding in a three-fold
series starting at 11.4 nM. For the rhPDGF-BB/OG 1448 interactions,
the rate constants were too fast to be reported with confidence
because of mass transport effects. The following K.sub.D constants
were observed:
TABLE-US-00010 K.sub.D (pM) OG1321 OG1448 Bevacizumab Aflibercept
Anti-PDGF rhVEGFA-165 9.8 .+-. 0.1 5.1 .+-. 0.1 9.6 .+-. 0.8 1.56
.+-. 0.2 (25.degree. C.) rhPDGF-BB 14 .+-. 3 17 .+-. 2 107 .+-. 3
(25.degree. C.)
Example 34. TAP--a Competitive Inhibitor of rhVEGFA-165 Binding to
rhVEGFR
[0398] As a measure of its potential potency on anti-VEOP activity,
binding activity of TAP (001448 and 001321) to VEOPA-165 was
evaluated in a competitive binding assay where TAP, at different
concentrations, was competing with immobilized rhVEOPR for binding
of rhVEOP. rhVEOPA-165 bound by the immobilized VEOPR was
determined by ELISA (data not shown).
[0399] Human VEOPR 1/Pc was coated onto the bottom of 96-well ELISA
plates at 1.0 .mu.g/mL. Various concentrations of TAP (001448 and
001321), ranging from 0.39 to 200 nM, were incubated with 0.1 nM of
biotinylated VEOPA-165 for 30 min before adding to the ELISA
plates. Biotinylated-rhVEOPA-165 bound to VEOPR 1 was detected by
streptavidin-HRP and followed by development with HRP substrates.
Ranibizumab (Lucentis) and bevacizumab (Avastin) were similarly
tested for competitive binding inhibition of VEOPA-165 to VEOPR
1.
[0400] 001321, 001448, ranibizumab and bevacizumab showed similar
1C.sub.S0S in inhibiting the binding of VEOP-165 to rhVEOPR
suggesting similar potential potency in anti-VEOP activity. These
results suggest that TAP (both 001448 and 001321) can be as potent
as the approved agents ranibizumab and bevacizumab, hence, suitable
for treating neovascular (i.e., wet) AMD.
TABLE-US-00011 IC.sub.50(nM) OG1321 OG1448 Ranibizumab Bevacizumab
Competitive 12.5 .+-. 1.2* 8.5 .+-. 1.1* 12.5 .+-. 1.2* 10.7 .+-.
0.9* binding to rhVEGFA-165 (vs VEGFR) *Mean and SD of at least
three trials.
Example 35. 001448--a Competitive Inhibitor of rhVEOPA-165 Binding
to rhVEOPR in the Presence of rhPDOP-BB
[0401] To evaluate whether 001448 can bind rhVEOPA-165 in the
presence of rhPDOP-BB, i.e., whether rhPDOP-BB binding to the
receptor decoy of TAP inhibits the ability of TAP to bind to
rhVEGPA-165, a similar binding study to Example 27 was conducted
but in the presence of various concentrations of rhPDGP-BB.
[0402] Human VEGPR 1/Pc was coated onto the bottom of 96 well ELISA
plates at 1.0 .mu.g/mL. Various concentrations of OG 1448 were
incubated with 0.1 nM of rhVEGPA-165 plus rhPDGP-BB at 0.4, 1.2 and
2.0 nM, respectively, for 30 minutes before adding to the ELISA
plates. rhVEGPA-165 binding to rhVEGPR 1 was detected by
biotinylated anti-VEGPA antibody, 0.4 .mu.g/mL, followed with
streptavidin HRP and HRP substrate. OG 1448 was found to have an
IC50 (nM) of 10.1. This is quite comparable to the IC50 observed
without rhPDGP-BB from example 28. The value for OG 1321 was not
determined in this assay but is expected to be similar to OG
1448.
Example 36. TAP--a Competitive Inhibitor of rhPDGP-BB Binding to
rhPDGPR
[0403] As a measure of its potential potency of anti-PDGP activity,
the binding activity of TAP (OG1448 and 001321) to rhPDGP-BB was
evaluated in a competitive binding assay where TAP, at different
concentrations, was competing with immobilized PDGPR for binding of
rhPDGPBB. rhPDGP-BB bound to immobilized PDGPR was determined by
ELISA assay.
[0404] Human PDGPR/Fc was coated onto the bottom of 96-well ELISA
plates at 0.4 .mu.g/mL. Various concentrations of OG 1448 and OG
1321, ranging from 1 pM to 20 nM, were incubated with 0.2 nM of
rhPDGP-BB for 30 minutes before adding to the ELISA plates.
rhPDGP-BB bound to rhPDGPR was detected by biotinylated anti-PDGPBB
antibody, 0.4 .mu.g/mL, followed with streptavidin-HRP and HRP
substrate.
[0405] OG 1448, OG 1321 and a reference anti-PDGP antibody showed
similar IC50s in inhibiting rhPDGPBB binding to PDGPR, as shown in
the following table, suggesting highly potent anti-PDGP
activity.
TABLE-US-00012 IC50 (pM) OG1321 OG1448 Anti-PDGFBB Competitive
Binding 46 .+-. 21* 54 .+-. 21 66 to rhPDGFBB (vs rhPDGFR) *Mean
and SD of at least 3 trials.
Example 37. OG 1448--a Competitive Inhibitor of rhPDGP-BB Binding
to rhPDGPR in the Presence of rhVEGPA-165
[0406] To evaluate whether OG 1448 can bind rhPDGF-BB in the
presence of rhVEGFA-165, a similar competitive inhibition of
binding study towards PDGF (as Example 29) was performed in the
presence and absence of rhVEGFA-165.
[0407] Human PDGFRb/Fc was coated onto the bottom of 96-well ELISA
plates at 0.4 .mu.g/mL. Various concentrations of OG 1448 were
incubated with 0.2 nM of PDGFBB and with 0.2 nM of PDGFRb plus
rhVEGFA-165 at 0.2 nM, 0.6 nM and 1.0 nM, respectively, for 30
minutes before adding to the ELISA plates. PDGF-BB bind to PDGFRb
was detected by biotinylated anti-PEGFBB antibody, 0.4 .mu.g/mL,
followed by streptavidin HRP and HRP substrate. The IC50 (pM) in
the presence of rhVEGFA-165 (25) was comparable to the figure
derived in Example 29. The figure for OG1321 in the presence of
rhVEGFA-165 was not determined but is expected to be similar.
Example 38. Inhibition of VEGF-induced Proliferation of Primary
Human Retinal Microvascular Endothelial Cells (HRMVEC)
[0408] Endothelial cell proliferation is a crucial step in
angiogenesis and hence in the pathogenesis of neovascular AMD. The
ability of OG 1448 to antagonize the proliferating action of VEGF
on primary human retinal microvascular endothelial cells can be a
measure of its bioactivity in treating neovascular AMD.
[0409] HRMVECs were stimulated with 1.3 nM of rhVEGF165-A for 3
days in the presence of various concentrations of TAP (OG 1448 and
OG 1321) and reference drugs. Cell proliferation was measured by
WST-1 cell proliferation detection reagent. Results are shown in
the table below:
TABLE-US-00013 IC50 (nM) OG1321 OG1448 Ranibizumab Bevacizumab
Aflibercept Inhibition of 0.43 .+-. 0.05* 0.49 .+-. 0.05* 0.98 .+-.
1.21* 0.81 .+-. 0.32* 0.55 .+-. 0.08* VEGF induced proliferation of
HRMVECs *Mean and SD of at least 3 trials.
[0410] OG 1448 and OG 1321 demonstrated an IC50 in this assay
comparable to other approved anti-VEGF therapies. These data show
that TAP (both OG1448 and 001321) has at least comparable potency
to inhibit VEGF-mediated retinal microvascular endothelial cell
proliferation activity as ranibizumab, bevacizumab and
aflibercept.
Example 39. Inhibition of PDGF-Induced Proliferation of Primary
Human Brain Vascular Pericytes (HBVP)
[0411] Pericyte migration and proliferation are crucial events in
angiogenesis and hence play important roles in the pathogenesis of
neovascular AMD. The ability of TAP (OG 1448 and OG 1321) to
antagonize the proliferating action of PDGF on human brain
pericytes can be a measure of its effectiveness in treating
neovascular AMD.
[0412] HBVPs were stimulated with 2.0 nM of PDGFBB for 3 days in
the presence of various concentrations of TAP (OG 1449 and OG 1321)
and a reference anti-PDGF-BB antibody (R&D Systems, Catalog #
AB-220-NA). Cell proliferation was measured by WST-1 cell
proliferation detection reagent.
TABLE-US-00014 IC50 (nM) OG1321 OG1448 Anti-PDGF Inhibition of 5.0
.+-. 2.0* 2.9 .+-. 1.4 5.4 PDGF induced proliferation of HPVPs
*Mean and SD of at least 3 trials.
[0413] From the various experiments above comparing OG 1321
(TAF443) to OG 1448 (TAF443 polymer conjugate), it can be seen that
conjugation to the HEMA-PC biopolymer does not negatively impact
protein activity.
[0414] OG 1448 and OG 1321 show a comparable IC50 to the anti-PDGF
antibody.
Example 40. Inhibition of Sprouting in Co-Culture of Human Retinal
Microvascular Endothelial Cells (HRMVEC) and Human Mesenchymal
Pericytes (HMPs)
[0415] To mimic in vivo conditions where endothelial cells and
pericytes coexist in blood vessels and proliferate and migrate
together during angiogenesis, events crucial in neovascular AMD, a
three dimensional co-culture of HRMVECs and HMPs was established
with the goal of evaluating the ability of OG 1448 to inhibit
angiogenesis in this complex model.
[0416] Vehicle, Avastin, an anti-PDGF-BB antibody (same as above),
Avastin in combination with the anti-PDGF-BB antibody and OG1448
were added to the co-cultures on day 7. On day 14,
immunohistochemical staining of CD31 (endothelial cells) and aSMA
(pericytes) was used to quantify the lengths of sprouts emanating
from established endothelial cell spheroids as compared across the
experimental groups.
[0417] OG 1448 was more effective in inhibiting
endothelial/pericyte sprouting in HRMVEC-HMP co-culture than
Avastin alone or anti-PDGF alone at two different concentrations.
Moreover, OG 1448 was also more effective in inhibiting sprouting
then a combination of Avastin and the anti-PDGF-BB antibody. This
demonstrates that OG 1448 is synergistic relative to Avastin and an
anti-PDGF-BB antibody. The results are shown in the table below and
in FIG. 40.
TABLE-US-00015 Mean Total Sprout Length S.D. Relative Drug (pix)
(pix) Angiogenesis % S.D. % Vehicle 6999 1266 100 18 Avastin-5 nM
4700 722 67 10 Avastin-25 nM 3763 909 54 13 Anti-PDGF- 4924 884 70
13 5 nM Anti-PDGF- 4461 1051 64 15 25 nM Avastin + anti- 5197 948
74 14 PDGF-5 nM Avastin + anti- 4287 822 61 12 PDGF-25 nM OG1448-5
nM 3584 478 51 7 OG1448-25 nM 2933 360 42 5
Example 41. Efficacy of OG 1448 on Inhibition of Laser-Induced
Choroidal Neovascularization in Cynomolgus Monkeys
[0418] The in vivo efficacy of OG 1448 was evaluated using the
laser-induced choroidal neovascularization (CNV) model in
cynomolgus monkeys, a well-recognized primate model of CNV. See,
e.g., Nork T M, Dubielzig R R, Christian B J, et al. 2011.
Prevention of experimental choroidal neovascularization and
resolution of active lesions by VEGF trap in nonhuman primates.
Arch Ophthalmol. 129: 1042-1052; Lloyd R L, Harris J, Wadhwa S,
Chambers W. 2008. Food and Drug Administration approval process for
ophthalmic drugs in the U.S. Curr Opin Ophthalmol. 19:190-194, both
of which are hereby incorporated by reference. In this model, laser
lesions are placed in the chorioretinal complex in the macula of
the monkey eye with evidence of Bruch's membrane breakage.
Choroidal neovascularization is developed in two to three weeks. At
various time points, fluorescein angiography is used to evaluate
the clinically relevant lesions (Grade IV) which show fluorescein
leakage beyond the primary lesion. This CNV model has been used
extensively for the study of CNV lesions and used as a benchmark
for all currently approved treatment for neovascular AMD. In this
model, all approved anti-VEGF agents for neovascular AMD are
effective in inhibiting the leakage from the clinically relevant
Grade IV lesions. The study was conducted at Covance, Madison,
Wis.
[0419] In summary, a dose-related response to a single intravitreal
injection of OG 1448 at 0.5 or 2.4 mg/eye (calculated based on
protein content) was observed in the animals in which CNV lesions
were allowed to develop for 14 days before treatment and evaluated
at subsequent time points using fluorescein angiography focusing on
the clinically relevant Grade IV lesions on the retina/choroid. At
0.5 mg/eye, the beneficial effect on the Grade IV lesions was
noticeable (p=0.019; generalized estimating equation [GEE] model;
0.5 mg treatment Group 7 vs PBS injected placebo Group 5). At 2.4
mg/eye OG 1448, a dose (in molar equivalence) within the
therapeutic dose of bevacizumab or aflibercept, was highly
effective (75% reduction in Grade IV-CNV like lesions on Day 43
from Day 15 versus 27% reduction in the PBS-treated group)
(p=0.0007; GEE model; 2.4 mg treatment Group 9 vs PBS injected
placebo Group 5) in ameliorating the leakage of Grade IV-CNV
lesions.
[0420] OG 1448 shows effectiveness in inhibiting the leakage from
the clinically relevant Grade IV lesion in this benchmark CNV
model.
[0421] The groups and study design are shown in the following
table. The study included groups for tolerability (Groups 1
thorough 4) however for purposes of this patent application only
the groups for pharmacological activity and a control group treated
with phosphate buffered saline (PBS) injection are shown.
TABLE-US-00016 No. Dose Level Dose of mg/left mg/right Concen- Fe-
eye/ eye/ mg/kg/ tration Group males Dose Route dose dose dose
(mg/ml) 5 6 Intravitreous.sup.a 0 0 NA 0 6 6 Intravitreous.sup.a
0.24 0.24 NA 5.9 7 6 Intravitreous.sup.b 0.51 0.51 NA 10.2 9 6
Intravitreous.sup.b 2.40 2.40 NA 26.6 NA = not applicable .sup.aat
days 1, 15, and 29 (a total of 3 doses): laser on day 8 of the
dosing phase. .sup.bonce; laser treatment on 15 days prior to
injection
[0422] Two treatment regimens were evaluated. In the prevention
regimen, OG 1448 was given intravitreally three times bilaterally
at 0.24 mg/eye/dose (dose content was based on protein content;
Group 6) or PBS (Group 5) on days 1, 15 and 29 with laser treatment
on day 8 of the dosing phase. Fluorescein angiograms on days 15,
21, 30, 37 and 43 of laser treatment (days 22, 28, 37, 44 and 50 of
the dosing phase) were used for evaluation of the clinically
relevant Grade IV lesions.
[0423] In the treatment regimen (Groups 7 [0.5 mg], 8 [0.5 mg] and
9 [2.4 mg], OG 1448 was administered intravitreally to both eyes of
6 animals at doses of 0.5 mg (Groups 7 and 9) or 2.4 mg/eye (Group
9) 15 days after laser induction when CNV lesions were established.
Fluorescein angiograms obtained at Days 15, 21, 30, 37 and 43 of
laser treatment were used for evaluation of the clinically relevant
Grade IV lesions.
[0424] Using generalized estimating equation (Gee) models (Halekoh,
U & Yan J (2006) The R Package geepack for Generalized
Estimating Equations Journal of Statistical Software 15, 2, pp
1-11), a dose-related response to OG 1448 was observed in the
intervention regimen. At 0.5 mg/eye, the effect was notable as
shown by the difference in the percent change in Grade IV lesions
as compared to the vehicle control (0.5 mg treatment Group 7 vs PBS
injected placebo Group 5; p=0.019, GEE). With 2.4 mg/eye OG 1448 (a
dose in molar equivalence within the therapeutic dose of
bevacizumab or aflibercept) a 75% reduction in percent change in
Grade IV lesions (2.4 mg treatment Group 9 vs PBS injected placebo
Group 5; p=0.0007, GEE) was observed on day 43 as compared to a 27%
reduction in CNV in the PBS control group. The data from the
various experiments in the monkey CNV model are shown in FIG.
41.
[0425] OG 1448 shows dose dependent effectiveness in inhibiting the
leakage from the clinically relevant Grade IV lesion in this CNV
model. These results are consistent with the studies described
above showing activity of OG 1448 against VEGF-mediated angiogenic
activities.
Example 42. Tissue Distribution and Pharmacokinetics
[0426] A tissue distribution and pharmacokinetic study using
125I-OG 1448 was conducted using male New Zealand Red White Fl
Cross pigmented rabbits. In summary, this study showed a vitreal
half-life of 16.1 days for OG 1448 in rabbits, approximately three
times that reported for aflibercept (4.5 days) and 5 times that of
ranibizumab (2.9 days) (Bakri S J, Snyder M R, Reid J M et al.
2007. Pharmacokinetics of Intravitreal Ranibizumab [Lucentis].
[0427] Ophthalmology 114:2179-2182) with little plasma exposure
(approximately 0.2% of that of vitreous exposure) and a plasma
half-life of 6.5 days (aflibercept reported 6.5 days) (Struble C,
Koehler-Stec E, Zimmer E, and Tu W. 2008. Pharmacokinetics and
ocular tissue penetration of VEGF Trap after intravitreal
injections in rabbits. EVER; Portorz, Slovenia).
[0428] The purpose of this study was to assess the ocular
distribution and pharmacokinetics of non-radiolabeled test articles
and radiolabeled test articles following an intravitreal or
intravenous dose administration to male New Zealand Red White Fl
rabbits. Treatment groups and the study design are shown in the
table below:
TABLE-US-00017 Groups and Study Design (Covance study) # of Dose
Test Dose Sample Group males Route Article (mg) Collected 1 14 IVT
.sup.1251-OG1448 0.25/eye (OU) Blood, ocular tissues 2 2 IV
.sup.1251-OG1448 0.25/animal Blood 3 6 IVT OG1448 0.25 (OD) Blood,
whole eyes for histology 4 6 IVT OG1448 0.25 (OD) Blood, vitreous
humor IVT: intravitreal; IV: intravenous; OU: Both eyes; OD: right
eye
[0429] PK parameters were obtained based on radioanalysis.
Clearance profiles from vitreous, retina and choroid were similar
to one another. This pattern is consistent with other established
CNV treatments such as ranibizumab or aflibercept. Set forth in the
table below are pharmacokinetic parameters in different ocular
tissues after single bilateral intravitreal injection of 0.25 mg
.sup.125I-OG 1448.
TABLE-US-00018 Exposure as % of C.sub.MAX T.sub.1/2
AUC.sub.0-.infin. vitreous Matrix (NG Eq./G) (day) (Day*NG EQ./G)
exposure Plasma 494 6.48 3,790 0.189 Aqueous humor 5,250 11.6
68,800 3.423 Choroid-RPE 4,170 32.8 134,000 6.667 Iris-ciliary body
12,100 42.6 235,000 11.692 Retina 13,500 30.4 309,000 15.373
Vitreous humor 112,000 16.1 2,010,000 100.00
[0430] The ocular tissue half-life of various VEGF inhibitors is
compared with OG 1448 in the table below and in FIG. 42, which
suggests that OG 1448 can stay above a pharmaceutically active
minimal inhibitory concentration of 0.1 .mu.g/ml for greater than
90 days, as opposed to 30 days for Lucentis and 50 days for
Eylea:
TABLE-US-00019 Ocular Tissue Elimination Half Life (Days) Vitreous
Retina Choroid Pegaptinib.sup.1 3.5 -- -- Ranibizumab.sup.1 2.9 2.9
-- Aflibercept.sup.1 4.5 5.5 4.8 OG1448.sup.2 16.1 30.5 32.9
.sup.1Based on publicly available data from 28-day rabbit studies:
Drolet D W, Nelson J, Tucker CE, et al. 2000. Pharmacokinetics and
safety of an anti-vascular endothelial growth factor Aptamer (NX
1828) following injection into the vitreous humor of rhesus
monkeys. Pharm Res. 17: 1503-1510; Gaudreault J, Fei D, Beyer J C
et al. 2007. Pharmacokinetics and retinal distribution of
ranibizumab, a humanized antibody fragment directed against VEGF-A,
following intravitreal administration in rabbits. Retina 27:
859-870; Bakri (2007), supra; Struble 2008, supra. .sup.2Based on
intravitreal injection of 250 .mu.g in the rabbit eye.
[0431] The study showed a vitreal half-life of 16.1 days for OG
1448 in rabbits, approximately three times the 4.5 day vitreal
half-life reported for aflibercept and five times the vitreal
half-life of ranibizumab (2.9 days) (Bakri 2007, supra) with low
plasma exposure (approximately 0.2% of that of vitreous exposure);
the plasma exposure is consistent to that of aflibercept (Sinapis C
I, Routsias J G, Sinapis A l, et al. 2011. Pharmacokinetics of
intravitreal bevacizumab [Avastin.RTM.] in rabbits. Clinical
Ophthalmology 5:697-704). Similar to the reported data for
ranibizumab and aflibercept, the vitreal, retinal and choroidal
clearance profiles are similar to one another.
Example 43. Toxicology
[0432] Two pilot non-GLP single dose ocular and systemic
tolerability studies on OG 1448 were conducted at Covance: (i) a
single dose 57-day intravitreal or intravenous tolerability study
in pigmented rabbits and (ii) a single dose tolerability study
after intravitreal (58-day study) or intravenous (28-day study)
administration in cynomolgus monkeys.
[0433] In brief, single dose intravitreal injection of 0.25 mg OG
1448/dose/eye in rabbits was initially well tolerated but was
associated with persistent anterior (mild to moderate conjunctiva!
hyperemia, mild to moderate aqueous flare and cells) and posterior
segment (mild to severe white vitreous cells, mild to moderate
vitreous haze and presence of vitreous floaters, and multifocal
grey-white subretinal inflammatory foci) inflammation which
developed approximately two weeks postdose (or later). This
inflammatory response improved with immune-suppressive and
anti-inflammatory therapy. The time of onset postdose and response
to treatment are consistent with an immune-mediated response
typical for intraocularly administered humanized biopharmaceuticals
in animals.
[0434] In contrast, a single intravitreal dose at 0.24 or 1.4 mg
001448/dose/eye was well tolerated in cynomolgus monkeys with no
adverse finding or evidence of immune reactions ophthalmologically,
clinically, and histopathologically.
[0435] In the efficacy study (discussed above), intravitreal
injections of 0.24 mg/eye/dose for three times at 14 days apart or
a single injection of 0.5 mg/eye/dose were well tolerated with at
least 40 days of follow-up as shown on ocular examinations. No
immune-related reactions were noted in the eyes of treated
animals.
[0436] These studies demonstrate that OG 1448 is well tolerated
when administered intravitreally or intravenously at the doses
evaluated.
Example 44. Single-Dose Tolerance in Cynomolgus Monkeys
[0437] The purpose of this part of the study was to evaluate
tolerability of OG 1448 after intravitreous or intravenous
administration in cynomolgus monkeys.
[0438] Ocular and systemic tolerability groups and study design are
shown in the table below:
TABLE-US-00020 Group and Study Design No. Dose Level.sup.a,b Dose
of .mu.g/left mg/right Concen- fe- Dose eye/ eye/ mg/Kg/ tration
Group males Route dose dose dose (mg/ml) 1 3 IVT 0 0.236 NA 5.9 2 3
IVT 0 1.36 NA 27.2 3 2 IV NA NA 0.235 9.4 4 2 IV NA NA 1.41 9.4 IVT
= intravitreal; IV = intravenous; NA = not applicable .sup.aThe
right eye of animals in Groups 1 and 2 received the test article
via intravitreous injection. Animals in Groups 3 and 4 received the
test article via colus intravenous injection. .sup.bThe left eye of
animals in Groups 1 and 2 animals received vehicle control only
(phosphate buffered saline, pH 74).
[0439] Ocular examinations by board certified veterinary
ophthalmologists were performed across all four groups predose and
(i) for intravitreal groups: on days 3, 8, 15, 29, 43 and 57, and
for intravenous groups: on days 3, 8, 15, and 29. Animals were
followed with clinical observations and clinical pathology on days
3, 8, 15, 29, 43 and 57 when applicable. Anatomic pathology was
also performed--macroscopic observation during necropsy for all
animals, and microscopic evaluations for ocular tissues for groups
1 and 2 (day 57) and for a standard list of systemic organs for
groups 3 and 4 (day 29).
[0440] There were no adverse or toxicologically meaningful findings
in any group. There were no findings in clinical observations and
body weight in any group. There were no OG 1448-related macroscopic
or microscopic findings from anatomic pathology for any group
(ocular tissues for intravitreally injected groups and standard
list of organs/tissues for intravenously injected groups).
[0441] Ophthalmic findings for intravitreal administration groups
were limited to injection-related events such as mild to moderate
and transient presence of aqueous and/or vitreous cells and scars
at the site of aqueous humor sampling.
Example 45. Synthesis of Polymer OG 1786
[0442] OG 1786 is the nine-arm initiator for polymer synthesis used
as a precursor in the synthesis of OG 1802. Each arm is terminated
with a 2-bromoisobutyrate which is capable of initiating
polymerization under ATRP. OG1786 is a salt of trifluoro acetic
acid (TFA) as shown in FIG. 30. OG 1786 is prepared as follows.
First, OG 1550 is reacted with TFA (trifluoro acetic acid) to
produce OG 1546 as depicted in FIG. 31.
[0443] In a 1 L round bottom flask equipped with a magnetic stir
bar and an addition funnel was added OG 1550 (14.8 g), methyl
tert-butyl ether (MTBE) (350 ml) and water (30 ml). The mixture was
stirred to dissolve the OG 1550, then cooled in an ice bath. To
this mixture was added a solution of trifluoroacetic acid (4.9 ml)
in water (90 ml) dropwise over 90 minutes. After addition is
complete the mixture was stirred an additional 15 minutes then
removed from the ice bath and allowed to warm to room temperature.
The mixture was stirred (after removal from the ice bath) for a
further 4-5 hours, until tlc showed -5% starting material
remaining, and the pH of the aqueous was between 3 and 4 (pH
paper).
[0444] The mixture was partitioned. The MTBE layer was washed with
water (30 ml). Combine aqueous layers then the aqueous extracted
with MTBE (150 ml). This second MTBE phase was washed with water
(30 ml). The combined aqueous layers were washed with a third
portion of MTBE (100 ml). The third MBTE phase was washed with
water (25 ml). The aqueous layers were again combined (-250 ml, pH
-4, by pH paper).
[0445] The product was collected by lyophilization. 11.5 g white
solid was obtained. This material is extremely hygroscopic, so best
handled under nitrogen. The product was confirmed by LCMS.
[0446] The prepared OG 1546 was then reacted with OG 1563 to yield
OG 1784 (as depicted in FIG. 32).
[0447] In a 250 ml flask under nitrogen equipped with a stir bar
was added OG 1546 (hygroscopic, 9.0 g), followed by
N,N-dimethylformamide (110 ml). The mixture was stirred at room
temperature until all OG 1546 dissolved (about 15 minutes), then OG
1563 (29.9 g) was added, and the mixture stirred a further 3
minutes until the OG 1563 had also been dissolved. The resulting
solution was cooled in an ice bath, and N,N-diisopropylethylamine
(37.6 ml) was added over 3 minutes, followed by propylphosphonic
anhydride (T3P), 50% in ethyl acetate (34.5 ml) dropwise over 5
minutes (T3P addition is exothermic). After T3P addition was
complete, the flask was removed from the cooling bath and allowed
to reach room temperature. Samples were then taken at 5 minute
intervals for LCMS analysis. The reaction showed very light
yellow/tan color.
[0448] After 20 minutes the reaction was cooled again in an ice
bath and 5 ml water added. The mixture was then removed from the
cooling bath and a further 50 ml water portion added, followed by
50 ml 0.5 M citric acid then isopropylacetate (300 ml). The mixture
was partitioned. The aqueous phase (-300 ml) was extracted with
additional isopropyl acetate (150 ml). The aqueous phase was AQ 1
for HPLC test. The combined organics were washed with aqueous
citric acid (115 ml, 65 mM, which was the mixture of 15 ml of 0.5 M
citric acid plus 100 ml water), and the aqueous phase was AQ2
(pH-3). The organic phase was washed with water/saturated sodium
chloride (100 ml/25 ml), and the aqueous phase was AQ3 (pH-3). The
organic phase was finally washed with saturated sodium chloride
(100 ml), and the aqueous phase was AQ4. None of the AQ fractions
contained any significant product (data not provided). The organic
phase confirmed the product via LCMS. The product was dried over
sodium sulfate (80 g), filtered and rinsed with isopropyl acetate
(75 ml), and concentrated on a rotary evaporator to a tan oil (33.2
g). The crude was stored overnight under nitrogen.
[0449] The next day the crude was allowed to come to room
temperature, then dissolved in acetonitrile/water (46 ml/12 ml) and
filtered using an HPLC filter disk (Cole-Parmer PTFE 0.2 .mu.m,
product number 02915-20). The filtrate was split into three equal
portions and purified in three runs.
[0450] Loaded onto a RediSep Rf Gold C18 column (275 g, SN
69-2203-339, Lot #24126-611Y) equilibrated with 50%
acetonitrile/water. The material was eluted at 100 ml/min using the
following gradient (solvent A: water, solvent B: acetonitrile). All
the relevant fractions were checked by HPLC. The fractions adjudged
to be pure enough were pooled (from all three runs) and
concentrated (bath temperature kept at about 20.degree. C.) on
rotovap, then partitioned between dichloromethane (100 ml) and
water (5 ml)/saturated sodium chloride (25 ml). The aqueous was
extracted twice more with dichloromethane (2.times.30 ml). The
combined organics were dried over sodium sulfate (35 g), filtered,
rinsed with DCM (30 ml), and concentrated. The product and purity
were confirmed by LCMS methods.
TABLE-US-00021 OG1784 lot R5172 R5228 OG1546 used 5.3 g 9.0 g
OG1563 used 17.6 g 29.9 g Isolated yield 53% 58% Purity (a/a 210
nm) 99.3% 100.0%
[0451] Next OG 1405 was prepared from OG 1784 as depicted in FIG.
33. In a 500 ml round bottom flask equipped with a magnetic stir
bar was added OG 1784 (20.9 g), followed by dichloromethane (50 ml)
then trifluoroacetic acid (20 ml). The mixture was stirred at room
temperature and HPLC analysis showed complete deprotection in 23
minutes. The mixture was concentrated on a rotary evaporator,
redissolved in dichloromethane (25 ml) and re-concentrated, then
redissolved in acetonitrile (25 ml) and re-concentrated. The
product was confirmed by LCMS. The material from above (OG 1405,
34.5 g, assume 21.0 g as quantitative yield) was used as a crude
oil in the next step. No purification is needed. Next, OG 1405 was
reacted with OG 1402 to prepare OG 1785 as set forth in FIG. 34. In
a 500 ml flask under nitrogen equipped with a stir bar was placed
OG 1402 (5.5 g), followed by acetonitrile (70 ml), then
N,N-diisopropylethylamine (26.3 ml) and T3P solution (see above)
(7.9 ml). The solution was stirred at room temperature for 30
minutes, then cooled in an ice water bath and a solution of OG 1405
(crude oil from above, 34.5 g) in acetonitrile (70 ml) added. The
mixture was warmed to room temperature. After 20 minutes the
reaction was cooled in an ice water bath and quenched with water (5
ml). The mixture was then concentrated under vacuum using a rotary
evaporator to half volume. Samples were taken for LCMS.
[0452] More water (50 ml), followed by 0.5 M citric acid (75 ml)
and isopropyl acetate (175 ml) was added. The mixture was
partitioned in 5 minutes. The aqueous was extracted with additional
isopropyl acetate (50 mL). The combined organics were washed with
aqueous citric acid (0.13 M, 30 ml, consist of 10 ml of 0.5 M
citric acid and 20 ml water). The organics were then washed with
the mixture of saturated sodium chloride (25 ml) and water (25 ml),
then finally washed with the saturated sodium chloride (25 ml).
They were then dried over sodium sulfate (124 g), filtered and
rinsed with isopropyl acetate (30 ml) and concentrated under rotary
evaporator to a tan oil (27.3 g). Samples were taken for LCMS
analysis.
[0453] The oil was dissolved in acetonitrile/water (3: 1, 15 ml/5
ml), filtered through an HPLC filter disk (Cole-Parmer PTFE
membrane 0.2 .mu.m, product number 02915-20) and split into three
equal portions, each of which were individually purified as
follows.
[0454] Portions were loaded onto Redi-Sep Gold C18 column (275 g,
SN-69-2203-339, Lot 241234-611W) equilibrated at 50% solvent B
(acetonitrile)/50% solvent A (water). The material was then
purified by reverse phase HPLC with a solvent A: water/solvent B:
acetonitrile gradient. Appropriate fractions were pooled and
partitioned between dichloromethane (150 ml) and water (5
ml)/saturated sodium chloride (25 ml). The aqueous was extracted
twice with dichloromethane (2.times.50 ml). Combined organics were
dried over sodium sulfate (60 g), filtered and rinsed with
dichloromethane (40 ml) and concentrated. Structure and purity were
confirmed by various analytics including LCMS: OG 1785 was isolated
as a foamy solid (R5329, 19.0 g, 83% yield, 95.1% purity (a/a 210
nm), stored under nitrogen at 4.degree. C.
[0455] Next, the tert-butyloxycarbonyl protecting group on OG 1785
was removed using trifluoroacetic acid (TFA) to produce OG 1786 as
depicted in FIG. 35.
Example 46. Synthesis of Polymer 1801
[0456] Compound OG 1802 is conjugated to a sulfhydryl group of
TAF443 to produce OG1448. Polymer OG1801 is made first from the
initiator OG1786. OG1801 has an amine functionality, which is more
stable (than maleimide) during polymer synthesis. To synthesize
polymer OG 1801, a modified version of ATRP is used wherein the
copper species (Cu(I)) is generated in situ by adding metallic
copper to Cu (II). Starting materials and reagents needed in the
reaction are calculated based on batch input of the monomer
(HEMA-PC) OG47, as well as the targeted molecular weight (MW).
[0457] Weighed 50 g monomer OG47 in glove box and added 200 mL of
degassed EtOH to dissolve the monomer at room temperature; sampled
for monomer concentration test. Weighed Cu (II), Bpy, Cu(O) in a
500 mL flask; purged with Argon, while adding monomer solution to
the flask; sealed the flask with stopper and vacuumed for 25 min
until no bubbles. The reaction changed color gradually from light
green to dark green, then to light brown; weighed -200 mg of
initiator OG 1786 in glove box, and dissolved in -2000 uL of DMF
under room temperature to make 100 mg/mL stock solution; Sampled
for initiator concentration and purity test; Added the initiator
solution to the flask under Argon. The reaction solution became
dark brown and started thickening over time; Sealed the system and
let the reaction occur over 2 days.
[0458] OG1801 was then prepared for addition of the maleimide and
catalyst (copper) was removed as follows: A prepacked RediSep.RTM.
Rf normal phase silica column is used to remove the catalyst. The
size of the column is chosen based on the copper amount in the
reaction mixture. For instance, a 330 g column (Cat. #69-2203-330,
Column size 330 g, CV=443 mL) was used for a 50 g batch of OG 1801.
Teflon tubing is used for all the connection as EtOH is the elute
solvent.
[0459] After copper removal, transferred all the fractions to a
round bottom flask in batches, and evaporated the EtOH by rotary
evaporator at 45-50.degree. C. at reduced pressure to dryness. In
this step, EtOH volume collected from condensation was monitored to
make sure EtOH removal was >90%. The polymer was dissolved in
250 mL of WFI and filtered using a 0.2 um filter. It resulted in a
clear to light yellow polymer solution at -150 mg/mL. The solution
could be stored at 2-8.degree. C. up to 3 month before use.
Example 47. Synthesis of Polymer OG 1802
[0460] Starting materials and reagents needed in the reaction is
calculated based on batch input of OG 1801. The linker is
3-maleimidopropionic acid, NHS ester. Added 30 ml of 0.5 M sodium
phosphate (in WFI, pH8) to 50 g polymer solution (-150 mg/mL). Let
stir for 1 min; pH was 8.0 by pH paper. Weighed 204.8 mg of linker
and dissolved in DMF 4.1 mL to make 50 mg/mL stock sln; Added
linker solution dropwise 815 uL per minute to the polymer sln with
strong stirring. Took 5 min to added 4095 uL of linker solution.
Reacted at room temperature for 30 min. Quenched reaction with 20
mL of 5% acetic acid to achieve a final pH of 5. Filtered the
solution using 1 L vacuum filter (0.2 um).
[0461] OG 1802 is then purified as follows: Milipore cross flow
cassettes was used for polymer purification in aqueous system.
Started with concentrating the polymer solution to 250 mL (-200
mg/mL). Added the fresh WFI from reservoir, and adjusted the flow
rate of the fresh WFI feed to the same as the permeate (-2 mL/min).
The UF/DF was set up at 2-8.degree. C. overnight. Typically 2.5 L
of WFI was used (10.times. volume ratio to the polymer solution). A
sample of retente was collected for purity test. The targeted
purity was >98%. Filtered the polymer solution by 0.2 .mu.M 1 L
filter bottle. The polymer solution could be stored at 2-8.degree.
C. for up to 3 month before conjugation.
Example 48. Formulations of OG 1448; Injectability
[0462] 27 0.2 mg/ml and 44.5 mg/ml solutions of OG 1448 were
prepared using 1.7 mM KH.sub.2PO.sub.4; 5 mM Na.sub.2HPO.sub.4; 150
mM NaCl in sterile water for injection. The OG 1448 conjugate was
concentrated by a Millipore Pellicon XL TFF cartridge (catalog #
PXB030A50, EMD Millipore), 30 kD MWCO or VIVACELL 100 spin
concentrator (catalog # VC1022, Sartorius), 30 kD MWCO, depending
on the volume. The 27.2 mg/ml solution of TAP was injected
intravitreally into the monkeys for the efficacy experiments
described above through a 30 gauge (G) V2 inch needle. Excessive
pressure was not required to push the OG 1448 through the needle.
The 44.5 mg/ml solution was tested for injectability in the
laboratory and was also capable of being pushed through the needle
without excessive pressure by a female operator.
Example 49. Storage Stability
[0463] An ongoing stability study was conducted using OG 1448
reference lot R5606 at 44.5 mg/ml in PBS at pH 7.4 (as described
above). Three temperatures were chosen for the study: room
temperature (RT), 4.degree. C. and -20.degree. C. Sampling
frequency is at 0, 14, 28, 91, 181 and 362 days. Samples were
evaluated by SDS-PAGE and analytical AE-HPLC for unreacted and
sequestered protein, and potential aggregates. It was observed
(data not shown) that OG 1448 demonstrates less than 5% protein
impurity by AE-HPLC at all three temperatures up to six months,
which is similar to the level at time 0. This study is ongoing.
Example 50. Alternative Phosphorylcholine Polymers
[0464] A HEA-PC polymer was synthesized as described below. HEA-PC
(2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate), which
is an acrylate as opposed to the methacrylate HEMA-PC described
above, has the following structure:
##STR00013##
[0465] HEA-PC was polymerized to the initiator shown in Example 23
as compound L.
TABLE-US-00022 Reactant Name Amount MW Initiator Compound L (see
above) 1.65 mg 2505.5 Monomer HEA-PC 0.461 g 281.24 Catalyst Cu (I)
Bromide 1.2 mg 143.45 Ligand Tris [2- 2.73 mg 230.39
(dimethylamino)ethyl]amine (Me6TREN) Solvent A
N,N-Dimethylformamide 21.85 .mu.l 73.09 (DMF) Solvent B Water 0.7
ml 18.02 Solvent C Methanol 0.7 ml 32.04
[0466] Prepared a stock solution of initiator at 200 mg/mL by
dissolving 2.2 mg of initiator in 11 .mu.l of dry DMF and a 200
mg/ml solution of ligand by dissolving 4.6 mg of Me6TREN in 23
.mu.L of dry DMF. Dispense 8.25 .mu.l of the stock solution of
initiator and 13.6 .mu.l of the ligand into a tube. Degas at
-78.degree. C. for 5 mn then refill with Argon and add 1.2 mg of
CuBr. Degas and refill with Argon. Add a stock solution of HEA-PC
in methanol (weigh out 0.461 g of HEA-PC and dissolve it in 0.5 mL
of methanol) to the solution inside the reactor at -78.degree. C.
Rinse the vial with 200 .mu.l of methanol and add it inside the
reactor at -78.degree. C. and then 0.5 mL of distilled water then
another 200 .mu.l of water. Degas thoroughly until no bubbling is
seen and all heterogeneity disappears (solid particulates dissolve
or disappear). Refill with 4 psi of Argon and let the reaction to
proceed at RT for an hour. The reaction was already viscous. The
reaction was allowed to proceed for about one hour. A solution of
bipyrindine in methanol (5 mg in 0.5 uL) was added. Another 2-3 ml
of methanol was added and the catalyst was allowed to oxidize
overnight at 4.degree. C. Conversion determined by 1H NMR was
estimated to be 94%.
[0467] The next day the polymer was dialyzed and subjected SEC/MALS
analysis using Shodex SB806M_HQ column (7.8.times.300 mm) in
1.times.PBS pH 7.4 at 1 ml/min, giving a PDI of 1.157, Mn of 723.5
kDa, Mp of 820.4 kDa and Mw of 837.2 kDa (before dialysis PDI is
1.12, Mn=695 kDa, Mp=778 kDa). Next a maleimide functionality was
added to the polymer so that it could be conjugate to a protein,
including TAF443.
[0468] Next, the maleimide Mal-PEG4-PFP (see Example 23 above)
ester was snapped on to the HEA-PC polymer as shown in Example 23.
The resulting maleimide functionalized HEA-PC polymer can then be
conjugated to sulfhydryl groups as discussed herein for HEMA-PC
polymers.
[0469] An acrylamide PC polymer was also made using the monomer
2-(acrylamyl)ethyl-2-(trimethylammonium)ethyl phosphate (Am-PC),
having the following structure:
##STR00014##
[0470] The Am-PC was used for polymerization employing a 3 arm
initiator (a TFA salt) having the structure:
##STR00015##
[0471] The synthesis of the Am-PC polymer was conducted as
follows:
TABLE-US-00023 Reactant Name/Identity Amount MW Initiator 3-arm
initiator (see above) 2.2 mg 885.35 Monomer Am-PC 0.5 g 280.26
Catalyst (I) Copper (I) Bromide 1 mg 143.45 Catalyst (II) Copper
(II) Bromide 0.2 mg 223.35 Ligand Tris[2- 3.94 mg 230.39
(dimethylamino)ethyl]amine (Me6TREN) Solvent A
N,N-Dimethylformamide 31.7 .mu.l 73.09 (DMF) Solvent B Water 1 ml
18.02 Solvent C Methanol 1 ml 32.04
[0472] A stock solution of ligand at 200 mg/mL was prepared by
dissolving 9 mg of Me6TREN in 45 uL of dry DMF. Add 19.7 uL of the
stock solution to a reaction vessel. Prepare a stock solution of
initiator at 200 mg/mL by dissolving 6.5 mg of material in 32.5 uL
of DMF. Add 11 uL of the initiator stock solution to the ligand
from above. Degas for 5 mn. Add 1 mg of CuBr. Prepared a stock
solution of CuBr.sub.2 at 200 mg/mL by dissolving 4 mg CuBr.sub.2
in 20 .mu.L of DMF. Add 0.5 g of monomer (AmPC) to 1 mL of methanol
(slow dissolution/viscous solution), followed by 1 uL of the stock
solution of CuBr.sub.2. Add the monomer solution dropwise to the
reaction mixture above. Rinse with 1 mL of water. Degas the
reaction mixture thoroughly (freeze-thaw). Let the reaction proceed
for 24 hours.
[0473] Afterwards the Am-PC polymer may be dialyzed. The molecular
weight of the above polymer was determined by SEC/MALS: Mn is 215
kDa, Mp: 250 kDa, PDI is 1.17. Conversion was estimated by 1H NMR
to be 94%. A maleimide functionality can be added to the Am-PC
polymer as discussed above for HEMA-PC and HEA-PC. Maleimide
functionalized Am-PC polymer can be conjugated to a protein, such
as TAF443, as described above.
Example 51. Reverse Ellman's Assay for Calculating Free Maleimide
in a Compound
[0474] After addition of the maleimide functionality to polymer OG
1801 to form OG 1802 (see above), an Ellman's assay is used to
determine the amount of functional maleimide (i.e. conjugatable) in
a sample. Thiol converts Ellman's reagent (DTNB) to TNB-then to
TNB2-in water at neutral and alkaline pH, which gives off a yellow
color (measured at 412 nm). A standard curve is established with
cysteine. Since the maleimide reacts with thiol, this assay
actually measures the thiol (cysteine) left. The inhibition is
calculated as the (original thiol-thiol left after maleimide
polymer addition)/(original thiol) and is expressed as a
percentage.
[0475] Reagents Employed in Assay: A standard curve was prepared
using the cysteine from 62.5 .mu.M to 2 .mu.M. Polymer stock
solutions were prepared by dissolving the powder in 1.times.PBS
pH7.4 (reaction buffer) and mixing thoroughly. An equal molar of
polymer and cysteine solutions were mixed and allowed to react at
27.degree. C. for 30 minutes. The 150 .mu.M of DTNB solution was
added into the cysteine standards and polymer/cysteine reactions
and the color was developed at 27.degree. C. for 5 minutes. OD at
412 nm was read on the Spectramax plate reader and percent
inhibition was calculated with the Softmax Pro software and the
cysteine standard curve.
[0476] All patent filings, websites, other publications, accession
numbers and the like cited above or below are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual item were specifically and individually
indicated to be so incorporated by reference. If different versions
of a sequence are associated with an accession number at different
times, the version associated with the accession number at the
effective filing date of this application is meant. The effective
filing date means the earlier of the actual filing date or filing
date of a priority application referring to the accession number if
applicable. Likewise if different versions of a publication,
website or the like are published at different times, the version
most recently published at the effective filing date of the
application is meant unless otherwise indicated. Any feature, step,
element, embodiment, or aspect of the invention can be used in
combination with any other unless specifically indicated otherwise.
Although the present invention has been described in some detail by
way of illustration and example for purposes of clarity and
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
TABLE-US-00024 SEQUENCES SEQ ID NO. 1 1 LVVTPPGPEL VLNVSSTFVL
TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG LDTGEYFCTH NDSRGLETDE
81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI TEITIPCRVT DPQLVVTLHE
KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161 TIGDREVDSD AYYVYRLQVS
SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK ESGRLVEPVT DFLLDMPYHI
241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD EKAINITVVE SGGGGGSGGG
GSDIQMTQSP SSLSASVGDR VTITCSASQD 321 ISNYLNWYQQ KPGKAPKVLI
YFTSSLHSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQYSTVPWTF GQGTKVEIKR
401 TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN
SQESVTEQDS KDSTYSLSST LTLSKADYEK 481 HKVYACEVTH QGLSSPVTKS FNRGEC
SEQ ID NO. 2 1 EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA
PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY 81 LQMNSLRAED
TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC
LVKDYFPEPV 161 TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG
TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL 241 LGGPSVFLFP
PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
VLTVLHQDWL 321 NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 401 PPVLDSDGSF
FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO. 3 1
LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGDIQMTQ SPSSLSASVG DRVTITCSAS QDISNYLNWY 321
QQKPGKAPKV LIYFTSSLHS GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQYSTVPW
TFGQGTKVEI KRTVAAPSVF 401 IFPPSDEQLK SGTASVVCLL NNFYPREAKV
QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV 481
THQGLSSPVT KSFNRGEC SEQ ID NO. 4 1 LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW
ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG LDTGEYFCTH NDSRGLETDE 81
RKRLYIFVPD PTVGFLPNDA EELFIFLTEI TEITIPCRVT DPQLVVTLHE KKGDVALPVP
YDHQRGFSGI FEDRSYICKT 161 TIGDREVDSD AYYVYRLQVS SINVSVNAVQ
TVVRQGENIT LMCIVIGNEV VNFEWTYPRK ESGRLVEPVT DFLLDMPYHI 241
RSILHIPSAE LEDSGTYTCN VTESVNDHQD EKAINITVVE SGGGGGSGGG GSEVQLVESG
GGLVQPGGSL RLSCAASGYT 321 FTNYGMNWVR QAPGKGLEWV GWINTYTGEP
TYAADFKRRF TFSLDTSKST AYLQMNSLRA EDTAVYYCAK YPHYYGSSHW 401
YFDVWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA
LTSGVHTFPA VLQSSGLYSL 481 SSVVTVPSSS LGTQTYICNV NHKPSNTKVD
KKVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV 561
VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKALPAPI EKTISKAKGQ 641 PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 721
FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO. 5 1 DIQMTQSPSS LSASVGDRVT
ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP
81 EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 161 ESVTEQDSKD STYSLSSTLT
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO. 6 1 LVVTPPGPEL
VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG LDTGEYFCTH
NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI TEITIPCRVT
DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161 TIGDREVDSD
AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK ESGRLVEPVT
DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD EKAINITVVE
SGGGEVQLVE SGGGLVQPGG SLRLSCAASG YTFTNYGMNW 321 VRQAPGKGLE
WVGWINTYTG EPTYAADFKR RFTFSLDTSK STAYLQMNSL RAEDTAVYYC AKYPHYYGSS
HWYFDVWGQG 401 TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF
PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 481 SSLGTQTYIC
NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSHED 561 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 641 LPPSREEMTK
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE 721 ALHNHYTQKS LSLSPGK SEQ ID NO. 7 1 EVQLVESGGG
LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF
SLDTSKSTAY 81 LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT
VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 161 TVSWNSGALT
SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH
TCPPCPAPEL 241 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 321 NGKEYKCKVS
NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN
GQPENNYKTT 401 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN
HYTQKSLSLS PGGGGGSGGG GSGGGGSGGG GSGLVVTPPG 481 PELVLNVSST
FVLTCSGSAP VVWERMSQEP PQEMAKAQDG TFSSVLTLTN LTGLDTGEYF CTHNDSRGLE
TDERKRLYIF 561 VPDPTVGFLP NDAEELFIFL TEITEITIPC RVTDPQLVVT
LHEKKGDVAL PVPYDHQRGF SGIFEDRSYI CKTTIGDREV 641 DSDAYYVYRL
QVSSINVSVN AVQTVVRQGE NITLMCIVIG NEVVNFEWTY PRKESGRLVE PVTDFLLDMP
YHIRSILHIP 721 SAELEDSGTY TCNVTESVND HQDEKAINIT VVESG SEQ ID NO. 8
1 LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCAASGYD 321
FTHYGMNWVR QAPGKGLEWV GWINTYTGEP TYAADFKRRF TFSLDTSKST AYLQMNSLRA
EDTAVYYCAK YPYYYGTSHW 401 YFDVWGQGTL VTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 481
SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP EAAGAPSVFL
FPPKPKDTLM ISRTPEVTCV 561 VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 641
PREPCVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSKLTV DKSRWQQGNV 721 FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO. 9
1 LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCAASGYD 321
FTHYGMNWVR QAPGKGLEWV GWINTYTGEP TYAADFKRRF TFSLDTSKST AYLQMNSLRA
EDTAVYYCAK YPYYYGTSHW 401 YFDVWGQGTL VTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 481
SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP EAAGAPSVFL
FPPKPKDTLM ISRTPEVTCV 561 VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 641
PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSKLTV DKSRWQQGNV 721 FSCSVMHEAL HNHYTQKSLS CSPGK SEQ ID NO. 10
1 DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS
RFSGSGSGTD FTLTISSLQP 81 EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 161
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID
NO. 11 1 MRLPGAMPAL ALKGELLLLS LLLLLEPQIS QGLVVTPPGP ELVLNVSSTF
VLTCSGSAPV VWERMSQEPP QEMAKAQDGT 81 FSSVLTLTNL TGLDTGEYFC
THNDSRGLET DERKRLYIFV PDPTVGFLPN DAEELFIFLT EITEITIPCR VTDPQLVVTL
161 HEKKGDVALP VPYDHQRGFS GIFEDRSYIC KTTIGDREVD SDAYYVYRLQ
VSSINVSVNA VQTVVRQGEN ITLMCIVIGN 241 EVVNFEWTYP RKESGRLVEP
VTDFLLDMPY HIRSILHIPS AELEDSGTYT CNVTESVNDH QDEKAINITV VESGYVRLLG
321 EVGTLQFAEL HRSRTLQVVF EAYPPPTVLW FKDNRTLGDS SAGEIALSTR
NVSETRYVSE LTLVRVKVAE AGHYTMRAFH 401 EDAEVQLSFQ LQINVPVRVL
ELSESHPDSG EQTVRCRGRG MPQPNIIWSA CRDLKRCPRE LPPTLLGNSS EEESQLETNV
481 TYWEEEQEFE VVSTLRLQHV DRPLSVRCTL RNAVGQDTQE VIVVPHSLPF
KVVVISAILA LVVLTIISLI ILIMLWQKKP
561 RYEIRWKVIE SVSSDGHEYI YVDPMQLPYD STWELPRDQL VLGRTLGSGA
FGQVVEATAH GLSHSQATMK VAVKMLKSTA 641 RSSEKQALMS ELKIMSHLGP
HLNVVNLLGA CTKGGPIYII TEYCRYGDLV DYLHRNKHTF LQHHSDKRRP PSAELYSNAL
721 PVGLPLPSHV SLTGESDGGY MDMSKDESVD YVPMLDMKGD VKYADIESSN
YMAPYDNYVP SAPERTCRAT LINESPVLSY 801 MDLVGFSYQV ANGMEFLASK
NCVHRDLAAR NVLICEGKLV KICDFGLARD IMRDSNYISK GSTFLPLKWM APESIFNSLY
881 TTLSDVWSFG ILLWEIFTLG GTPYPELPMN EQFYNAIKRG YRMAQPAHAS
DEIYEIMQKC WEEKFEIRPP FSQLVLLLER 961 LLGEGYKKKY QQVDEEFLRS
DHPAILRSQA RLPGFHGLRS PLDTSSVLYT AVQPNEGDND YIIPLPDPKP EVADEGPLEG
1041 SPSLASSTLN EVNTSSTISC DSPLEPQDEP EPEPQLELQV EPEPELEQLP
DSGCPAPRAE AEDSFL SEQ ID NO. 12 1 DIQLTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP 81
EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY
PREAKVQWKV DNALQSGNSQ 161 ESVTEQDSKD STYSLSSTLT LSKADYEKHK
VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO. 13 1 EVQLVESGGG LVQPGGSLRL
SCAASGYDFT HYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY
81 LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSSASTKGPS
VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 161 TVSWNSGALT SGVHTFPAVL
QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH L SEQ ID NO.
14 1 MVSYWDTGVL LCALLSCLLL TGSSSGSKLK DPELSLKGTQ HIMQAGQTLH
LQCRGEAAHK WSLPEMVSKE SERLSITKSA 81 CGRNGKQFCS TLTLNTAQAN
HTGFYSCKYL AVPTSKKKET ESAIYIFISD TGRPFVEMYS EIPEIIHMTE GRELVIPCRV
161 TSPNITVTLK KFPLDTLIPD GKRIIWDSRK GFIISNATYK EIGLLTCEAT
VNGHLYKTNY LTHRQTNTII DVQISTPRPV 241 KLLRGHTLVL NCTATTPLNT
RVQMTWSYPD EKNKRASVRR RIDQSNSHAN IFYSVLTIDK MQNKDKGLYT CRVRSGPSFK
321 SVNTSVHIYD KAFITVKHRK QQVLETVAGK RSYRLSMKVK AFPSPEVVWL
KDGLPATEKS ARYLTRGYSL IIKDVTEEDA 401 GNYTILLSIK QSNVFKNLTA
TLIVNVKPQI YEKAVSSFPD PALYPLGSRQ ILTCTAYGIP QPTIKWFWHP CNHNHSEARC
481 DFCSNNEESF ILDADSNMGN RIESITQRMA IIEGKNKMAS TLVVADSRIS
GIYICIASNK VGTVGRNISF YITDVPNGFH 561 VNLEKMPTEG EDLKLSCTVN
KFLYRDVTWI LLRTVNNRTM HYSISKQKMA ITKEHSITLN LTIMNVSLQD SGTYACRARN
641 VYTGEEILQK KEITIRDQEA PYLLRNLSDH TVAISSSTTL DCHANGVPEP
QITWFKNNHK IQQEPGIILG PGSSTLFIER 721 VTEEDEGVYH CKATNQKGSV
ESSAYLTVQG TSDKSNLELI TLTCTCVAAT LFWLLLTLFI RKMKRSSSEI KTDYLSIIMD
801 PDEVPLDEQC ERLPYDASKW EFARERLKLG KSLGRGAFGK VVQASAFGIK
KSPTCRTVAV KMLKEGATAS EYKALMTELK 881 ILTHIGHHLN VVNLLGACTK
QGGPLMVIVE YCKYGNLSNY LKSKRDLFFL NKDAALHMEP KKEKMEPGLE QGKKPRLDSV
961 TSSESFASSG FQEDKSLSDV EEEEDSDGFY KEPITMEDLI SYSFQVARGM
EFLSSRKCIH RDLAARNILL SENNVVKICD 1041 FGLARDIYKN PDYVRKGDTR
LPLKWMAPES IFDKIYSTKS DVWSYGVLLW EIFSLGGSPY PGVQMDEDFC SRLREGMRMR
1121 APEYSTPEIY QIMLDCWHRD PKERPRFAEL VEKLGDLLQA NVQQDGKDYI
PINAILTGNS GFTYSTPAFS EDFFKESISA 1201 PKFNSGSSDD VRYVNAFKFM
SLERIKTFEE LLPNATSMFD DYQGDSSTLL ASPMLKRFTW TDSKPKASLK IDLRVTSKSK
1281 ESGLSDVSRP SFCHSSCGHV SEGKRRFTYD HAELERKIAC CSPPPDYNSV
VLYSTPPI SEQ ID NO. 15 1 MQSKVLLAVA LWLCVETRAA SVGLPSVSLD
LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS 81
DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE
NKNKTVVIPC LGSISNLNVS 161 LCARYPEKRF VPDGNRISWD SKKGFTIPSY
MISYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE 241
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST 321 FVRVHEKPFV AFGSGMESLV EATVGERVRI
PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL 401
TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW
QLEEECANEP SQAVSVTNPY 481 PCEEWRSVED FQGGNKIEVN KNQFALIEGK
NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ 561
PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS
TNDILIMELK NASLQDQGDY 641 VCLAQDRKTK KRHCVVRQLT VLERVAPTIT
GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR 721
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFIIEGAQEK TNLEIIILVG TAVIAMFFWL
LLVIILRTVK RANGGELKTG 801 YLSIVMDPDE LPLDEHCERL PYDASKWEFP
RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR 881
ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT
KGARFRQGKD YVGAIPVDLK 961 RRLDSITSSQ SSASSGFVEE KSLSDVEEEE
APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN 1041
VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS
LGASPYPGVK IDEEFCRRLK 1121 EGTRMRAPDY TTPEMYQTML DCWHGEPSQR
PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS 1201
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS
GMVLASEELK TLEDRTKLSP 1281 SFGGMVPSKS RESVASEGSN QTSGYQSGYH
SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV SEQ ID NO. 16 1
MQRGAALCLR LWLCLGLLDG LVSGYSMTPP TLNITEESHV IDTGDSLSIS CRGQHPLEWA
WPGAQEAPAT GDKDSEDTGV 81 VRDCEGTDAR PYCKVLLLHE VHANDTGSYV
CYYKYIKARI EGTTAASSYV FVRDFEQPFI NKPDTLLVNR KDAMWVPCLV 161
SIPGLNVTLR SQSSVLWPDG QEVVWDDRRG MLVSTPLLHD ALYLQCETTW GDQDFLSNPF
LVHITGNELY DIQLLPRKSL 241 ELLVGEKLVL NCTVWAEFNS GVTFDWDYPG
KQAERGKWVP ERRSQQTHTE LSSILTIHNV SQHDLGSYVC KANNGIQRFR 321
ESTEVIVHEN PFISVEWLKG PILEATAGDE LVKLPVKLAA YPPPEFQWYK DGKALSGRHS
PHALVLKEVT EASTGTYTLA 401 LWNSAAGLRR NISLELVVNV PPQIHEKEAS
SPSIYSRHSR QALTCTAYGV PLPLSIQWHW RPWTPCKMFA QRSLRRRQQQ 481
DLMPQCRDWR AVTTQDAVNP IESLDTWTEF VEGKNKTVSK LVIQNANVSA MYKCVVSNKV
GQDERLIYFY VTTIPDGFTI 561 ESKPSEELLE GQPVLLSCQA DSYKYEHLRW
YRLNLSTLHD AHGNPLLLDC KNVHLFATPL AASLEEVAPG ARHATLSLSI 641
PRVAPEHEGH YVCEVQDRRS HDKHCHKKYL SVQALEAPRL TQNLTDLLVN VSDSLEMQCL
VAGAHAPSIV WYKDERLLEE 721 KSGVDLADSN QKLSIQRVRE EDAGRYLCSV
CNAKGCVNSS ASVAVEGSED KGSMEIVILV GTGVIAVFFW VLLLLIFCNM 801
RRPAHADIKT GYLSIIMDPG EVPLEEQCEY LSYDASQWEF PRERLHLGRV LGYGAFGKVV
EASAFGIHKG SSCDTVAVKM 881 LKEGATASEH RALMSELKIL IHIGNHLNVV
NLLGACTKPQ GPLMVIVEFC KYGNLSNFLR AKRDAFSPCA EKSPEQRGRF 961
RAMVELARLD RRRPGSSDRV LFARFSKTEG GARRASPDQE AEDLWLSPLT MEDLVCYSFQ
VARGMEFLAS RKCIHRDLAA 1041 RNILLSESDV VKICDFGLAR DIYKDPDYVR
KGSARLPLKW MAPESIFDKV YTTQSDVWSF GVLLWEIFSL GASPYPGVQI 1121
NEEFCQRLRD GTRMRAPELA TPAIRRIMLN CWSGDPKARP AFSELVEILG DLLQGRGLQE
EEEVCMAPRS SQSSEEGSFS 1201 QVSTMALHIA QADAEDSPPS LQRHSLAARY
YNWVSFPGCL ARGAETRGSS RMKTFEEFPM TPTTYKGSVD NQTDSGMVLA 1281
SEEFEQIESR HRQESGFSCK GPGQNVAVTR AHPDSQGRRR RPERGARGGQ VFYNSEYGEL
SEPSEEDHCS PSARVTFFTD 1361 NSY SEQ ID NO. 17 1 ASTKGPSVFP
LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSSLGTQT 81 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG
PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW 161 YVDGVEVHNA
KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ
VYTLPPSRDE 241 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV
LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT 321 QKSLSLSPGK SEQ ID
NO. 18 1 TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN
SQESVTEQDS KDSTYSLSST LTLSKADYEK 81 HKVYACEVTH QGLSSPVTKS FNRGEC
SEQ ID NO. 19 1 LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE
MAKAQDGTFS SVLTLTNLTG LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD
PTVGFLPNDA EELFIFLTEI TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI
FEDRSYICKT 161 TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT
LMCIVIGNEV VNFEWTYPRK ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE
LEDSGTYTCN VTESVNDHQD EKAINITVVE SGGGGGSGGG GSDIQMTQSP SSLSASVGDR
VTITCSASQD 321 ISNYLNWYQQ KPGKAPKVLI YFTSSLHSGV PSRFSGSGSG
TDFTLTISSL QPEDFATYYC QQYSTVPWTF GQGTKVEIKR 401 TVAAPSVFIF
PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST
LTLSKADYEK 481 HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO. 21 1
EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY
AADFKRRFTF SLDTSKSTAY 81 LQMNSLRAED TAVYYCAKYP HYYGSSHWYF
DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 161
TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK
VEPKSCDKTH T SEQ ID NO. 22
1 LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCAASGYT 321
FTNYGMNWVR QAPGKGLEWV GWINTYTGEP TYAADFKRRF TFSLDTSKST AYLQMNSLRA
EDTAVYYCAK YPHYYGSSHW 401 YFDVWGQGTL VTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 481
SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THT SEQ ID NO. 23 1
LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGEVQLVE SGGGLVQPGG SLRLSCAASG YTFTNYGMNW 321
VRQAPGKGLE WVGWINTYTG EPTYAADFKR RFTFSLDTSK STAYLQMNSL RAEDTAVYYC
AKYPHYYGSS HWYFDVWGQG 401 TLVTVSSAST KGPSVFPLAP SSKSTSGGTA
ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 481
SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHT SEQ ID NO. 24 1 EVQLVESGGG
LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF
SLDTSKSTAY 81 LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT
VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 161 TVSWNSGALT
SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH
TGGGGGSGGG 241 GSGGGGSGGG GSGLVVTPPG PELVLNVSST FVLTCSGSAP
VVWERMSQEP PQEMAKAQDG TFSSVLTLTN LTGLDTGEYF 321 CTHNDSRGLE
TDERKRLYIF VPDPTVGFLP NDAEELFIFL TEITEITIPC RVTDPQLVVT LHEKKGDVAL
PVPYDHQRGF 401 SGIFEDRSYI CKTTIGDREV DSDAYYVYRL QVSSINVSVN
AVQTVVRQGE NITLMCIVIG NEVVNFEWTY PRKESGRLVE 481 PVTDFLLDMP
YHIRSILHIP SAELEDSGTY TCNVTESVND HQDEKAINIT VVESG SEQ ID NO. 25 1
LVVTPPGPEL VLNVSSTFVL TCSGSAPVVW ERMSQEPPQE MAKAQDGTFS SVLTLTNLTG
LDTGEYFCTH NDSRGLETDE 81 RKRLYIFVPD PTVGFLPNDA EELFIFLTEI
TEITIPCRVT DPQLVVTLHE KKGDVALPVP YDHQRGFSGI FEDRSYICKT 161
TIGDREVDSD AYYVYRLQVS SINVSVNAVQ TVVRQGENIT LMCIVIGNEV VNFEWTYPRK
ESGRLVEPVT DFLLDMPYHI 241 RSILHIPSAE LEDSGTYTCN VTESVNDHQD
EKAINITVVE SGGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCAASGYD 321
FTHYGMNWVR QAPGKGLEWV GWINTYTGEP TYAADFKRRF TFSLDTSKST AYLQMNSLRA
EDTAVYYCAK YPYYYGTSHW 401 YFDVWGQGTL VTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 481
SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THL SEQ ID NO. 26 1
LVVTPPGPE LVLNVSSTFV LTCSGSAPVV WERMSQEPPQ EMAKAQDGTF SSVLTLTNLT
GLDTGEYFCT HNDSRGLETD 80 ERKRLYIFVP DPTVGFLPND AEELFIFLTE
ITEITIPCRV TDPQLVVTLH EKKGDVALPV PYDHQRGFSG IFEDRSYICK 160
TTIGDREVDS DAYYVYRLQV SSINVSVNAV QTVVRQGENI TLMCIVIGNE VVNFEWTYPR
KESGRLVEPV TDFLLDMPYH 240 IRSILHIPSA ELEDSGTYTC NVTESVNDHQ
DEKAINITVV ESGEVQLVES GGGLVQPGGS LRLSCAASGY TFTNYGMNWV 320
RQAPGKGLEW VGWINTYTGE PTYAADFKRR FTFSLDTSKS TAYLQMNSLR AEDTAVYYCA
KYPHYYGSSH WYFDVWGQGT 400 LVTVSSASTK GPSVFPLAPS SKSTSGGTAA
LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS 480
SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL
MISRTPEVTC VVVDVSHEDP 560 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR
VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 640
PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT
VDKSRWQQGN VFSCSVMHEA 720 LHNHYTQKSL SLSPGK SEQ ID NO. 27 1
VGFLPNDAEE LFIFLTEITE ITIPCRVTDP QLVVTLHEKK GDVALPVPYD HQRGFSGIFE
DRSYICKTTI GDREVDSDAY 81 YVYRLQVSSI NVSVNAVQTV VRQGENITLM
CIVIGNEVVN FEWTYPRKES GRLVEPVTDF LLDMPYHIRS ILHIPSAELE 161
DSGTYTCNVT ESVNDHQDEK AINITVVESG EVQLVESGGG LVQPGGSLRL SCAASGYTFT
NYGMNWVRQA PGKGLEWVGW 241 INTYTGEPTY AADFKRRFTF SLDTSKSTAY
LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS 321
VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS
VVTVPSSSLG TQTYICNVNH 401 KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL
LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV 481
HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
EPQVYTLPPS REEMTKNQVS 561 LTCLVKGFYP SDIAVEWESN GQPENNYKTT
PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS 641 PGK SEQ
ID NO. 28 1 VGFLPNDAEE LFIFLTEITE ITIPCRVTDP QLVVTLHEKK GDVALPVPYD
HQRGFSGIFE DRSYICKTTI GDREVDSDAY 81 YVYRLQVSSI NVSVNAVQTV
VRQGENITLM CIVIGNEVVN FEWTYPRKES GRLVEPVTDF LLDMPYHIRS ILHIPSAELE
161 DSGTYTCNVT ESVNDHQDEK AINITVVESG EVQLVESGGG LVQPGGSLRL
SCAASGYTFT NYGMNWVRQA PGKGLEWVGW 241 INTYTGEPTY AADFKRRFTF
SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS
321 VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL
QSSGLYSLSS VVTVPSSSLG TQTYICNVNH 401 KPSNTKVDKK VEPKSCDKTH T SEQ ID
NO. 29 1 VGFLPNDAEE LFIFLTEITE ITIPCRVTDP QLVVTLHEKK GDVALPVPYD
HQRGFSGIFE DRSYICKTTI GDREVDSDAY 81 YVYRLQVSSI NVSVNAVQTV
VRQGENITLM CIVIGNEVVN FEWTYPRKES GRLVEPVTDF LLDMPYHIRS ILHIPSAELE
161 DSGTYTCNVT ESVNDHQDEK AINITVVESG GGGSGGGGSG GGGSGGGGSG
GGGSGGGGSE VQLVESGGGL VQPGGSLRLS 241 CAASGYTFTN YGMNWVRQAP
GKGLEWVGWI NTYTGEPTYA ADFKRRFTFS LDTSKSTAYL QMNSLRAEDT AVYYCAKYPH
321 YYGSSHWYFD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL
VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 401 SSGLYSLSSV VTVPSSSLGT
QTYICNVNHK PSNTKVDKKV EPKSCDKTHT SEQ ID NO. 30 1 EVQLVESGGG
LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF
SLDTSKSTAY 81 LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT
VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 161 TVSWNSGALT
SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH
TGGGSGGGGS 241 GGGGSGGGGS GGGGSGGGGS VGFLPNDAEE LFIFLTEITE
ITIPCRVTDP QLVVTLHEKK GDVALPVPYD HQRGFSGIFE 321 DRSYICKTTI
GDREVDSDAY YVYRLQVSSI NVSVNAVQTV VRQGENITLM CIVIGNEVVN FEWTYPRKES
GRLVEPVTDF 401 LLDMPYHIRS ILHIPSAELE DSGTYTCNVT ESVNDHQDEK
AINITVVESG SEQ ID NO: 31 Nucleic acid encoding heavy chain
anti-VEGF-PDGFR fusion
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG
CATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCA
AAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTA
GGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTA
GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA
TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT
ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA
TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT
TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG
ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT
CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC
TCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGG
AGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGT
GTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCCatgaaagctgtggtgctggccg
tggctctggtcttcctgacagggagccaggctctggtcgtcacacccccggggccagagcttg
tcctcaatgtctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaac
ggatgtcccaggagcccccacaggaaatggccaaggcccaggatggcaccttctccagcg
tgctcacactgaccaacctcactgggctagacacgggagaatacttttgcacccacaatgact
cccgggactggagaccgatgagcggaaacggctctacatctttgtgccagatcccaccgtggg
cttcctccctaatgatgccgaggaactattcatctttctcacggaaataactgagatcaccat
tccatgccgagtaacagacccacagctggtggtgacactgcacgagaagaaaggggacgttgc
actgcctgtcccctatgatcaccaacgtggcttttctggtatctttgaggacagaagctacat
ctgcaaaaccaccattggggacagggaggtggattctgatgcctactatgtctacagactcca
ggtgtcatccatcaacgtctctgtgaacgcagtgcagactgtggtccgccagggtgagaacat
caccctcatgtgcattgtgatcgggaatgaggtggtcaacttcgagtggacatacccccgcaa
agaaagtgggcggctggtggagccggtgactgacttcctcttggatatgccttaccacatccg
ctccatcctgcacatccccagtgccgagttagaagactcggggacctacacctgcaatgtgac
ggagagtgtgaatgaccatcaggatgaaaaggccatcaacatcaccgtggttgagagcggcgg
tggtggcggctccggtggaggcggaagcgaggtgcagctggtggaatccggcggaggcctggt
ccagcctggcggatccctgagactgtcctgtgccgcctccggctacgacttcacccattacgg
catgaactgggtccgacaggcccctggcaagggcctggaatgggtcggatggatcaacaccta
caccggcgagcccacctacgccgccgacttcaagcggcggttcaccttctccctggacacctc
caagtccaccgcctacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactg
cgccaagtacccctactactacggcacctcccactggtacttcgacgtgtggggccagggcac
cctggtcaccgtgtcctccgcctctaccaagggcccctccgtgttccctctggccccctccag
caagtccacctctggcggcaccgccgctctgggctgcctggtcaaggactacttccccgagcc
cgtgaccgtgtcctggaactctggcgccctgacctccggcgtgcacacctttccagccgtgct
gcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctccagctctctgggcac
ccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagaaggtgga
acccaagtcctgcgacaagacccacacctgtcccccctgccctgcccctgaagcagccggtgc
acccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccga
agtgacctgcgtggtggtggacgtgtcccacgaggaccctgaagtgaagttcaattggtacgt
ggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagtacaactccaccta
ccgggtggtgtccgtgctgaccgtgctgcatcaggactggctgaacggcaaagagtacaagtg
caaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggcca
gccccgcgagcctcaggtgtacacactgccacccagccgggaagagatgaccaagaaccaggt
ctccctgacctgtctggtcaagggcttctacccctccgatatcgccgtcgaatgggagtccaa
cggccagcccgagaacaactacaagaccaccccccctgtgctggactccgacggctcattctt
cctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgctc
cgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtcctgcagccccggcaa
gtgataaTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA
GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGT
CCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG
GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA
TGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCA
CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTAC
ACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC
CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG
GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA
GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAAC
TGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC
GGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAAT
GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATG
CATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATG
CAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC
CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCA
GAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGC
CTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACA
GGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG
GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTG
TTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTG
AATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCA
GCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGG
CAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATG
CGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATC
GAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCAT
CAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGAT
CTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT
GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC
CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATC
GCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGA
CTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCA
CCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCC
TCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATA
ATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT
CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTA
GCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA
TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCT
AACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC
TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTT
CCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA
AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAG
GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC
CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT
AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC
TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT
GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG
ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTG
CTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCT
GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA
CCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC
AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG
GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA
GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCA
GTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCG
TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG
ACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA
GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAG
TAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT
CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT
GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA
AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA
TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAA
CTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGC
TGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTT
TCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG
CGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG
GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC
CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC SEQ ID NO: 32: Light chain
encoding anti-VEGF light chain
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGC
CGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCG
AGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTA
GGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATT
ATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG
TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC
CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG
TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG
CCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT
ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC
CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGA
TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG
ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACG
GTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT
ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCT
TGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGT
GGCGGCCGCCatgggatggagctgtatcatcctcttcttggtggcaacagctacaggcgtg
cactccgacatccagctgacccagtccccctccagcctgtccgcctctgtgggcgacagag
tgaccatcacctgttccgccagccaggacatctccaactacctgaactggtatcagcagaa
gcccggcaaggcccccaaggtgctgatctacttcacctcctccctgcactccggcgtgccc
tccagattctccggctctggctccggcaccgactttaccctgaccatctccagcctgcagc
ccgaggacttcgccacctactactgccagcagtactccaccgtgccctggaccttcggcca
gggcaccaaggtggaaatcaagcggaccgtggccgctccctccgtgttcatcttcccaccc
tccgacgagcagctgaagtccggaaccgcctccgtcgtgtgcctgctgaacaacttctacc
cccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaactcccagga
atccgtcaccgagcaggactccaaggacagcacctactccctgtccagcaccctgaccctg
tccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctca
gctccccagtgaccaagtccttcaaccggggcgagtgctagtaaTCTAGAGGGCCCGTTTA
AACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCC
CCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG
AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGA
CAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG
GCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCG
GCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC
CCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCC
CGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCG
ACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGT
TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA
ACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGG
CCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAAT
GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCA
TGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAG
TATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC
CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA
TTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTT
TTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCT
GATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTT
CTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTG
CTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACC
GACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCA
CGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT
GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAA
GTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCAT
TCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGT
CGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGG
CTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGC
CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGT
GGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGC
GAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCG
CCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGAC
CAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGT
TGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT
GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC
AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGT
CCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGC
GTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC
ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT
TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA
ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG
CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG
CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGG
CCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC
CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC
TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT
GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC
TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG
AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC
GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG
TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA
CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC
TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT
GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA
GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG
TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT
CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC
TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCA
ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA
TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCG
CAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA
TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAG
CGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACT
CATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT
GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT
CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT
CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT
TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT
CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA
ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT
CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA
CATTTCCCCGAAAAGTGCCACCTGACGTC
Sequence CWU 1
1
561506PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val
Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala
Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met
Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr
Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn
Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr
Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu 115 120
125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln
130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys
Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala
Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser
Val Asn Ala Val Gln Thr Val 180 185 190Val Arg Gln Gly Glu Asn Ile
Thr Leu Met Cys Ile Val Ile Gly Asn 195 200 205Glu Val Val Asn Phe
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 210 215 220Leu Val Glu
Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile225 230 235
240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr
245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp
Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val Glu Ser Gly Gly Gly
Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser 290 295 300Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Ser Ala Ser Gln Asp305 310 315 320Ile Ser Asn Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 325 330 335Lys Val Leu
Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser 340 345 350Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 355 360
365Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser
370 375 380Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg385 390 395 400Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 405 410 415Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 420 425 430Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 435 440 445Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 450 455 460Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys465 470 475
480His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
485 490 495Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 500
5052453PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser
Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235
240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360
365Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
4503498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val
Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala
Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met
Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr
Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn
Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr
Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu 115 120
125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln
130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys
Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala
Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser
Val Asn Ala Val Gln Thr Val 180 185 190Val Arg Gln Gly Glu Asn Ile
Thr Leu Met Cys Ile Val Ile Gly Asn 195 200 205Glu Val Val Asn Phe
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 210 215 220Leu Val Glu
Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile225 230 235
240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr
245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp
Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val Glu Ser Gly Gly Gly
Asp Ile Gln Met 275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 290 295 300Ile Thr Cys Ser Ala Ser Gln Asp
Ile Ser Asn Tyr Leu Asn Trp Tyr305 310 315 320Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser 325 330 335Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360
365Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln
370 375 380Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val Phe385 390 395 400Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val 405 410 415Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp 420 425 430Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr 435 440 445Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 450 455 460Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val465 470 475
480Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
485 490 495Glu Cys4745PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Leu Val Val Thr Pro Pro
Gly Pro Glu Leu Val Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr
Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu
Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser
Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr
Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75
80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu
85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr
Glu 100 105 110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val
Val Thr Leu 115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val
Pro Tyr Asp His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp
Arg Ser Tyr Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu
Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser
Ser Ile Asn Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg
Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200
205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg
210 215 220Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr
His Ile225 230 235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu
Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val
Asn Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val
Glu Ser Gly Gly Gly Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 290 295 300Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr305 310 315
320Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
325 330 335Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro
Thr Tyr 340 345 350Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu
Asp Thr Ser Lys 355 360 365Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala 370 375 380Val Tyr Tyr Cys Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp385 390 395 400Tyr Phe Asp Val Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 405 410 415Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 420 425 430Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 435 440
445Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
450 455 460Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu465 470 475 480Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr 485 490 495Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys 500 505 510Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro 515 520 525Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 530 535 540Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val545 550 555
560Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
565 570 575Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 580 585 590Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 595 600 605Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 610 615 620Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln625 630 635 640Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 645 650 655Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 660 665 670Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 675 680
685Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
690 695 700Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val705 710 715 720Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln 725 730 735Lys Ser Leu Ser Leu Ser Pro Gly Lys
740 7455214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 2106737PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Leu Val Val Thr Pro Pro Gly Pro Glu
Leu Val Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly
Ser Ala Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln
Glu Met Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr
Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg
Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn
Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100 105
110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu
115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp
His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr
Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp Ser
Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile Asn
Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg Gln Gly Glu
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200 205Glu Val Val
Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 210 215 220Leu
Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile225 230
235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly
Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln
Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val Glu Ser Gly Gly
Gly Glu Val Gln Leu 275 280 285Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu 290 295 300Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr Gly Met Asn Trp305 310 315 320Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn 325 330 335Thr Tyr
Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe 340 345
350Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn
355 360 365Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys
Tyr Pro 370 375 380His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
Trp Gly Gln Gly385 390 395 400Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 405 410 415Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 420 425 430Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 435 440 445Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 450 455 460Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser465 470
475 480Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 485 490 495Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 500 505 510Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 515 520 525Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 530 535 540Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp545 550 555 560Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 565 570 575Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 580 585
590Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
595 600 605Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys 610 615 620Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr625 630 635 640Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr 645 650 655Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 660 665 670Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 675 680 685Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 690 695 700Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu705 710
715 720Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 725 730 735Lys7755PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200
205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460Gly Gly Ser Gly Gly Gly Gly Ser Gly Leu Val Val Thr Pro
Pro Gly465 470 475 480Pro Glu Leu Val Leu Asn Val Ser Ser Thr Phe
Val Leu Thr Cys Ser 485 490 495Gly Ser Ala Pro Val Val Trp Glu Arg
Met Ser Gln Glu Pro Pro Gln 500 505 510Glu Met Ala Lys Ala Gln Asp
Gly Thr Phe Ser Ser Val Leu Thr Leu 515 520 525Thr Asn Leu Thr Gly
Leu Asp Thr Gly Glu Tyr Phe Cys Thr His Asn 530 535 540Asp Ser Arg
Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe545 550 555
560Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu
565 570 575Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
Arg Val 580 585 590Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys
Lys Gly Asp Val 595 600 605Ala Leu Pro Val Pro Tyr Asp His Gln Arg
Gly Phe Ser Gly Ile Phe 610 615 620Glu Asp Arg Ser Tyr Ile Cys Lys
Thr Thr Ile Gly Asp Arg Glu Val625 630 635 640Asp Ser Asp Ala Tyr
Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn 645 650 655Val Ser Val
Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile 660 665 670Thr
Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp 675 680
685Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp
690 695 700Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
Ile Pro705 710 715 720Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr
Cys Asn Val Thr Glu 725 730 735Ser Val Asn Asp His Gln Asp Glu Lys
Ala Ile Asn Ile Thr Val Val 740 745 750Glu Ser Gly
7558745PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val
Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala
Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met
Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr
Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn
Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr
Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu 115 120
125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln
130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys
Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala
Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser
Val Asn Ala Val Gln Thr Val 180 185 190Val Arg Gln Gly Glu Asn Ile
Thr Leu Met Cys Ile Val Ile Gly Asn 195 200 205Glu Val Val Asn Phe
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 210 215 220Leu Val Glu
Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile225 230 235
240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr
245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln Asp
Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val Glu Ser Gly Gly Gly
Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val 290 295 300Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Asp305 310 315 320Phe Thr His Tyr Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 325 330 335Leu Glu Trp
Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr 340 345 350Ala
Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys 355 360
365Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
370 375 380Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser
His Trp385 390 395 400Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala 405 410 415Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser 420 425 430Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe 435 440 445Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 450 455 460Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu465 470 475
480Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
485 490 495Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys 500 505 510Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro 515 520 525Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
Phe Leu Phe Pro Pro Lys 530 535 540Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val545 550 555 560Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 565 570 575Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 580 585 590Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 595 600
605Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
610 615 620Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln625 630 635 640Pro Arg Glu Pro Cys Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 645 650 655Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 660 665 670Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn 675 680 685Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 690 695 700Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val705 710 715
720Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
725 730 735Lys Ser Leu Ser Leu Ser Pro Gly Lys 740
7459745PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val
Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala
Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met
Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr
Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn
Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr
Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu 115 120
125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln
130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys
Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala
Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser
Val Asn Ala Val Gln Thr Val
180 185 190Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile
Gly Asn 195 200 205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys
Glu Ser Gly Arg 210 215 220Leu Val Glu Pro Val Thr Asp Phe Leu Leu
Asp Met Pro Tyr His Ile225 230 235 240Arg Ser Ile Leu His Ile Pro
Ser Ala Glu Leu Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn
Ile Thr Val Val Glu Ser Gly Gly Gly Gly Gly Ser Gly 275 280 285Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 290 295
300Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Asp305 310 315 320Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly 325 330 335Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr
Thr Gly Glu Pro Thr Tyr 340 345 350Ala Ala Asp Phe Lys Arg Arg Phe
Thr Phe Ser Leu Asp Thr Ser Lys 355 360 365Ser Thr Ala Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 370 375 380Val Tyr Tyr Cys
Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp385 390 395 400Tyr
Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 405 410
415Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
420 425 430Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe 435 440 445Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly 450 455 460Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu465 470 475 480Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr 485 490 495Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 500 505 510Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 515 520 525Ala
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys 530 535
540Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val545 550 555 560Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 565 570 575Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 580 585 590Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His 595 600 605Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 610 615 620Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln625 630 635 640Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 645 650
655Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
660 665 670Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 675 680 685Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 690 695 700Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val705 710 715 720Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 725 730 735Lys Ser Leu Ser Cys
Ser Pro Gly Lys 740 74510214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 10Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210111106PRTHomo sapiens 11Met Arg Leu
Pro Gly Ala Met Pro Ala Leu Ala Leu Lys Gly Glu Leu1 5 10 15Leu Leu
Leu Ser Leu Leu Leu Leu Leu Glu Pro Gln Ile Ser Gln Gly 20 25 30Leu
Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser 35 40
45Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg
50 55 60Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly
Thr65 70 75 80Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu
Asp Thr Gly 85 90 95Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu
Glu Thr Asp Glu 100 105 110Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp
Pro Thr Val Gly Phe Leu 115 120 125Pro Asn Asp Ala Glu Glu Leu Phe
Ile Phe Leu Thr Glu Ile Thr Glu 130 135 140Ile Thr Ile Pro Cys Arg
Val Thr Asp Pro Gln Leu Val Val Thr Leu145 150 155 160His Glu Lys
Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln 165 170 175Arg
Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr 180 185
190Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg
195 200 205Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln
Thr Val 210 215 220Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile
Val Ile Gly Asn225 230 235 240Glu Val Val Asn Phe Glu Trp Thr Tyr
Pro Arg Lys Glu Ser Gly Arg 245 250 255Leu Val Glu Pro Val Thr Asp
Phe Leu Leu Asp Met Pro Tyr His Ile 260 265 270Arg Ser Ile Leu His
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr 275 280 285Tyr Thr Cys
Asn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys 290 295 300Ala
Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly305 310
315 320Glu Val Gly Thr Leu Gln Phe Ala Glu Leu His Arg Ser Arg Thr
Leu 325 330 335Gln Val Val Phe Glu Ala Tyr Pro Pro Pro Thr Val Leu
Trp Phe Lys 340 345 350Asp Asn Arg Thr Leu Gly Asp Ser Ser Ala Gly
Glu Ile Ala Leu Ser 355 360 365Thr Arg Asn Val Ser Glu Thr Arg Tyr
Val Ser Glu Leu Thr Leu Val 370 375 380Arg Val Lys Val Ala Glu Ala
Gly His Tyr Thr Met Arg Ala Phe His385 390 395 400Glu Asp Ala Glu
Val Gln Leu Ser Phe Gln Leu Gln Ile Asn Val Pro 405 410 415Val Arg
Val Leu Glu Leu Ser Glu Ser His Pro Asp Ser Gly Glu Gln 420 425
430Thr Val Arg Cys Arg Gly Arg Gly Met Pro Gln Pro Asn Ile Ile Trp
435 440 445Ser Ala Cys Arg Asp Leu Lys Arg Cys Pro Arg Glu Leu Pro
Pro Thr 450 455 460Leu Leu Gly Asn Ser Ser Glu Glu Glu Ser Gln Leu
Glu Thr Asn Val465 470 475 480Thr Tyr Trp Glu Glu Glu Gln Glu Phe
Glu Val Val Ser Thr Leu Arg 485 490 495Leu Gln His Val Asp Arg Pro
Leu Ser Val Arg Cys Thr Leu Arg Asn 500 505 510Ala Val Gly Gln Asp
Thr Gln Glu Val Ile Val Val Pro His Ser Leu 515 520 525Pro Phe Lys
Val Val Val Ile Ser Ala Ile Leu Ala Leu Val Val Leu 530 535 540Thr
Ile Ile Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys Pro545 550
555 560Arg Tyr Glu Ile Arg Trp Lys Val Ile Glu Ser Val Ser Ser Asp
Gly 565 570 575His Glu Tyr Ile Tyr Val Asp Pro Met Gln Leu Pro Tyr
Asp Ser Thr 580 585 590Trp Glu Leu Pro Arg Asp Gln Leu Val Leu Gly
Arg Thr Leu Gly Ser 595 600 605Gly Ala Phe Gly Gln Val Val Glu Ala
Thr Ala His Gly Leu Ser His 610 615 620Ser Gln Ala Thr Met Lys Val
Ala Val Lys Met Leu Lys Ser Thr Ala625 630 635 640Arg Ser Ser Glu
Lys Gln Ala Leu Met Ser Glu Leu Lys Ile Met Ser 645 650 655His Leu
Gly Pro His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr 660 665
670Lys Gly Gly Pro Ile Tyr Ile Ile Thr Glu Tyr Cys Arg Tyr Gly Asp
675 680 685Leu Val Asp Tyr Leu His Arg Asn Lys His Thr Phe Leu Gln
His His 690 695 700Ser Asp Lys Arg Arg Pro Pro Ser Ala Glu Leu Tyr
Ser Asn Ala Leu705 710 715 720Pro Val Gly Leu Pro Leu Pro Ser His
Val Ser Leu Thr Gly Glu Ser 725 730 735Asp Gly Gly Tyr Met Asp Met
Ser Lys Asp Glu Ser Val Asp Tyr Val 740 745 750Pro Met Leu Asp Met
Lys Gly Asp Val Lys Tyr Ala Asp Ile Glu Ser 755 760 765Ser Asn Tyr
Met Ala Pro Tyr Asp Asn Tyr Val Pro Ser Ala Pro Glu 770 775 780Arg
Thr Cys Arg Ala Thr Leu Ile Asn Glu Ser Pro Val Leu Ser Tyr785 790
795 800Met Asp Leu Val Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu
Phe 805 810 815Leu Ala Ser Lys Asn Cys Val His Arg Asp Leu Ala Ala
Arg Asn Val 820 825 830Leu Ile Cys Glu Gly Lys Leu Val Lys Ile Cys
Asp Phe Gly Leu Ala 835 840 845Arg Asp Ile Met Arg Asp Ser Asn Tyr
Ile Ser Lys Gly Ser Thr Phe 850 855 860Leu Pro Leu Lys Trp Met Ala
Pro Glu Ser Ile Phe Asn Ser Leu Tyr865 870 875 880Thr Thr Leu Ser
Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile 885 890 895Phe Thr
Leu Gly Gly Thr Pro Tyr Pro Glu Leu Pro Met Asn Glu Gln 900 905
910Phe Tyr Asn Ala Ile Lys Arg Gly Tyr Arg Met Ala Gln Pro Ala His
915 920 925Ala Ser Asp Glu Ile Tyr Glu Ile Met Gln Lys Cys Trp Glu
Glu Lys 930 935 940Phe Glu Ile Arg Pro Pro Phe Ser Gln Leu Val Leu
Leu Leu Glu Arg945 950 955 960Leu Leu Gly Glu Gly Tyr Lys Lys Lys
Tyr Gln Gln Val Asp Glu Glu 965 970 975Phe Leu Arg Ser Asp His Pro
Ala Ile Leu Arg Ser Gln Ala Arg Leu 980 985 990Pro Gly Phe His Gly
Leu Arg Ser Pro Leu Asp Thr Ser Ser Val Leu 995 1000 1005Tyr Thr
Ala Val Gln Pro Asn Glu Gly Asp Asn Asp Tyr Ile Ile 1010 1015
1020Pro Leu Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu
1025 1030 1035Glu Gly Ser Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu
Val Asn 1040 1045 1050Thr Ser Ser Thr Ile Ser Cys Asp Ser Pro Leu
Glu Pro Gln Asp 1055 1060 1065Glu Pro Glu Pro Glu Pro Gln Leu Glu
Leu Gln Val Glu Pro Glu 1070 1075 1080Pro Glu Leu Glu Gln Leu Pro
Asp Ser Gly Cys Pro Ala Pro Arg 1085 1090 1095Ala Glu Ala Glu Asp
Ser Phe Leu 1100 110512214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21013231PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His
Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys
Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr
Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Leu225
230141338PRTHomo sapiens 14Met Val Ser Tyr Trp Asp Thr Gly Val Leu
Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser
Gly Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu Lys Gly Thr Gln
His Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu Gln Cys Arg Gly
Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met Val Ser Lys Glu
Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn
Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95Ala Gln Ala
Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val
100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile
Phe Ile 115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser
Glu Ile Pro Glu 130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu
Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp Gly
Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile Ser
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205Ala
Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215
220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro
Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys
Thr Ala Thr Thr 245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp
Ser Tyr Pro Asp Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg Arg
Arg Ile Asp Gln Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr Ser
Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly Leu
Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser
Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330
335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser
340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu
Val Val 355 360 365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser
Ala Arg Tyr Leu 370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp
Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu
Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415Asn Leu Thr Ala Thr
Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430Lys Ala Val
Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445Arg
Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455
460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg
Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu
Asp Ala Asp Ser 485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr
Gln Arg Met Ala Ile Ile 500 505 510Glu Gly Lys Asn Lys Met Ala Ser
Thr Leu Val Val Ala Asp Ser Arg 515 520 525Ile Ser Gly Ile Tyr Ile
Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540Gly Arg Asn Ile
Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His545 550 555 560Val
Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570
575Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu
580 585 590Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys
Gln Lys 595 600 605Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn
Leu Thr Ile Met 610 615 620Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr
Ala Cys Arg Ala Arg Asn625 630 635 640Val Tyr Thr Gly Glu Glu Ile
Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655Asp Gln Glu Ala Pro
Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670Ala Ile Ser
Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685Glu
Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695
700Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile Glu
Arg705 710 715 720Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys
Ala Thr Asn Gln 725 730 735Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu
Thr Val Gln Gly Thr Ser 740 745 750Asp Lys Ser Asn Leu Glu Leu Ile
Thr Leu Thr Cys Thr Cys Val Ala 755 760 765Ala Thr Leu Phe Trp Leu
Leu Leu Thr Leu Phe Ile Arg Lys Met Lys 770 775 780Arg Ser Ser Ser
Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp785 790 795 800Pro
Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp 805 810
815Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly Lys Ser
820 825 830Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser Ala
Phe Gly 835 840 845Ile Lys Lys Ser Pro Thr Cys Arg Thr Val Ala Val
Lys Met Leu Lys 850 855 860Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala
Leu Met Thr Glu Leu Lys865 870 875 880Ile Leu Thr His Ile Gly His
His Leu Asn Val Val Asn Leu Leu Gly 885 890 895Ala Cys Thr Lys Gln
Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905 910Lys Tyr Gly
Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915 920 925Phe
Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys 930 935
940Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu Asp Ser
Val945 950 955 960Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Gln
Glu Asp Lys Ser 965 970 975Leu Ser Asp Val Glu Glu Glu Glu Asp Ser
Asp Gly Phe Tyr Lys Glu 980 985 990Pro Ile Thr Met Glu Asp Leu Ile
Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005Gly Met Glu Phe Leu
Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020Ala Ala Arg
Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025 1030 1035Cys
Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr 1040 1045
1050Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met Ala Pro
1055 1060 1065Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp
Val Trp 1070 1075 1080Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser
Leu Gly Gly Ser 1085 1090 1095Pro Tyr Pro Gly Val Gln Met Asp Glu
Asp Phe Cys Ser Arg Leu 1100 1105 1110Arg Glu Gly Met Arg Met Arg
Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125Ile Tyr Gln Ile Met
Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140Arg Pro Arg
Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145 1150 1155Gln
Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn 1160 1165
1170Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala
1175 1180 1185Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro
Lys Phe 1190 1195 1200Asn Ser Gly Ser Ser Asp Asp Val Arg Tyr Val
Asn Ala Phe Lys 1205 1210 1215Phe Met Ser Leu Glu Arg Ile Lys Thr
Phe Glu Glu Leu Leu Pro 1220 1225 1230Asn Ala Thr Ser Met Phe Asp
Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245Leu Leu Ala Ser Pro
Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260Lys Pro Lys
Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265 1270 1275Ser
Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys 1280 1285
1290His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr
1295 1300 1305Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala Cys Cys
Ser Pro 1310 1315 1320Pro Pro Asp Tyr Asn Ser Val Val Leu Tyr Ser
Thr Pro Pro Ile 1325 1330 1335151356PRTHomo sapiens 15Met Gln Ser
Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg
Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro 20 25 30Arg
Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr 35 40
45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro
50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys
Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val
Ile Gly Asn 85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr
Asp Leu Ala Ser 100 105 110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg
Ser Pro Phe Ile Ala Ser 115 120 125Val Ser Asp Gln His Gly Val Val
Tyr Ile Thr Glu Asn Lys Asn Lys 130 135 140Thr Val Val Ile Pro Cys
Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala
Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg 165 170 175Ile
Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile 180 185
190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser
195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg
Ile Tyr 210 215 220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu
Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg
Thr Glu Leu Asn Val Gly Ile 245 250 255Asp Phe Asn Trp Glu Tyr Pro
Ser Ser Lys His Gln His Lys Lys Leu 260 265 270Val Asn Arg Asp Leu
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 275 280 285Leu Ser Thr
Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 290 295 300Tyr
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310
315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly
Met 325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg
Ile Pro Ala 340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys
Trp Tyr Lys Asn Gly 355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile
Lys Ala Gly His Val Leu Thr 370 375 380Ile Met Glu Val Ser Glu Arg
Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile
Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val 405 410 415Val Tyr
Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val 420 425
430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr
435 440 445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu
Glu Glu 450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val
Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu
Asp Phe Gln Gly Gly Asn Lys 485 490 495Ile Glu Val Asn Lys Asn Gln
Phe Ala Leu Ile Glu Gly Lys Asn Lys 500 505 510Thr Val Ser Thr Leu
Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr 515 520 525Lys Cys Glu
Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser 530 535 540Phe
His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550
555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg
Ser 565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln
Pro Leu Pro 580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys
Lys Asn Leu Asp Thr 595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe
Ser Asn Ser Thr Asn Asp Ile 610 615 620Leu Ile Met Glu Leu Lys Asn
Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala
Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val 645 650 655Arg Gln
Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn 660 665
670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys
675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys
Asp Asn 690 695 700Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys
Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile Arg Arg Val Arg Lys
Glu Asp Glu Gly Leu Tyr Thr 725 730 735Cys Gln Ala Cys Ser Val Leu
Gly Cys Ala Lys Val Glu Ala Phe Phe 740 745 750Ile Ile Glu Gly Ala
Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu 755 760 765Val Gly Thr
Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile 770 775 780Ile
Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly785 790
795 800Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu
His 805 810 815Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe
Pro Arg Asp 820 825 830Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly
Ala Phe Gly Gln Val 835 840 845Ile Glu Ala Asp Ala Phe Gly Ile Asp
Lys Thr Ala Thr Cys Arg Thr 850 855 860Val Ala Val Lys Met Leu Lys
Glu Gly Ala Thr His Ser Glu His Arg865 870 875 880Ala Leu Met Ser
Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu 885 890 895Asn Val
Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu 900 905
910Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu
915 920 925Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly
Ala Arg 930 935 940Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro
Val Asp Leu Lys945 950 955 960Arg Arg Leu Asp Ser Ile Thr Ser Ser
Gln Ser Ser Ala Ser Ser Gly 965 970 975Phe Val Glu Glu Lys Ser Leu
Ser Asp Val Glu Glu Glu Glu Ala Pro 980 985 990Glu Asp Leu Tyr Lys
Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr 995 1000 1005Ser Phe
Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys 1010 1015
1020Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu
1025 1030 1035Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg
Asp Ile 1040 1045 1050Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp
Ala Arg Leu Pro 1055 1060 1065Leu Lys Trp Met Ala Pro Glu Thr Ile
Phe Asp Arg Val Tyr Thr 1070 1075 1080Ile Gln Ser Asp Val Trp Ser
Phe Gly Val Leu Leu Trp Glu Ile 1085 1090 1095Phe Ser Leu Gly Ala
Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu 1100 1105 1110Glu Phe Cys
Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro 1115 1120 1125Asp
Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp 1130 1135
1140His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser Glu Leu Val Glu
1145 1150 1155His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp
Gly Lys 1160 1165 1170Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu
Ser Met Glu Glu 1175 1180 1185Asp Ser Gly Leu Ser Leu Pro Thr Ser
Pro Val Ser Cys Met Glu 1190 1195 1200Glu Glu Glu Val Cys Asp Pro
Lys Phe His Tyr Asp Asn Thr Ala 1205 1210 1215Gly Ile Ser Gln Tyr
Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro 1220
1225 1230Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro
Glu 1235 1240 1245Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser
Gly Met Val 1250 1255 1260Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu
Asp Arg Thr Lys Leu 1265 1270 1275Ser Pro Ser Phe Gly Gly Met Val
Pro Ser Lys Ser Arg Glu Ser 1280 1285 1290Val Ala Ser Glu Gly Ser
Asn Gln Thr Ser Gly Tyr Gln Ser Gly 1295 1300 1305Tyr His Ser Asp
Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu 1310 1315 1320Ala Glu
Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser 1325 1330
1335Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser
1340 1345 1350Pro Pro Val 1355161363PRTHomo sapiens 16Met Gln Arg
Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly1 5 10 15Leu Leu
Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu 20 25 30Asn
Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser 35 40
45Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala
50 55 60Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly
Val65 70 75 80Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys
Lys Val Leu 85 90 95Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser
Tyr Val Cys Tyr 100 105 110Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly
Thr Thr Ala Ala Ser Ser 115 120 125Tyr Val Phe Val Arg Asp Phe Glu
Gln Pro Phe Ile Asn Lys Pro Asp 130 135 140Thr Leu Leu Val Asn Arg
Lys Asp Ala Met Trp Val Pro Cys Leu Val145 150 155 160Ser Ile Pro
Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu 165 170 175Trp
Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu 180 185
190Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr
195 200 205Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val
His Ile 210 215 220Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro
Arg Lys Ser Leu225 230 235 240Glu Leu Leu Val Gly Glu Lys Leu Val
Leu Asn Cys Thr Val Trp Ala 245 250 255Glu Phe Asn Ser Gly Val Thr
Phe Asp Trp Asp Tyr Pro Gly Lys Gln 260 265 270Ala Glu Arg Gly Lys
Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His 275 280 285Thr Glu Leu
Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp 290 295 300Leu
Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg305 310
315 320Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val
Glu 325 330 335Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp
Glu Leu Val 340 345 350Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro
Pro Glu Phe Gln Trp 355 360 365Tyr Lys Asp Gly Lys Ala Leu Ser Gly
Arg His Ser Pro His Ala Leu 370 375 380Val Leu Lys Glu Val Thr Glu
Ala Ser Thr Gly Thr Tyr Thr Leu Ala385 390 395 400Leu Trp Asn Ser
Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu 405 410 415Val Val
Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro 420 425
430Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr
435 440 445Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro
Trp Thr 450 455 460Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg
Arg Gln Gln Gln465 470 475 480Asp Leu Met Pro Gln Cys Arg Asp Trp
Arg Ala Val Thr Thr Gln Asp 485 490 495Ala Val Asn Pro Ile Glu Ser
Leu Asp Thr Trp Thr Glu Phe Val Glu 500 505 510Gly Lys Asn Lys Thr
Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val 515 520 525Ser Ala Met
Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu 530 535 540Arg
Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile545 550
555 560Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu
Leu 565 570 575Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg
Trp Tyr Arg 580 585 590Leu Asn Leu Ser Thr Leu His Asp Ala His Gly
Asn Pro Leu Leu Leu 595 600 605Asp Cys Lys Asn Val His Leu Phe Ala
Thr Pro Leu Ala Ala Ser Leu 610 615 620Glu Glu Val Ala Pro Gly Ala
Arg His Ala Thr Leu Ser Leu Ser Ile625 630 635 640Pro Arg Val Ala
Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln 645 650 655Asp Arg
Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val 660 665
670Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu
675 680 685Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala
Gly Ala 690 695 700His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg
Leu Leu Glu Glu705 710 715 720Lys Ser Gly Val Asp Leu Ala Asp Ser
Asn Gln Lys Leu Ser Ile Gln 725 730 735Arg Val Arg Glu Glu Asp Ala
Gly Arg Tyr Leu Cys Ser Val Cys Asn 740 745 750Ala Lys Gly Cys Val
Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser 755 760 765Glu Asp Lys
Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val 770 775 780Ile
Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met785 790
795 800Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile
Ile 805 810 815Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu
Tyr Leu Ser 820 825 830Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu
Arg Leu His Leu Gly 835 840 845Arg Val Leu Gly Tyr Gly Ala Phe Gly
Lys Val Val Glu Ala Ser Ala 850 855 860Phe Gly Ile His Lys Gly Ser
Ser Cys Asp Thr Val Ala Val Lys Met865 870 875 880Leu Lys Glu Gly
Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu 885 890 895Leu Lys
Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu 900 905
910Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu
915 920 925Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys
Arg Asp 930 935 940Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln
Arg Gly Arg Phe945 950 955 960Arg Ala Met Val Glu Leu Ala Arg Leu
Asp Arg Arg Arg Pro Gly Ser 965 970 975Ser Asp Arg Val Leu Phe Ala
Arg Phe Ser Lys Thr Glu Gly Gly Ala 980 985 990Arg Arg Ala Ser Pro
Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro 995 1000 1005Leu Thr
Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg 1010 1015
1020Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu
1025 1030 1035Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val
Lys Ile 1040 1045 1050Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys
Asp Pro Asp Tyr 1055 1060 1065Val Arg Lys Gly Ser Ala Arg Leu Pro
Leu Lys Trp Met Ala Pro 1070 1075 1080Glu Ser Ile Phe Asp Lys Val
Tyr Thr Thr Gln Ser Asp Val Trp 1085 1090 1095Ser Phe Gly Val Leu
Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser 1100 1105 1110Pro Tyr Pro
Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu 1115 1120 1125Arg
Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala 1130 1135
1140Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala
1145 1150 1155Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu Gly Asp
Leu Leu 1160 1165 1170Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val
Cys Met Ala Pro 1175 1180 1185Arg Ser Ser Gln Ser Ser Glu Glu Gly
Ser Phe Ser Gln Val Ser 1190 1195 1200Thr Met Ala Leu His Ile Ala
Gln Ala Asp Ala Glu Asp Ser Pro 1205 1210 1215Pro Ser Leu Gln Arg
His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp 1220 1225 1230Val Ser Phe
Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly 1235 1240 1245Ser
Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr 1250 1255
1260Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val
1265 1270 1275Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His
Arg Gln 1280 1285 1290Glu Ser Gly Phe Ser Cys Lys Gly Pro Gly Gln
Asn Val Ala Val 1295 1300 1305Thr Arg Ala His Pro Asp Ser Gln Gly
Arg Arg Arg Arg Pro Glu 1310 1315 1320Arg Gly Ala Arg Gly Gly Gln
Val Phe Tyr Asn Ser Glu Tyr Gly 1325 1330 1335Glu Leu Ser Glu Pro
Ser Glu Glu Asp His Cys Ser Pro Ser Ala 1340 1345 1350Arg Val Thr
Phe Phe Thr Asp Asn Ser Tyr 1355 136017330PRTHomo sapiens 17Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33018106PRTHomo sapiens 18Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10519506PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser1
5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu
Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp
Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu
Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu
Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp
Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile
Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr Ile Pro Cys Arg Val
Thr Asp Pro Gln Leu Val Val Thr Leu 115 120 125His Glu Lys Lys Gly
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln 130 135 140Arg Gly Phe
Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr145 150 155
160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg
165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln
Thr Val 180 185 190Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile
Val Ile Gly Asn 195 200 205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro
Arg Lys Glu Ser Gly Arg 210 215 220Leu Val Glu Pro Val Thr Asp Phe
Leu Leu Asp Met Pro Tyr His Ile225 230 235 240Arg Ser Ile Leu His
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys
Asn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys 260 265 270Ala
Ile Asn Ile Thr Val Val Glu Ser Gly Gly Gly Gly Gly Ser Gly 275 280
285Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
290 295 300Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser
Gln Asp305 310 315 320Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro 325 330 335Lys Val Leu Ile Tyr Phe Thr Ser Ser
Leu His Ser Gly Val Pro Ser 340 345 350Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 355 360 365Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser 370 375 380Thr Val Pro
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg385 390 395
400Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
405 410 415Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 420 425 430Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 435 440 445Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 450 455 460Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys465 470 475 480His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 485 490 495Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 500 50520498PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptide
20Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser1
5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu
Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp
Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu
Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu
Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp
Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile
Phe Leu Thr Glu Ile Thr Glu 100 105 110Ile Thr Ile Pro Cys Arg Val
Thr Asp Pro Gln Leu Val Val Thr Leu 115 120 125His Glu Lys Lys Gly
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln 130 135 140Arg Gly Phe
Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr145 150 155
160Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg
165 170 175Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln
Thr Val 180 185 190Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile
Val Ile Gly Asn 195 200 205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro
Arg Lys Glu Ser Gly Arg 210 215 220Leu Val Glu Pro Val Thr Asp Phe
Leu Leu Asp Met Pro Tyr His Ile225 230 235 240Arg Ser Ile Leu His
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys
Asn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys 260 265 270Ala
Ile Asn Ile Thr Val Val Glu Ser Gly Gly Gly Asp Ile Gln Met 275 280
285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
290 295 300Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
Trp Tyr305 310 315 320Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu
Ile Tyr Phe Thr Ser 325 330 335Ser Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360 365Thr Tyr Tyr Cys Gln
Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln 370 375 380Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe385 390 395
400Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
405 410 415Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp 420 425 430Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr 435 440 445Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr 450 455 460Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val465 470 475 480Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 485 490 495Glu
Cys21231PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser
Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr225 23022523PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 22Leu Val Val Thr Pro Pro
Gly Pro Glu Leu Val Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr
Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu
Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser
Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr
Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75
80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu
85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr
Glu 100 105 110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val
Val Thr Leu 115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val
Pro Tyr Asp His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp
Arg Ser Tyr Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu
Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser
Ser Ile Asn Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg
Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200
205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg
210 215 220Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr
His Ile225 230 235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu
Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val
Asn Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val
Glu Ser Gly Gly Gly Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 290 295 300Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr305 310 315
320Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
325 330 335Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro
Thr Tyr 340 345 350Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu
Asp Thr Ser Lys 355 360 365Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala 370 375 380Val Tyr Tyr Cys Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp385 390 395 400Tyr Phe Asp Val Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 405 410 415Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 420 425 430Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 435 440
445Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
450 455 460Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu465 470 475 480Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr 485 490 495Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys 500 505 510Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 515 52023515PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Leu Val Val Thr Pro Pro
Gly Pro Glu Leu Val Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr
Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu
Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser
Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr
Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75
80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu
85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr
Glu 100 105 110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val
Val Thr Leu 115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val
Pro Tyr Asp His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp
Arg Ser Tyr Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu
Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser
Ser Ile Asn Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg
Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200
205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg
210 215 220Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr
His Ile225 230 235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu
Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val
Asn Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val
Glu Ser Gly Gly Gly Glu Val Gln Leu 275 280 285Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 290 295 300Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp305 310 315
320Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn
325 330 335Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg
Arg Phe 340 345 350Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
Leu Gln Met Asn 355 360 365Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Lys Tyr Pro 370 375 380His Tyr Tyr Gly Ser Ser His Trp
Tyr Phe Asp Val Trp Gly Gln Gly385 390 395 400Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 405 410 415Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 420 425 430Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 435 440
445Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
450 455 460Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser465 470 475 480Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 485 490 495Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 500 505 510Thr His Thr
51524535PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser
Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly225 230 235
240Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Leu Val Val
245 250 255Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser Thr
Phe Val 260 265 270Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu
Arg Met Ser Gln 275 280 285Glu Pro Pro Gln Glu Met Ala Lys Ala Gln
Asp Gly Thr Phe Ser Ser 290 295 300Val Leu Thr Leu Thr Asn Leu Thr
Gly Leu Asp Thr Gly Glu Tyr Phe305 310 315 320Cys Thr His Asn Asp
Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg 325 330 335Leu Tyr Ile
Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp 340 345 350Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile 355 360
365Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys
370 375 380Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg
Gly Phe385 390 395 400Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys
Lys Thr Thr Ile Gly 405 410 415Asp Arg Glu Val Asp Ser Asp Ala Tyr
Tyr Val Tyr Arg Leu Gln Val 420 425 430Ser Ser Ile Asn Val Ser Val
Asn Ala Val Gln Thr Val Val Arg Gln 435 440 445Gly Glu Asn Ile Thr
Leu Met Cys Ile Val Ile Gly Asn Glu Val Val 450 455 460Asn Phe Glu
Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu465 470 475
480Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile
485 490 495Leu His Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr
Thr Cys 500 505 510Asn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu
Lys Ala Ile Asn 515 520 525Ile Thr Val Val Glu Ser Gly 530
53525523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val
Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr Cys Ser Gly Ser Ala
Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu Pro Pro Gln Glu Met
Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser Val Leu Thr Leu Thr
Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr Phe Cys Thr His Asn
Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75 80Arg Lys Arg Leu Tyr
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 85 90 95Pro Asn Asp Ala
Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 100
105 110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr
Leu 115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr
Asp His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser
Tyr Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu Val Asp
Ser Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser Ser Ile
Asn Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg Gln Gly
Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200 205Glu Val
Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 210 215
220Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr His
Ile225 230 235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu
Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val Asn
Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val Glu
Ser Gly Gly Gly Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val 290 295 300Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp305 310 315 320Phe
Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 325 330
335Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr
340 345 350Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr
Ser Lys 355 360 365Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala 370 375 380Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr
Tyr Gly Thr Ser His Trp385 390 395 400Tyr Phe Asp Val Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala 405 410 415Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 420 425 430Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 435 440 445Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 450 455
460Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu465 470 475 480Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr 485 490 495Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys 500 505 510Val Glu Pro Lys Ser Cys Asp Lys
Thr His Leu 515 52026735PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Leu Val Val Thr Pro Pro
Gly Pro Glu Leu Val Leu Asn Val Ser Ser1 5 10 15Thr Phe Val Leu Thr
Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 20 25 30Met Ser Gln Glu
Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 35 40 45Phe Ser Ser
Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 50 55 60Glu Tyr
Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu65 70 75
80Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu
85 90 95Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr
Glu 100 105 110Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val
Val Thr Leu 115 120 125His Glu Lys Lys Gly Asp Val Ala Leu Pro Val
Pro Tyr Asp His Gln 130 135 140Arg Gly Phe Ser Gly Ile Phe Glu Asp
Arg Ser Tyr Ile Cys Lys Thr145 150 155 160Thr Ile Gly Asp Arg Glu
Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg 165 170 175Leu Gln Val Ser
Ser Ile Asn Val Ser Val Asn Ala Val Gln Thr Val 180 185 190Val Arg
Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn 195 200
205Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg
210 215 220Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr
His Ile225 230 235 240Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu
Glu Asp Ser Gly Thr 245 250 255Tyr Thr Cys Asn Val Thr Glu Ser Val
Asn Asp His Gln Asp Glu Lys 260 265 270Ala Ile Asn Ile Thr Val Val
Glu Ser Gly Glu Val Gln Leu Val Glu 275 280 285Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg305 310 315
320Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr
325 330 335Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe
Thr Phe 340 345 350Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln
Met Asn Ser Leu 355 360 365Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Lys Tyr Pro His Tyr 370 375 380Tyr Gly Ser Ser His Trp Tyr Phe
Asp Val Trp Gly Gln Gly Thr Leu385 390 395 400Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 405 410 415Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 420 425 430Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 435 440
445Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
450 455 460Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser465 470 475 480Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 485 490 495Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His 500 505 510Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val 515 520 525Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 530 535 540Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu545 550 555
560Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
565 570 575Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 580 585 590Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 595 600 605Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile 610 615 620Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro625 630 635 640Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 645 650 655Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 660 665 670Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 675 680
685Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
690 695 700Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu705 710 715 720His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 725 730 73527643PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 27Val Gly Phe Leu Pro
Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr1 5 10 15Glu Ile Thr Glu
Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu 20 25 30Val Val Thr
Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro 35 40 45Tyr Asp
His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr 50 55 60Ile
Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr65 70 75
80Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala
85 90 95Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys
Ile 100 105 110Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr
Pro Arg Lys 115 120 125Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp
Phe Leu Leu Asp Met 130 135 140Pro Tyr His Ile Arg Ser Ile Leu His
Ile Pro Ser Ala Glu Leu Glu145 150 155 160Asp Ser Gly Thr Tyr Thr
Cys Asn Val Thr Glu Ser Val Asn Asp His 165 170 175Gln Asp Glu Lys
Ala Ile Asn Ile Thr Val Val Glu Ser Gly Glu Val 180 185 190Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 195 200
205Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met
210 215 220Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Gly Trp225 230 235 240Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe Lys Arg 245 250 255Arg Phe Thr Phe Ser Leu Asp Thr Ser
Lys Ser Thr Ala Tyr Leu Gln 260 265 270Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Lys 275 280 285Tyr Pro His Tyr Tyr
Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly 290 295 300Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser305 310 315
320Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
325 330 335Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 340 345 350Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 355 360 365Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 370 375 380Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His385 390 395 400Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 405 410 415Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 420 425 430Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 435 440
445Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
450 455 460Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val465 470 475 480His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 485 490 495Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 500 505 510Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 515 520 525Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 530 535 540Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser545 550 555
560Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
565 570 575Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 580 585 590Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 595 600 605Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 610 615 620His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser625 630 635 640Pro Gly
Lys28421PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu
Phe Ile Phe Leu Thr1 5 10 15Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg
Val Thr Asp Pro Gln Leu 20 25 30Val Val Thr Leu His Glu Lys Lys Gly
Asp Val Ala Leu Pro Val Pro 35 40 45Tyr Asp His Gln Arg Gly Phe Ser
Gly Ile Phe Glu Asp Arg Ser Tyr 50 55 60Ile Cys Lys Thr Thr Ile Gly
Asp Arg Glu Val Asp Ser Asp Ala Tyr65 70 75 80Tyr Val Tyr Arg Leu
Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala 85 90 95Val Gln Thr Val
Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile 100 105 110Val Ile
Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys 115 120
125Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met
130 135 140Pro Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu
Leu Glu145 150 155 160Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu
Ser Val Asn Asp His 165 170 175Gln Asp Glu Lys Ala Ile Asn Ile Thr
Val Val Glu Ser Gly Glu Val 180 185 190Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu 195 200 205Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 210 215 220Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp225 230 235
240Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg
245 250 255Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
Leu Gln 260 265 270Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Lys 275 280 285Tyr Pro His Tyr Tyr Gly Ser Ser His Trp
Tyr Phe Asp Val Trp Gly 290 295 300Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser305 310 315 320Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 325 330 335Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 340 345 350Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 355 360
365Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
370 375 380Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His385 390 395 400Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys 405 410 415Asp Lys Thr His Thr
42029450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Val Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu
Phe Ile Phe Leu Thr1 5 10 15Glu Ile Thr Glu Ile Thr Ile Pro Cys Arg
Val Thr Asp Pro Gln Leu 20 25 30Val Val Thr Leu His Glu Lys Lys Gly
Asp Val Ala Leu Pro Val Pro 35 40 45Tyr Asp His Gln Arg Gly Phe Ser
Gly Ile Phe Glu Asp Arg Ser Tyr 50 55 60Ile Cys Lys Thr Thr Ile Gly
Asp Arg Glu Val Asp Ser Asp Ala Tyr65 70 75 80Tyr Val Tyr Arg Leu
Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala 85 90 95Val Gln Thr Val
Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile 100 105 110Val Ile
Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys 115 120
125Glu Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met
130 135 140Pro Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu
Leu Glu145 150 155 160Asp Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu
Ser Val Asn Asp His 165 170 175Gln Asp Glu Lys Ala Ile Asn Ile Thr
Val Val Glu Ser Gly Gly Gly 180 185 190Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 195 200 205Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 210 215 220Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser225 230 235
240Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val
245 250 255Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile
Asn Thr 260 265 270Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys
Arg Arg Phe Thr 275 280 285Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala
Tyr Leu Gln Met Asn Ser 290 295 300Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Lys Tyr Pro His305 310 315 320Tyr Tyr Gly Ser Ser
His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 325 330 335Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 340 345 350Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 355 360
365Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
370 375 380Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln385 390 395 400Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser 405 410 415Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 420 425 430Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr 435 440 445His Thr
45030450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser
Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro
His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Gly Gly Gly Ser Gly Gly Gly Gly Ser225 230 235
240Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
245 250 255Gly Gly Gly Ser Val Gly Phe Leu Pro Asn Asp Ala Glu Glu
Leu Phe 260 265 270Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro
Cys Arg Val Thr 275 280 285Asp Pro Gln Leu Val Val Thr Leu His Glu
Lys Lys Gly Asp Val Ala 290 295 300Leu Pro Val Pro Tyr Asp His Gln
Arg Gly Phe Ser Gly Ile Phe Glu305 310 315 320Asp Arg Ser Tyr Ile
Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp 325 330 335Ser Asp Ala
Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val 340 345 350Ser
Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr 355 360
365Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr
370 375 380Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val Thr
Asp Phe385 390 395 400Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile
Leu His Ile Pro Ser 405 410 415Ala Glu Leu Glu Asp Ser Gly Thr Tyr
Thr Cys Asn Val Thr Glu Ser 420 425 430Val Asn Asp His Gln Asp Glu
Lys Ala Ile Asn Ile Thr Val Val Glu 435 440 445Ser Gly
450317719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 31gacggatcgg gagatctccc gatcccctat
ggtgcactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg
cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca
acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag
gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctagc 900gtttaaactt aagcttggta ccgagctcgg atccactagt
ccagtgtggt ggaattctgc 960agatatccag cacagtggcg gccgccatga
aagctgtggt gctggccgtg gctctggtct 1020tcctgacagg gagccaggct
ctggtcgtca cacccccggg gccagagctt gtcctcaatg 1080tctccagcac
cttcgttctg acctgctcgg gttcagctcc ggtggtgtgg gaacggatgt
1140cccaggagcc cccacaggaa atggccaagg cccaggatgg caccttctcc
agcgtgctca 1200cactgaccaa cctcactggg ctagacacgg gagaatactt
ttgcacccac aatgactccc 1260gtggactgga gaccgatgag cggaaacggc
tctacatctt tgtgccagat cccaccgtgg 1320gcttcctccc taatgatgcc
gaggaactat tcatctttct cacggaaata actgagatca 1380ccattccatg
ccgagtaaca gacccacagc tggtggtgac actgcacgag aagaaagggg
1440acgttgcact gcctgtcccc tatgatcacc aacgtggctt ttctggtatc
tttgaggaca 1500gaagctacat ctgcaaaacc accattgggg acagggaggt
ggattctgat gcctactatg 1560tctacagact ccaggtgtca tccatcaacg
tctctgtgaa cgcagtgcag actgtggtcc 1620gccagggtga gaacatcacc
ctcatgtgca ttgtgatcgg gaatgaggtg gtcaacttcg 1680agtggacata
cccccgcaaa gaaagtgggc ggctggtgga gccggtgact gacttcctct
1740tggatatgcc ttaccacatc cgctccatcc tgcacatccc cagtgccgag
ttagaagact 1800cggggaccta cacctgcaat gtgacggaga gtgtgaatga
ccatcaggat gaaaaggcca 1860tcaacatcac cgtggttgag agcggcggtg
gtggcggctc cggtggaggc ggaagcgagg 1920tgcagctggt ggaatccggc
ggaggcctgg tccagcctgg cggatccctg agactgtcct 1980gtgccgcctc
cggctacgac ttcacccatt acggcatgaa ctgggtccga caggcccctg
2040gcaagggcct ggaatgggtc ggatggatca acacctacac cggcgagccc
acctacgccg 2100ccgacttcaa gcggcggttc accttctccc tggacacctc
caagtccacc gcctacctgc 2160agatgaactc cctgcgggcc gaggacaccg
ccgtgtacta ctgcgccaag tacccctact 2220actacggcac ctcccactgg
tacttcgacg tgtggggcca gggcaccctg gtcaccgtgt 2280cctccgcctc
taccaagggc ccctccgtgt tccctctggc cccctccagc aagtccacct
2340ctggcggcac cgccgctctg ggctgcctgg tcaaggacta cttccccgag
cccgtgaccg 2400tgtcctggaa ctctggcgcc ctgacctccg gcgtgcacac
ctttccagcc gtgctgcagt 2460cctccggcct gtactccctg tcctccgtcg
tgaccgtgcc ctccagctct ctgggcaccc 2520agacctacat ctgcaacgtg
aaccacaagc cctccaacac caaggtggac aagaaggtgg 2580aacccaagtc
ctgcgacaag acccacacct gtcccccctg ccctgcccct gaagcagccg
2640gtgcacccag cgtgttcctg ttccccccaa agcccaagga caccctgatg
atctcccgga 2700cccccgaagt gacctgcgtg gtggtggacg tgtcccacga
ggaccctgaa gtgaagttca 2760attggtacgt ggacggcgtg gaagtgcaca
atgccaagac caagcccaga gaggaacagt 2820acaactccac ctaccgggtg
gtgtccgtgc tgaccgtgct gcatcaggac tggctgaacg 2880gcaaagagta
caagtgcaag gtctccaaca aggccctgcc tgcccccatc gaaaagacca
2940tctccaaggc caagggccag ccccgcgagc ctcaggtgta cacactgcca
cccagccggg 3000aagagatgac caagaaccag gtctccctga cctgtctggt
caagggcttc tacccctccg 3060atatcgccgt cgaatgggag tccaacggcc
agcccgagaa caactacaag accacccccc 3120ctgtgctgga ctccgacggc
tcattcttcc tgtactccaa gctgaccgtg gacaagtccc 3180ggtggcagca
gggcaacgtg ttctcctgct ccgtgatgca cgaggccctg cacaaccact
3240acacccagaa gtccctgtcc tgcagccccg gcaagtgata atctagaggg
cccgtttaaa 3300cccgctgatc agcctcgact gtgccttcta gttgccagcc
atctgttgtt tgcccctccc 3360ccgtgccttc cttgaccctg gaaggtgcca
ctcccactgt cctttcctaa taaaatgagg 3420aaattgcatc gcattgtctg
agtaggtgtc attctattct ggggggtggg gtggggcagg 3480acagcaaggg
ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
3540tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
gcgccctgta 3600gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
cgtgaccgct acacttgcca 3660gcgccctagc gcccgctcct ttcgctttct
tcccttcctt tctcgccacg ttcgccggct 3720ttccccgtca agctctaaat
cgggggctcc ctttagggtt ccgatttagt gctttacggc 3780acctcgaccc
caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
3840agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
ctcttgttcc 3900aaactggaac aacactcaac cctatctcgg tctattcttt
tgatttataa gggattttgc 3960cgatttcggc ctattggtta aaaaatgagc
tgatttaaca aaaatttaac gcgaattaat 4020tctgtggaat gtgtgtcagt
tagggtgtgg aaagtcccca ggctccccag caggcagaag 4080tatgcaaagc
atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
4140agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
tcccgcccct 4200aactccgccc atcccgcccc taactccgcc cagttccgcc
cattctccgc cccatggctg 4260actaattttt tttatttatg cagaggccga
ggccgcctct gcctctgagc tattccagaa 4320gtagtgagga ggcttttttg
gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 4380atccattttc
ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
4440tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
atgactgggc 4500acaacagaca atcggctgct ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc 4560ggttcttttt gtcaagaccg acctgtccgg
tgccctgaat gaactgcagg acgaggcagc 4620gcggctatcg tggctggcca
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 4680tgaagcggga
agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
4740tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
ggctgcatac 4800gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
catcgcatcg agcgagcacg 4860tactcggatg gaagccggtc ttgtcgatca
ggatgatctg gacgaagagc atcaggggct 4920cgcgccagcc gaactgttcg
ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 4980cgtgacccat
ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
5040attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
cgttggctac 5100ccgtgatatt gctgaagagc ttggcggcga atgggctgac
cgcttcctcg tgctttacgg 5160tatcgccgct cccgattcgc agcgcatcgc
cttctatcgc cttcttgacg agttcttctg 5220agcgggactc tggggttcga
aatgaccgac caagcgacgc ccaacctgcc atcacgagat 5280ttcgattcca
ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
5340ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
ccccaacttg 5400tttattgcag cttataatgg ttacaaataa agcaatagca
tcacaaattt cacaaataaa 5460gcattttttt cactgcattc tagttgtggt
ttgtccaaac tcatcaatgt atcttatcat 5520gtctgtatac cgtcgacctc
tagctagagc ttggcgtaat catggtcata gctgtttcct 5580gtgtgaaatt
gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
5640aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc 5700gctttccagt cgggaaacct gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg 5760agaggcggtt tgcgtattgg gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg 5820gtcgttcggc tgcggcgagc
ggtatcagct cactcaaagg cggtaatacg gttatccaca 5880gaatcagggg
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
5940cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
cgagcatcac 6000aaaaatcgac gctcaagtca gaggtggcga aacccgacag
gactataaag ataccaggcg 6060tttccccctg gaagctccct cgtgcgctct
cctgttccga ccctgccgct taccggatac 6120ctgtccgcct ttctcccttc
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 6180ctcagttcgg
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
6240cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
aagacacgac 6300ttatcgccac tggcagcagc cactggtaac aggattagca
gagcgaggta tgtaggcggt 6360gctacagagt tcttgaagtg gtggcctaac
tacggctaca ctagaagaac agtatttggt 6420atctgcgctc tgctgaagcc
agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6480aaacaaacca
ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa
6540aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
gtggaacgaa 6600aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
ggatcttcac ctagatcctt 6660ttaaattaaa aatgaagttt taaatcaatc
taaagtatat atgagtaaac ttggtctgac 6720agttaccaat gcttaatcag
tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6780atagttgcct
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
6840cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
atcagcaata 6900aaccagccag ccggaagggc cgagcgcaga agtggtcctg
caactttatc cgcctccatc 6960cagtctatta attgttgccg ggaagctaga
gtaagtagtt cgccagttaa tagtttgcgc 7020aacgttgttg ccattgctac
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 7080ttcagctccg
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
7140gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
agtgttatca 7200ctcatggtta tggcagcact gcataattct cttactgtca
tgccatccgt aagatgcttt 7260tctgtgactg gtgagtactc aaccaagtca
ttctgagaat agtgtatgcg gcgaccgagt 7320tgctcttgcc cggcgtcaat
acgggataat accgcgccac atagcagaac tttaaaagtg 7380ctcatcattg
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
7440tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
tactttcacc 7500agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
caaaaaaggg aataagggcg 7560acacggaaat gttgaatact catactcttc
ctttttcaat attattgaag catttatcag 7620ggttattgtc tcatgagcgg
atacatattt gaatgtattt agaaaaataa acaaataggg 7680gttccgcgca
catttccccg aaaagtgcca cctgacgtc 7719326129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
32gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctagc 900gtttaaactt aagcttggta
ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960agatatccag
cacagtggcg gccgccatgg gatggagctg tatcatcctc ttcttggtgg
1020caacagctac aggcgtgcac tccgacatcc agctgaccca gtccccctcc
agcctgtccg 1080cctctgtggg cgacagagtg accatcacct gttccgccag
ccaggacatc tccaactacc 1140tgaactggta tcagcagaag cccggcaagg
cccccaaggt gctgatctac ttcacctcct 1200ccctgcactc cggcgtgccc
tccagattct ccggctctgg ctccggcacc gactttaccc 1260tgaccatctc
cagcctgcag cccgaggact tcgccaccta ctactgccag cagtactcca
1320ccgtgccctg gaccttcggc cagggcacca aggtggaaat caagcggacc
gtggccgctc 1380cctccgtgtt catcttccca ccctccgacg agcagctgaa
gtccggaacc gcctccgtcg 1440tgtgcctgct gaacaacttc tacccccgcg
aggccaaggt gcagtggaag gtggacaacg 1500ccctgcagag cggcaactcc
caggaatccg tcaccgagca ggactccaag gacagcacct 1560actccctgtc
cagcaccctg accctgtcca aggccgacta cgagaagcac aaggtgtacg
1620cctgcgaagt gacccaccag ggcctcagct ccccagtgac caagtccttc
aaccggggcg 1680agtgctagta atctagaggg cccgtttaaa cccgctgatc
agcctcgact gtgccttcta 1740gttgccagcc atctgttgtt tgcccctccc
ccgtgccttc cttgaccctg gaaggtgcca 1800ctcccactgt cctttcctaa
taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 1860attctattct
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
1920gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
accagctggg 1980gctctagggg gtatccccac gcgccctgta gcggcgcatt
aagcgcggcg ggtgtggtgg 2040ttacgcgcag cgtgaccgct acacttgcca
gcgccctagc gcccgctcct ttcgctttct 2100tcccttcctt tctcgccacg
ttcgccggct ttccccgtca agctctaaat cgggggctcc 2160ctttagggtt
ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
2220atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
acgttggagt 2280ccacgttctt taatagtgga ctcttgttcc aaactggaac
aacactcaac cctatctcgg 2340tctattcttt tgatttataa gggattttgc
cgatttcggc ctattggtta aaaaatgagc 2400tgatttaaca aaaatttaac
gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg 2460aaagtcccca
ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
2520aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
gcatgcatct 2580caattagtca gcaaccatag tcccgcccct aactccgccc
atcccgcccc taactccgcc 2640cagttccgcc cattctccgc cccatggctg
actaattttt tttatttatg cagaggccga 2700ggccgcctct gcctctgagc
tattccagaa gtagtgagga ggcttttttg gaggcctagg 2760cttttgcaaa
aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
2820atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
cggccgcttg
2880ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
ctgatgccgc 2940cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
gtcaagaccg acctgtccgg 3000tgccctgaat gaactgcagg acgaggcagc
gcggctatcg tggctggcca cgacgggcgt 3060tccttgcgca gctgtgctcg
acgttgtcac tgaagcggga agggactggc tgctattggg 3120cgaagtgccg
gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
3180catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
cattcgacca 3240ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
gaagccggtc ttgtcgatca 3300ggatgatctg gacgaagagc atcaggggct
cgcgccagcc gaactgttcg ccaggctcaa 3360ggcgcgcatg cccgacggcg
aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3420tatcatggtg
gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
3480ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
ttggcggcga 3540atgggctgac cgcttcctcg tgctttacgg tatcgccgct
cccgattcgc agcgcatcgc 3600cttctatcgc cttcttgacg agttcttctg
agcgggactc tggggttcga aatgaccgac 3660caagcgacgc ccaacctgcc
atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 3720ttgggcttcg
gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
3780atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
ttacaaataa 3840agcaatagca tcacaaattt cacaaataaa gcattttttt
cactgcattc tagttgtggt 3900ttgtccaaac tcatcaatgt atcttatcat
gtctgtatac cgtcgacctc tagctagagc 3960ttggcgtaat catggtcata
gctgtttcct gtgtgaaatt gttatccgct cacaattcca 4020cacaacatac
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
4080ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
gtcgtgccag 4140ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc 4200gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct 4260cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 4320tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
4380cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga 4440aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct cgtgcgctct 4500cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc gggaagcgtg 4560gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 4620ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
4680cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
cactggtaac 4740aggattagca gagcgaggta tgtaggcggt gctacagagt
tcttgaagtg gtggcctaac 4800tacggctaca ctagaagaac agtatttggt
atctgcgctc tgctgaagcc agttaccttc 4860ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggttttttt 4920gtttgcaagc
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
4980tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga 5040ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
aatgaagttt taaatcaatc 5100taaagtatat atgagtaaac ttggtctgac
agttaccaat gcttaatcag tgaggcacct 5160atctcagcga tctgtctatt
tcgttcatcc atagttgcct gactccccgt cgtgtagata 5220actacgatac
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
5280cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
cgagcgcaga 5340agtggtcctg caactttatc cgcctccatc cagtctatta
attgttgccg ggaagctaga 5400gtaagtagtt cgccagttaa tagtttgcgc
aacgttgttg ccattgctac aggcatcgtg 5460gtgtcacgct cgtcgtttgg
tatggcttca ttcagctccg gttcccaacg atcaaggcga 5520gttacatgat
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
5580gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
gcataattct 5640cttactgtca tgccatccgt aagatgcttt tctgtgactg
gtgagtactc aaccaagtca 5700ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat acgggataat 5760accgcgccac atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5820aaactctcaa
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
5880aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg 5940caaaatgccg caaaaaaggg aataagggcg acacggaaat
gttgaatact catactcttc 6000ctttttcaat attattgaag catttatcag
ggttattgtc tcatgagcgg atacatattt 6060gaatgtattt agaaaaataa
acaaataggg gttccgcgca catttccccg aaaagtgcca 6120cctgacgtc
6129331106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Met Arg Leu Pro Gly Ala Met Pro Ala Leu Ala
Leu Lys Gly Glu Leu1 5 10 15Leu Leu Leu Ser Leu Leu Leu Leu Leu Glu
Pro Gln Ile Ser Gln Gly 20 25 30Leu Val Val Thr Pro Pro Gly Pro Glu
Leu Val Leu Asn Val Ser Ser 35 40 45Thr Phe Val Leu Thr Cys Ser Gly
Ser Ala Pro Val Val Trp Glu Arg 50 55 60Met Ser Gln Glu Pro Pro Gln
Glu Met Ala Lys Ala Gln Asp Gly Thr65 70 75 80Phe Ser Ser Val Leu
Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 85 90 95Glu Tyr Phe Cys
Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu 100 105 110Arg Lys
Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 115 120
125Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu
130 135 140Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val
Thr Leu145 150 155 160His Glu Lys Lys Gly Asp Val Ala Leu Pro Val
Pro Tyr Asp His Gln 165 170 175Arg Gly Phe Ser Gly Ile Phe Glu Asp
Arg Ser Tyr Ile Cys Lys Thr 180 185 190Thr Ile Gly Asp Arg Glu Val
Asp Ser Asp Ala Tyr Tyr Val Tyr Arg 195 200 205Leu Gln Val Ser Ser
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val 210 215 220Val Arg Gln
Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn225 230 235
240Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg
245 250 255Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr
His Ile 260 265 270Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu
Asp Ser Gly Thr 275 280 285Tyr Thr Cys Asn Val Thr Glu Ser Val Asn
Asp His Gln Asp Glu Lys 290 295 300Ala Ile Asn Ile Thr Val Val Glu
Ser Gly Tyr Val Arg Leu Leu Gly305 310 315 320Glu Val Gly Thr Leu
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu 325 330 335Gln Val Val
Phe Glu Ala Tyr Pro Pro Pro Thr Val Leu Trp Phe Lys 340 345 350Asp
Asn Arg Thr Leu Gly Asp Ser Ser Ala Gly Glu Ile Ala Leu Ser 355 360
365Thr Arg Asn Val Ser Glu Thr Arg Tyr Val Ser Glu Leu Thr Leu Val
370 375 380Arg Val Lys Val Ala Glu Ala Gly His Tyr Thr Met Arg Ala
Phe His385 390 395 400Glu Asp Ala Glu Val Gln Leu Ser Phe Gln Leu
Gln Ile Asn Val Pro 405 410 415Val Arg Val Leu Glu Leu Ser Glu Ser
His Pro Asp Ser Gly Glu Gln 420 425 430Thr Val Arg Cys Arg Gly Arg
Gly Met Pro Gln Pro Asn Ile Ile Trp 435 440 445Ser Ala Cys Arg Asp
Leu Lys Arg Cys Pro Arg Glu Leu Pro Pro Thr 450 455 460Leu Leu Gly
Asn Ser Ser Glu Glu Glu Ser Gln Leu Glu Thr Asn Val465 470 475
480Thr Tyr Trp Glu Glu Glu Gln Glu Phe Glu Val Val Ser Thr Leu Arg
485 490 495Leu Gln His Val Asp Arg Pro Leu Ser Val Arg Cys Thr Leu
Arg Asn 500 505 510Ala Val Gly Gln Asp Thr Gln Glu Val Ile Val Val
Pro His Ser Leu 515 520 525Pro Phe Lys Val Val Val Ile Ser Ala Ile
Leu Ala Leu Val Val Leu 530 535 540Thr Ile Ile Ser Leu Ile Ile Leu
Ile Met Leu Trp Gln Lys Lys Pro545 550 555 560Arg Tyr Glu Ile Arg
Trp Lys Val Ile Glu Ser Val Ser Ser Asp Gly 565 570 575His Glu Tyr
Ile Tyr Val Asp Pro Met Gln Leu Pro Tyr Asp Ser Thr 580 585 590Trp
Glu Leu Pro Arg Asp Gln Leu Val Leu Gly Arg Thr Leu Gly Ser 595 600
605Gly Ala Phe Gly Gln Val Val Glu Ala Thr Ala His Gly Leu Ser His
610 615 620Ser Gln Ala Thr Met Lys Val Ala Val Lys Met Leu Lys Ser
Thr Ala625 630 635 640Arg Ser Ser Glu Lys Gln Ala Leu Met Ser Glu
Leu Lys Ile Met Ser 645 650 655His Leu Gly Pro His Leu Asn Val Val
Asn Leu Leu Gly Ala Cys Thr 660 665 670Lys Gly Gly Pro Ile Tyr Ile
Ile Thr Glu Tyr Cys Arg Tyr Gly Asp 675 680 685Leu Val Asp Tyr Leu
His Arg Asn Lys His Thr Phe Leu Gln His His 690 695 700Ser Asp Lys
Arg Arg Pro Pro Ser Ala Glu Leu Tyr Ser Asn Ala Leu705 710 715
720Pro Val Gly Leu Pro Leu Pro Ser His Val Ser Leu Thr Gly Glu Ser
725 730 735Asp Gly Gly Tyr Met Asp Met Ser Lys Asp Glu Ser Val Asp
Tyr Val 740 745 750Pro Met Leu Asp Met Lys Gly Asp Val Lys Tyr Ala
Asp Ile Glu Ser 755 760 765Ser Asn Tyr Met Ala Pro Tyr Asp Asn Tyr
Val Pro Ser Ala Pro Glu 770 775 780Arg Thr Cys Arg Ala Thr Leu Ile
Asn Glu Ser Pro Val Leu Ser Tyr785 790 795 800Met Asp Leu Val Gly
Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe 805 810 815Leu Ala Ser
Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val 820 825 830Leu
Ile Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala 835 840
845Arg Asp Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe
850 855 860Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser
Leu Tyr865 870 875 880Thr Thr Leu Ser Asp Val Trp Ser Phe Gly Ile
Leu Leu Trp Glu Ile 885 890 895Phe Thr Leu Gly Gly Thr Pro Tyr Pro
Glu Leu Pro Met Asn Glu Gln 900 905 910Phe Tyr Asn Ala Ile Lys Arg
Gly Tyr Arg Met Ala Gln Pro Ala His 915 920 925Ala Ser Asp Glu Ile
Tyr Glu Ile Met Gln Lys Cys Trp Glu Glu Lys 930 935 940Phe Glu Ile
Arg Pro Pro Phe Ser Gln Leu Val Leu Leu Leu Glu Arg945 950 955
960Leu Leu Gly Glu Gly Tyr Lys Lys Lys Tyr Gln Gln Val Asp Glu Glu
965 970 975Phe Leu Arg Ser Asp His Pro Ala Ile Leu Arg Ser Gln Ala
Arg Leu 980 985 990Pro Gly Phe His Gly Leu Arg Ser Pro Leu Asp Thr
Ser Ser Val Leu 995 1000 1005Tyr Thr Ala Val Gln Pro Asn Glu Gly
Asp Asn Asp Tyr Ile Ile 1010 1015 1020Pro Leu Pro Asp Pro Lys Pro
Glu Val Ala Asp Glu Gly Pro Leu 1025 1030 1035Glu Gly Ser Pro Ser
Leu Ala Ser Ser Thr Leu Asn Glu Val Asn 1040 1045 1050Thr Ser Ser
Thr Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp 1055 1060 1065Glu
Pro Glu Pro Glu Pro Gln Leu Glu Leu Gln Val Glu Pro Glu 1070 1075
1080Pro Glu Leu Glu Gln Leu Pro Asp Ser Gly Cys Pro Ala Pro Arg
1085 1090 1095Ala Glu Ala Glu Asp Ser Phe Leu 1100
1105341338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 34Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu
Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly
Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu Lys Gly Thr Gln His
Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu Gln Cys Arg Gly Glu
Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met Val Ser Lys Glu Ser
Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn Gly
Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95Ala Gln Ala Asn
His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110Pro Thr
Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120
125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys
Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp Gly Lys Arg Ile Ile
Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile Ser Asn Ala Thr Tyr
Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205Ala Thr Val Asn Gly
His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220Gln Thr Asn
Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val225 230 235
240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr
245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp
Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln
Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile
Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly Leu Tyr Thr Cys Arg
Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser Val Asn Thr Ser
Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335Lys His Arg
Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350Tyr
Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360
365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu
370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu
Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln
Ser Asn Val Phe Lys 405 410 415Asn Leu Thr Ala Thr Leu Ile Val Asn
Val Lys Pro Gln Ile Tyr Glu 420 425 430Lys Ala Val Ser Ser Phe Pro
Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445Arg Gln Ile Leu Thr
Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460Lys Trp Phe
Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys465 470 475
480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser
485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala
Ile Ile 500 505 510Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val
Ala Asp Ser Arg 515 520 525Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser
Asn Lys Val Gly Thr Val 530 535 540Gly Arg Asn Ile Ser Phe Tyr Ile
Thr Asp Val Pro Asn Gly Phe His545 550 555 560Val Asn Leu Glu Lys
Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575Cys Thr Val
Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590Arg
Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600
605Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met
610 615 620Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala
Arg Asn625 630 635 640Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys
Glu Ile Thr Ile Arg 645 650 655Asp Gln Glu Ala Pro Tyr Leu Leu Arg
Asn Leu Ser Asp His Thr Val 660 665 670Ala Ile Ser Ser Ser Thr Thr
Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685Glu Pro Gln Ile Thr
Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700Pro Gly Ile
Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile Glu Arg705 710 715
720Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln
725 730 735Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly
Thr Ser 740 745 750Asp Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr Cys
Thr Cys Val Ala 755 760 765Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu
Phe Ile Arg Lys Met Lys 770 775 780Arg Ser Ser Ser Glu Ile Lys Thr
Asp Tyr Leu Ser Ile Ile Met Asp785 790 795
800Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp
805 810 815Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly
Lys Ser 820 825 830Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala
Ser Ala Phe Gly 835 840 845Ile Lys Lys Ser Pro Thr Cys Arg Thr Val
Ala Val Lys Met Leu Lys 850 855 860Glu Gly Ala Thr Ala Ser Glu Tyr
Lys Ala Leu Met Thr Glu Leu Lys865 870 875 880Ile Leu Thr His Ile
Gly His His Leu Asn Val Val Asn Leu Leu Gly 885 890 895Ala Cys Thr
Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905 910Lys
Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915 920
925Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys
930 935 940Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu Asp
Ser Val945 950 955 960Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe
Gln Glu Asp Lys Ser 965 970 975Leu Ser Asp Val Glu Glu Glu Glu Asp
Ser Asp Gly Phe Tyr Lys Glu 980 985 990Pro Ile Thr Met Glu Asp Leu
Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005Gly Met Glu Phe
Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020Ala Ala
Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025 1030
1035Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr
1040 1045 1050Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met
Ala Pro 1055 1060 1065Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys
Ser Asp Val Trp 1070 1075 1080Ser Tyr Gly Val Leu Leu Trp Glu Ile
Phe Ser Leu Gly Gly Ser 1085 1090 1095Pro Tyr Pro Gly Val Gln Met
Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110Arg Glu Gly Met Arg
Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125Ile Tyr Gln
Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140Arg
Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145 1150
1155Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn
1160 1165 1170Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr
Pro Ala 1175 1180 1185Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser
Ala Pro Lys Phe 1190 1195 1200Asn Ser Gly Ser Ser Asp Asp Val Arg
Tyr Val Asn Ala Phe Lys 1205 1210 1215Phe Met Ser Leu Glu Arg Ile
Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230Asn Ala Thr Ser Met
Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245Leu Leu Ala
Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260Lys
Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265 1270
1275Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys
1280 1285 1290His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg
Phe Thr 1295 1300 1305Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala
Cys Cys Ser Pro 1310 1315 1320Pro Pro Asp Tyr Asn Ser Val Val Leu
Tyr Ser Thr Pro Pro Ile 1325 1330 1335351356PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1
5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu
Pro 20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn
Thr Thr 35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp
Leu Trp Pro 50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val
Thr Glu Cys Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile
Pro Lys Val Ile Gly Asn 85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr
Arg Glu Thr Asp Leu Ala Ser 100 105 110Val Ile Tyr Val Tyr Val Gln
Asp Tyr Arg Ser Pro Phe Ile Ala Ser 115 120 125Val Ser Asp Gln His
Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys 130 135 140Thr Val Val
Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155
160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg
165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr
Met Ile 180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile
Asn Asp Glu Ser 195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val
Val Gly Tyr Arg Ile Tyr 210 215 220Asp Val Val Leu Ser Pro Ser His
Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn
Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 245 250 255Asp Phe Asn
Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu 260 265 270Val
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 275 280
285Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu
290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn
Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala
Phe Gly Ser Gly Met 325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly
Glu Arg Val Arg Ile Pro Ala 340 345 350Lys Tyr Leu Gly Tyr Pro Pro
Pro Glu Ile Lys Trp Tyr Lys Asn Gly 355 360 365Ile Pro Leu Glu Ser
Asn His Thr Ile Lys Ala Gly His Val Leu Thr 370 375 380Ile Met Glu
Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395
400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val
405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser
Pro Val 420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr
Cys Thr Val Tyr 435 440 445Ala Ile Pro Pro Pro His His Ile His Trp
Tyr Trp Gln Leu Glu Glu 450 455 460Glu Cys Ala Asn Glu Pro Ser Gln
Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp
Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys 485 490 495Ile Glu Val
Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys 500 505 510Thr
Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr 515 520
525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser
530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp
Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys
Thr Ala Asp Arg Ser 565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys
Leu Gly Pro Gln Pro Leu Pro 580 585 590Ile His Val Gly Glu Leu Pro
Thr Pro Val Cys Lys Asn Leu Asp Thr 595 600 605Leu Trp Lys Leu Asn
Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile 610 615 620Leu Ile Met
Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635
640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val
645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr
Gly Asn 660 665 670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile
Glu Val Ser Cys 675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile
Met Trp Phe Lys Asp Asn 690 695 700Glu Thr Leu Val Glu Asp Ser Gly
Ile Val Leu Lys Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile Arg
Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr 725 730 735Cys Gln Ala
Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe 740 745 750Ile
Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu 755 760
765Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile
770 775 780Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys
Thr Gly785 790 795 800Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu
Pro Leu Asp Glu His 805 810 815Cys Glu Arg Leu Pro Tyr Asp Ala Ser
Lys Trp Glu Phe Pro Arg Asp 820 825 830Arg Leu Lys Leu Gly Lys Pro
Leu Gly Arg Gly Ala Phe Gly Gln Val 835 840 845Ile Glu Ala Asp Ala
Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr 850 855 860Val Ala Val
Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg865 870 875
880Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu
885 890 895Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly
Pro Leu 900 905 910Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu
Ser Thr Tyr Leu 915 920 925Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr
Lys Thr Lys Gly Ala Arg 930 935 940Phe Arg Gln Gly Lys Asp Tyr Val
Gly Ala Ile Pro Val Asp Leu Lys945 950 955 960Arg Arg Leu Asp Ser
Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly 965 970 975Phe Val Glu
Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro 980 985 990Glu
Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr 995
1000 1005Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg
Lys 1010 1015 1020Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu
Leu Ser Glu 1025 1030 1035Lys Asn Val Val Lys Ile Cys Asp Phe Gly
Leu Ala Arg Asp Ile 1040 1045 1050Tyr Lys Asp Pro Asp Tyr Val Arg
Lys Gly Asp Ala Arg Leu Pro 1055 1060 1065Leu Lys Trp Met Ala Pro
Glu Thr Ile Phe Asp Arg Val Tyr Thr 1070 1075 1080Ile Gln Ser Asp
Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile 1085 1090 1095Phe Ser
Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu 1100 1105
1110Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro
1115 1120 1125Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp
Cys Trp 1130 1135 1140His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser
Glu Leu Val Glu 1145 1150 1155His Leu Gly Asn Leu Leu Gln Ala Asn
Ala Gln Gln Asp Gly Lys 1160 1165 1170Asp Tyr Ile Val Leu Pro Ile
Ser Glu Thr Leu Ser Met Glu Glu 1175 1180 1185Asp Ser Gly Leu Ser
Leu Pro Thr Ser Pro Val Ser Cys Met Glu 1190 1195 1200Glu Glu Glu
Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala 1205 1210 1215Gly
Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro 1220 1225
1230Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu
1235 1240 1245Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly
Met Val 1250 1255 1260Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp
Arg Thr Lys Leu 1265 1270 1275Ser Pro Ser Phe Gly Gly Met Val Pro
Ser Lys Ser Arg Glu Ser 1280 1285 1290Val Ala Ser Glu Gly Ser Asn
Gln Thr Ser Gly Tyr Gln Ser Gly 1295 1300 1305Tyr His Ser Asp Asp
Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu 1310 1315 1320Ala Glu Leu
Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser 1325 1330 1335Thr
Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser 1340 1345
1350Pro Pro Val 1355361363PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 36Met Gln Arg Gly Ala Ala
Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly1 5 10 15Leu Leu Asp Gly Leu
Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu 20 25 30Asn Ile Thr Glu
Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser 35 40 45Ile Ser Cys
Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala 50 55 60Gln Glu
Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val65 70 75
80Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu
85 90 95Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys
Tyr 100 105 110Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala
Ala Ser Ser 115 120 125Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe
Ile Asn Lys Pro Asp 130 135 140Thr Leu Leu Val Asn Arg Lys Asp Ala
Met Trp Val Pro Cys Leu Val145 150 155 160Ser Ile Pro Gly Leu Asn
Val Thr Leu Arg Ser Gln Ser Ser Val Leu 165 170 175Trp Pro Asp Gly
Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu 180 185 190Val Ser
Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr 195 200
205Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile
210 215 220Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys
Ser Leu225 230 235 240Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn
Cys Thr Val Trp Ala 245 250 255Glu Phe Asn Ser Gly Val Thr Phe Asp
Trp Asp Tyr Pro Gly Lys Gln 260 265 270Ala Glu Arg Gly Lys Trp Val
Pro Glu Arg Arg Ser Gln Gln Thr His 275 280 285Thr Glu Leu Ser Ser
Ile Leu Thr Ile His Asn Val Ser Gln His Asp 290 295 300Leu Gly Ser
Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg305 310 315
320Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu
325 330 335Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu
Leu Val 340 345 350Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro
Glu Phe Gln Trp 355 360 365Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg
His Ser Pro His Ala Leu 370 375 380Val Leu Lys Glu Val Thr Glu Ala
Ser Thr Gly Thr Tyr Thr Leu Ala385 390 395 400Leu Trp Asn Ser Ala
Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu 405 410 415Val Val Asn
Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro 420 425 430Ser
Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr 435 440
445Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr
450 455 460Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln
Gln Gln465 470 475 480Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala
Val Thr Thr Gln Asp 485 490 495Ala Val Asn Pro Ile Glu Ser Leu Asp
Thr Trp Thr Glu Phe Val Glu 500 505 510Gly Lys Asn Lys Thr Val Ser
Lys Leu Val Ile Gln Asn Ala Asn Val 515 520 525Ser Ala Met Tyr Lys
Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu 530 535
540Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr
Ile545 550 555 560Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln
Pro Val Leu Leu 565 570 575Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu
His Leu Arg Trp Tyr Arg 580 585 590Leu Asn Leu Ser Thr Leu His Asp
Ala His Gly Asn Pro Leu Leu Leu 595 600 605Asp Cys Lys Asn Val His
Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu 610 615 620Glu Glu Val Ala
Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile625 630 635 640Pro
Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln 645 650
655Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val
660 665 670Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp
Leu Leu 675 680 685Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu
Val Ala Gly Ala 690 695 700His Ala Pro Ser Ile Val Trp Tyr Lys Asp
Glu Arg Leu Leu Glu Glu705 710 715 720Lys Ser Gly Val Asp Leu Ala
Asp Ser Asn Gln Lys Leu Ser Ile Gln 725 730 735Arg Val Arg Glu Glu
Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn 740 745 750Ala Lys Gly
Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser 755 760 765Glu
Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val 770 775
780Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn
Met785 790 795 800Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr
Leu Ser Ile Ile 805 810 815Met Asp Pro Gly Glu Val Pro Leu Glu Glu
Gln Cys Glu Tyr Leu Ser 820 825 830Tyr Asp Ala Ser Gln Trp Glu Phe
Pro Arg Glu Arg Leu His Leu Gly 835 840 845Arg Val Leu Gly Tyr Gly
Ala Phe Gly Lys Val Val Glu Ala Ser Ala 850 855 860Phe Gly Ile His
Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met865 870 875 880Leu
Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu 885 890
895Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu
900 905 910Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile
Val Glu 915 920 925Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg
Ala Lys Arg Asp 930 935 940Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro
Glu Gln Arg Gly Arg Phe945 950 955 960Arg Ala Met Val Glu Leu Ala
Arg Leu Asp Arg Arg Arg Pro Gly Ser 965 970 975Ser Asp Arg Val Leu
Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala 980 985 990Arg Arg Ala
Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro 995 1000
1005Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg
1010 1015 1020Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg
Asp Leu 1025 1030 1035Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp
Val Val Lys Ile 1040 1045 1050Cys Asp Phe Gly Leu Ala Arg Asp Ile
Tyr Lys Asp Pro Asp Tyr 1055 1060 1065Val Arg Lys Gly Ser Ala Arg
Leu Pro Leu Lys Trp Met Ala Pro 1070 1075 1080Glu Ser Ile Phe Asp
Lys Val Tyr Thr Thr Gln Ser Asp Val Trp 1085 1090 1095Ser Phe Gly
Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser 1100 1105 1110Pro
Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu 1115 1120
1125Arg Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala
1130 1135 1140Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro
Lys Ala 1145 1150 1155Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu
Gly Asp Leu Leu 1160 1165 1170Gln Gly Arg Gly Leu Gln Glu Glu Glu
Glu Val Cys Met Ala Pro 1175 1180 1185Arg Ser Ser Gln Ser Ser Glu
Glu Gly Ser Phe Ser Gln Val Ser 1190 1195 1200Thr Met Ala Leu His
Ile Ala Gln Ala Asp Ala Glu Asp Ser Pro 1205 1210 1215Pro Ser Leu
Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp 1220 1225 1230Val
Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly 1235 1240
1245Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr
1250 1255 1260Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly
Met Val 1265 1270 1275Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser
Arg His Arg Gln 1280 1285 1290Glu Ser Gly Phe Ser Cys Lys Gly Pro
Gly Gln Asn Val Ala Val 1295 1300 1305Thr Arg Ala His Pro Asp Ser
Gln Gly Arg Arg Arg Arg Pro Glu 1310 1315 1320Arg Gly Ala Arg Gly
Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly 1325 1330 1335Glu Leu Ser
Glu Pro Ser Glu Glu Asp His Cys Ser Pro Ser Ala 1340 1345 1350Arg
Val Thr Phe Phe Thr Asp Asn Ser Tyr 1355 1360375PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gly
Gly Gly Gly Ser1 5384PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Gly Gly Gly
Ser1395PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Gly Gly Gly Glu Ser1 54010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 104121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10
15Gly Gly Gly Ser Gly 204210PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Gly Tyr Asp Phe Thr His Tyr
Gly Met Asn1 5 104317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 43Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Ala Ala Asp Phe Lys1 5 10 15Arg4414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val1 5
104511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5
10467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Phe Thr Ser Ser Leu His Ser1 5479PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Gln
Gln Tyr Ser Thr Val Pro Trp Thr1 54810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Gly
Tyr Thr Phe Thr Asn Tyr Gly Met Asn1 5 104914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Tyr
Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val1 5
105020PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidemisc_feature(1)..(20)This sequence may encompass
1-4 'Gly-Gly-Gly- Gly-Ser' repeating units 50Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
20514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidemisc_feature(1)..(4)This sequence may encompass
1-4 'Gly' residues 51Gly Gly Gly Gly1528PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptidemisc_feature(1)..(8)This sequence may encompass 1-4
'Gly-Gly' repeating units 52Gly Gly Gly Gly Gly Gly Gly Gly1
55316PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidemisc_feature(1)..(16)This sequence may encompass
1-4 'Gly-Gly-Gly- Ser' repeating units 53Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 155420PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptidemisc_feature(1)..(20)This sequence may encompass 1-4
'Gly-Gly-Gly- Glu-Ser' repeating units 54Gly Gly Gly Glu Ser Gly
Gly Gly Glu Ser Gly Gly Gly Glu Ser Gly1 5 10 15Gly Gly Glu Ser
20555PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Ser Gly Gly Gly Cys1 55629PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly1 5 10
15Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
* * * * *