U.S. patent application number 13/392509 was filed with the patent office on 2012-10-18 for coagulation factor vii compositions and methods of making and using same.
Invention is credited to Nathan C. Geething, Volker Schellenberger, Joshua Silverman, Benjamin Spink, Willem P. Stemmer, Wayne To, Chia-wei Wang.
Application Number | 20120263701 13/392509 |
Document ID | / |
Family ID | 43605834 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120263701 |
Kind Code |
A1 |
Schellenberger; Volker ; et
al. |
October 18, 2012 |
COAGULATION FACTOR VII COMPOSITIONS AND METHODS OF MAKING AND USING
SAME
Abstract
The present invention relates to compositions comprising factor
VII coagulation factors linked to extended recombinant polypeptide
(XTEN), isolated nucleic acids encoding the compositions and
vectors and host cells containing the same, and methods of making
and using such compositions in treatment of coagulation
factor-related diseases, disorders, and conditions.
Inventors: |
Schellenberger; Volker; (Los
Gatos, CA) ; Stemmer; Willem P.; (Los Gatos, CA)
; Geething; Nathan C.; (San Juan, PR) ; To;
Wayne; (Fremont, CA) ; Silverman; Joshua;
(Sunnyvale, CA) ; Wang; Chia-wei; (Milpitas,
CA) ; Spink; Benjamin; (San Carlos, CA) |
Family ID: |
43605834 |
Appl. No.: |
13/392509 |
Filed: |
August 2, 2010 |
PCT Filed: |
August 2, 2010 |
PCT NO: |
PCT/US10/02147 |
371 Date: |
June 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61236493 |
Aug 24, 2009 |
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61236836 |
Aug 25, 2009 |
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61280956 |
Nov 10, 2009 |
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61280955 |
Nov 10, 2009 |
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Current U.S.
Class: |
424/94.3 ;
435/188 |
Current CPC
Class: |
A61P 7/04 20180101; C12N
15/70 20130101; C12N 9/644 20130101; C12N 9/6437 20130101; C07K
2319/50 20130101; C07K 14/001 20130101; C07K 2319/31 20130101; C07K
2319/00 20130101; A61P 7/02 20180101; A61K 38/00 20130101; C12Y
304/21022 20130101 |
Class at
Publication: |
424/94.3 ;
435/188 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61P 7/04 20060101 A61P007/04; C12N 9/96 20060101
C12N009/96 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under SBIR
grant 2R44GM079873-02 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Feb 3, 2010 |
US |
12699761 |
Claims
1. An isolated factor VII polypeptide comprising an extended
recombinant polypeptide (XTEN), said XTEN comprising at least 200
amino acid residues, wherein said factor VII polypeptide exhibits a
terminal half-life that is longer than about 12 hours when
administered to a subject.
2. The isolated factor VII polypeptide of claim 1, wherein the
factor VII polypeptide exhibits at least 90% sequence identity
compared to a sequence selected from Table 2 when optimally
aligned.
3. The isolated factor VII polypeptide of claim 1 or 2, wherein
said factor VII is linked at its C-terminus to the XTEN.
4. The isolated factor VII polypeptide of any of proceeding claims
that is linked to the XTEN via a cleavage sequence that is
cleavable by a mammalian protease selected from the group
consisting of factor XIa, factor XIIa, kallikrein, factor VIIa,
factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP13,
MMP-17 and MMP-20.
5. The isolated factor VII polypeptide of claim 4, wherein cleavage
at the cleavage sequence by the mammalian protease releases said
XTEN from said factor VII polypeptide.
6. The isolated factor VII polypeptide of any of proceeding claims,
wherein said XTEN is incorporated between any two adjacent domains
contained in said factor VII, wherein said two adjacent domains are
selected from the group consisting of Gla, EGF1, EGF2, activated
peptide (AP), and peptidase S1 (Pro).
7. The isolated factor VII polypeptide of any of proceeding claims,
wherein said XTEN is characterized in that: (a) the cumulative
total of XTEN amino acid residues is greater than 200 to about 3000
amino acid residues: (b) the sum of asparagine and glutamine
residues is less than 10% of the total amino acid sequence of the
XTEN; (c) the sum of methionine and tryptophan residues is less
than 2% of the total amino acid sequence of the XTEN; (d) the XTEN
sequence has a subsequence score less than 10; (e) the XTEN
sequence has greater than 90% random coil formation as determined
by GOR algorithm; and (f) the XTEN sequence has less than 2% alpha
helices and 2% beta-sheets as determined by Chou-Fasman
algorithm.
8. The isolated factor VII polypeptide of any of proceeding claims
exhibiting an apparent molecular weight factor of at least about
4.
9. The isolated factor VII polypeptide of any of proceeding claims,
wherein said XTEN exhibits at least 90% sequence identity to a
comparable length of an amino acid sequence selected from Table 4,
Table 9, Table 10, Table 11, Table 12, or Table 13.
10. The isolated factor VII polypeptide of any of proceeding claims
that is configured according to formula VII:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(AP1)-(XTEN).sub.w-(AP2)-(X-
TEN).sub.x-(Pro)-(S).sub.y--(XTEN).sub.z VII wherein independently
for each occurrence, (a) Gla is a Gla domain of factor VII; (b)
EGF1 is an EGF1 domain of factor VII; (c) EGF2 is an EFG2 domain of
factor VII; (d) AP1 is a portion of an activator peptide domain of
factor VII; (e) AP2 is a portion of an activator peptide domain of
factor IX that includes at least a first cleavage sequence; (f) PRO
is a protease domain of factor VII; (g) S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence; (h) XTEN is an extended
recombinant polypeptide that exhibits at least 90% sequence
identity to a comparable length of an amino acid sequence selected
from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13,
(i) u is either 0 or 1; (j) v is either 0 or 1; (k) x is either 0
or 1; (l) y is either 0 or 1; and (m) z is either 0 or 1, with the
proviso that u+v+x+y+z.gtoreq.1.
11. The isolated factor VII polypeptide of any of proceeding claims
comprising more than one XTEN.
12. An isolated factor VII polypeptide comprising at least one
heterologous sequence that is cleavable by a pro-coagulant protease
that does not activate a wildtype factor VII, wherein upon cleavage
heterologous sequence, said factor VII polypeptide is
activated.
13. The isolated factor VII polypeptide of claim 12 that is
activatable in the absence of a tissue factor.
14. The isolated factor VII polypeptide of claim 12 or 13, wherein
the heterologous sequence is cleavable by activated factor XI.
15. The isolated factor VII polypeptide of any of claims 12-14
comprising at least two heterologous sequences, each of which is
cleavable by the same or different pro-coagulant proteases.
16. The isolated factor VII polypeptide of any of claims 12-15
comprising an extended recombinant polypeptide (XTEN).
17. The isolated factor VII polypeptide of any of claims 12-16,
wherein upon cleavage of the at least one heterologous sequence,
the XTEN is also cleaved.
18. The isolated factor VII polypeptide of any of claims 12-17,
wherein said heterologous sequence exhibits at least 90% sequence
identity to the sequence
KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV.
19. The isolated factor VII polypeptide of any of claims 12-18,
wherein said heterologous sequence exhibits at least 90% sequence
identity to a sequence selected from TSKLTRAETVFP and FNDFTRV when
optimally aligned.
20. The isolated factor VII polypeptide of any of claims 12-19,
wherein said factor VII polypeptide does not contain a component
selected the group consisting of polyethylene glycol (PEG),
albumin, antibody, and an antibody fragment.
21. The isolated factor VII polypeptide of any of claims 12-10
exhibiting an apparent molecular weight factor of at least about
4.
22. A method of treating coagulopathy in a subject, comprising
administering to said subject a composition comprising a
therapeutically effective amount of factor VII of any of proceeding
claims.
23. The method of claim 22, wherein said coagulopathy is hemophilia
A or hemophilia B.
24. The method of claim 22 or 23 wherein the therapeutically
effective amount of factor VII is sufficient to bypass the need for
exogenously administered factor VIII and/or factor IX to yield a
comparable therapeutic effect.
25. A method of treating a bleeding episode in a subject
comprising: administering to said subject a composition comprising
a therapeutically effective amount of the factor VII of any of
claims 1-21.
26. A method of treating a subject deficient in a clotting protein,
comprising: administering to said subject a composition comprising
a therapeutically effective amount of the factor VII of any of
claims 1-21.
27. The method of claim 26, wherein the clotting protein
substitutes wildtype factor VII, factor IX, or factor XI.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. Nos. 61/236,493 filed Aug. 24, 2009,
61/236,836, filed Aug. 25, 2009, 61/280,955, filed Nov. 10, 2009,
61/280,956 filed Nov. 10, 2009, and U.S. application Ser. No.
12/699,761 filed Feb. 3, 2010, all of which, and co-pending
application under attorney docket number 32808-726.201 filed
herewith, are incorporated herein by reference in their entirety
for all purposes.
BACKGROUND OF THE INVENTION
[0003] In hemophilia, the clotting of blood is disturbed by a lack
of certain plasma blood clotting factors. Human factor IX (FIX) is
a zymogen of a serine protease that is an important component of
the intrinsic pathway of the blood coagulation cascade. In
individuals who do not have FIX deficiency, the average half-life
of FIX is short, approximately 18-24 hours. A deficiency of
functional FIX, due to an X-linked disorder that occurs in about
one in 30,000 males, results in hemophilia B, also known as
Christmas disease, named after a young boy named Stephen Christmas
who was found to be lacking this factor. Over 100 mutations of
factor IX have been described; some cause no symptoms, but many
lead to a significant bleeding disorder. When untreated, hemophilia
B is associated with uncontrolled bleeding into muscles, joints,
and body cavities following injury, and may result in death.
Previously, treatments for the disease included administration of
FIX prepared from human plasma derived from donor pools, which
carried attendant risks of infection with blood-borne viruses
including human immunodeficiency virus (HIV) and hepatitis C virus
(HCV). More recently, recombinant FIX products have become
commercially available.
[0004] The in vivo activity of exogenously supplied factor IX is
limited both by protein half-life and inhibitors of coagulation,
including antithrombin III. Factor IX compositions typically have
short half-lives, requiring frequent injections. Also, current
FIX-based therapeutics requires intravenous administration due to
poor bioavailability. Thus, there is a need for improved factor IX
compositions with extended half-life and retention of activity when
administered as part of a preventive and/or therapeutic regimen for
hemophilia, including hemophilia B.
[0005] Factor VII is a coagulation factor protein synthesized in
the liver and secreted into the blood as a single chain zymogen
with a molecular weight of approximately 50 kDa. The FVII zymogen
is converted into an activated form (FVIIa) by proteolytic
cleavage, and the activated form, when complexed with tissue factor
(TF), is able to convert both factor IX and factor X into their
activated forms, leading to rapid thrombin generation and fibrin
formation. Because the circulating half-life of rFVIIa is about 2.3
hours ("Summary Basis for Approval for NovoSeven.COPYRGT.", FDA
reference number 96-0597), multiple and frequent administrations
are required for the treatment of bleeding disorders in
hemophiliacs and subjects with factor VII deficiency.
[0006] Chemical modifications to a therapeutic protein can reduce
its in vivo clearance rate and subsequent increase serum half-life.
One example of a common modification is the addition of a
polyethylene glycol (PEG) moiety, typically coupled to the protein
via an aldehyde or N-hydroxysuccinimide (NHS) group on the PEG
reacting with an amine group (e.g. lysine side chain or the
N-terminus). However, the conjugation step can result in the
formation of heterogeneous product mixtures that need to be
separated, leading to significant product loss and complexity of
manufacturing and does not result in a completely
chemically-uniform product. Also, the pharmacologic function of the
therapeutics protein may be hampered if amino acid side chains in
the vicinity of its binding site are modified by the PEGylation
process. Fusing an Fc domain to the therapeutic protein is another
approach to increases the size of the therapeutic protein, hence
reducing the rate of clearance through the kidney. Additionally,
the Fc domain confers the ability to bind to, and be recycled from
lysosomes by, the FcRn receptor, which results in increased
pharmacokinetic half-life. Unfortunately, the Fc domain does not
fold efficiently during recombinant expression, and tends to form
insoluble precipitates known as inclusion bodies. These inclusion
bodies must be solubilized and functional protein must be renatured
from the misfolded aggregate. Such process is time-consuming,
inefficient, and expensive. Accordingly, there remains a need for
improved coagulation factor compositions with increased half-life
which can be administered less frequently, and/or be produced by a
simpler process at a cheaper cost.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to compositions and
methods for the treatment or improvement of a condition or the
enhancement of a parameter associated with the administration of
coagulations factors IX and/or VII. In particular, the present
invention provides compositions of fusion proteins comprising one
or more extended recombinant polypeptides (XTEN). A subject XTEN is
typically a non-repetitive sequence and unstructured conformation.
XTEN is linked to a coagulation factor ("CF") selected from factor
IX ("FIX"), factor VII ("FVII"), factor VII-factor IX hybrids, and
sequence variants thereof, resulting in a coagulation factor-XTEN
fusion protein ("CFXTEN"). In part, the present disclosure is
directed to pharmaceutical compositions comprising the fusion
proteins and the uses thereof for treating coagulation
factor-related diseases, disorders or conditions. The CFXTEN
compositions have enhanced pharmacokinetic properties compared to
CF not linked to XTEN, which may permit more convenient dosing and
improved efficacy. In some embodiments, the CFXTEN compositions of
the invention do not have a component selected the group consisting
of: polyethylene glycol (PEG), albumin, antibody, and an antibody
fragment.
[0008] In some embodiments, the invention provides an isolated
factor IX fusion protein, comprising a factor IX sequence that is
at least about 90%, or about 95%, or about 96%, or about 97%, or
about 98%, or about 99% identical to an amino acid sequence
selected from Table 1. The factor IX having such sequence idendity
is further linked to an extended recombinant polypeptide (XTEN)
having at least about 100 to about 3000 amino acid residues. In one
embodiment, the XTEN is linked to the C-terminus of the FIX or the
FVII CF. In some embodiments, the invention provides an isolated
factor VII fusion protein, comprising a factor VII that is at least
about 90%, or about 95%, or about 96%, or about 97%, or about 98%,
or about 99% identical to an amino acid sequence selected from
Table 2. The factor VII having such sequence is linked to an
extended recombinant polypeptide (XTEN).
[0009] Non-limiting examples of CFXTEN with a single FIX or a
single FVII linked to a single XTEN are presented in Table 41. In
one embodiment, the invention provides a CFXTEN composition has at
least about 80% sequence identity compared to a CFXTEN from Table
41, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
about 100% sequence identity as compared to a CFXTEN from Table 41.
In some embodiments, the CF and the XTEN components of the fusion
protein are linked via a cleavage sequence that is cleavable by a
protease, including endogenous mammalian proteases. Examples of
such protease include, but are not limited to, FXIa, FXIIa,
kallikrein, FVIIa, FIXa, FXa, thrombin, elastase-2, granzyme B,
MMP-12, MMP-13, MMP-17 or MMP-20, TEV, enterokinase, rhinovirus 3C
protease, and sortase A, or a sequence selected from Table 7. In
one embodiment, a CFXTEN composition with a cleavage sequence has a
sequence having at least about 80% sequence identity compared to a
CFXTEN from Table 42, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or about 100% sequence identity as compared to a
CFXTEN from Table 42. However, the invention also provides
substitution of any of the CF sequences of Table 1 or Table 2 for a
CF in a sequence of Table 42, and substitution of any XTEN sequence
of Table 4 for an XTEN in a sequence of Table 42, and substitution
of any cleavage sequence of Table 7 for a cleavage sequence in a
sequence of Table 42. In CFXTEN embodiments having cleavage
sequences, cleavage of the cleavage sequence by the protease
releases the XTEN from the CF. In some embodiments of the
foregoing, the CF component becomes biologically active or has an
increase in activity upon its release from the XTEN by cleavage of
the cleavage sequence, wherein the pro-coagulant activity is at
least about 30%, or at least about 40%, or at least about 50%, or
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 90% compared to the corresponding FIX or FVII not
linked to XTEN.
[0010] The invention provides isolated CFXTEN fusion proteins that
comprise a second XTEN of about 36 to about 3000 amino acid
residues, which can be identical or can be different from the first
XTEN, wherein the second XTEN can be incorporated between any two
adjacent domains of the CF, i.e., between the Gla, EFG1, EGF2,
activating peptide and protease domains, or is incorporated within
the sequence of an existing loop domain of a domain sequence of the
CF, as described more fully in the Examples. In one embodiment, the
first and the second XTEN can be an amino acid sequence selected
from any one of Tables 4, or 9-13, or can exhibit at least at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least about 99% sequence identity
compared to a sequence selected from Tables 4 and 9-13. In another
embodiment, the isolated fusion protein comprises a second XTEN of
about 36 to about 3000 amino acid residues. The fusion protein can
adopt a multiple-XTEN configuration of Table 6, or a variation
thereof.
[0011] The invention provides CFXTEN compositions comprising XTEN
linked to a factor VII comprising one or more heterologous cleavage
sequences cleavable by the same or different pro-coagulant
proteases. In some embodiments of the foregoing, the factor VII
comprises a heterologous sequence of factor XI incorporated into or
substituted for portions of the FVII sequence, resulting in factor
VII-factor IX hybrid sequence variants. In some embodiments, a
portion or the entirety of the sequence from the activation peptide
domain of FIX is incorporated or substituted for FVII sequences
bridging the region between the EFG2 and protease domains of the
FVII component, resulting in compositions that can be activated as
part of the intrinsic system of the coagulation cascade (e.g.,
activated factor XI). In such case, the factor VII-factor IX CFXTEN
composition can be activated by a pro-coagulant protease in the
absence of tissue factor, such that the CFXTEN can serve as a
by-pass of factors VIII and IX in the intrinsic coagulation pathway
when such factors are deficient (e.g., in hemophilia A or B) or
when inhibitors to these factors are present. In one embodiment,
the FVII-FIX sequence variant incorporates the full-length FIX AP
domain plus at least about 2, or at least about 3, or at least
about 4, or at least about 5, or at least about 6, or at least
about 7, or at least about 8, or at least about 9, or at least
about 10, or at least about 11, or at least about 12 or more amino
acids flanking adjacent amino acid residues on one or both sides of
the R145-A146 and R180-V181 cleavage sites of the FIX AP domain
(e.g., the sequence
RVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE in the case of
12 flanking amino acids on the N-terminus side and 5 flanking amino
acids on the C-terminus side).
[0012] In another embodiment, the CFXTEN FVII-FIX sequence variant
comprises a heterologous FIX sequence exhibiting at least at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or 100% identity compared to the sequence:
KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV, when optimally
aligned.
[0013] In other embodiments, the CFXTEN comprises FVII-FIX sequence
variants that incorporate a portion of the FIX AP that includes a
sequence of at least about 2, or at least about 3, or at least
about 4, or at least about 5, or more amino acids that flank one or
both sides of the R145-A146 cleavage site (e.g., the sequence
TSKLTRAETVFP in the case of 6 flanking amino acids on either side
of the cleavage site) or a sequence of at least about 2, or at
least about 3, or at least about 4, or at least about 5 or more
amino acids that flank one or both sides of the R180-V181 cleavage
site (e.g., the sequence and DFTRV in the case of 4 amino acids on
the N-terminal flank and valine as the C-terminus of the cleavage
site from FIX). In one embodiment of the foregoing, the CFXTEN
FVII-FIX sequence variant comprises a heterologous FIX sequence
exhibiting at least at least about 80%, or at least about 90%, or
at least about 91%, or at least about 92%, or at least about 93%,
or at least about 94%, or at least about 95%, or at least about
96%, or at least about 97%, or at least about 98%, or 100% identity
compared to a sequence selected from TSKLTRAETVFP and FNDFTRV, when
optimally aligned.
[0014] In another embodiment, the CFXTEN comprises a FVII-FIX
sequence variant disclosed above that further includes the same AP
cleavage sequence as a linker between the C-terminus of the FVII
component and the XTEN component of the fusion protein, e.g., an N-
to C-terminus configuration of FVII variant-AP sequence-XTEN,
thereby permitting the release of the FVII variant component from
the CFXTEN fusion protein when cleaved by the same pro-coagulant
protease as per that of the FVII to FVIIa transition. In one
embodiment, the FVII-FIX CFXTEN of any of the foregoing embodiments
includes the factor XI cleavage sequence KLTRAET as the linker
between the FVII-FIX sequence and the XTEN, thereby permitting the
release of the FVII variant component from the CFXTEN fusion
protein by the initiation of the intrinsic coagulation cascade. In
one embodiment, the invention provides a CFXTEN with a FVII-FIX
hybrid sequence that exhibits at least about 80%, or at least about
85%, or at least about 90%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, sequence identity compared to a sequence from
Table 43. In other embodiments, the invention provides a FVII-FIX
sequence variant with incorporated FIX-derived AP cleavage sequence
that is not linked to an XTEN. In one embodiment, the FVII-FIX
sequence without an XTEN exhibits at least about 80%, or at least
about 85%, or at least about 90%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or
at least about 99% sequence identity as compared with a sequence
from Table 43 without an XTEN.
[0015] In one embodiment of the CFXTEN composition, the invention
provides a fusion protein of formula I:
(XTEN).sub.x--CF--(XTEN).sub.y I
wherein independently for each occurrence, CF is a coagulation
factor; x is either 0 or 1 and y is either 0 or 1 wherein
x+y.gtoreq.1; and XTEN is an extended recombinant polypeptide.
[0016] In another embodiment of the CFXTEN composition, the
invention provides a fusion protein of formula II:
(XTEN).sub.x--(CF)--(S).sub.y--(XTEN).sub.y II
wherein independently for each occurrence, CF is a coagulation
factor a; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence; x is
either 0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN
is an extended recombinant polypeptide.
[0017] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein, wherein the fusion
protein is of formula III:
(XTEN).sub.x(S).sub.x--(CF)--(S).sub.y--(XTEN).sub.y III
wherein independently for each occurrence, CF is a coagulation
factor; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence; x is
either 0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN
is an extended recombinant polypeptide.
[0018] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula IV:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(XTEN).sub.w-(AP)--(XTEN).-
sub.x-(Pro)-(S).sub.y--(XTEN).sub.z IV
wherein independently for each occurrence, Gla is a Gla domain of
FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX;
AP is an activator peptide of FIX; PRO is a protease domain of FIX;
S is a spacer sequence having between 1 to about 50 amino acid
residues that can optionally include a cleavage sequence; u is
either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either
0 or 1; z is either 0 or 1 with the proviso that
u+v+w+x+z.gtoreq.1; and XTEN is an extended recombinant
polypeptide.
[0019] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula V:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(AP1)-(XTEN).sub.w-(AP2)-(-
XTEN).sub.x-(Pro)-(S).sub.y--(XTEN).sub.z V
wherein independently for each occurrence, Gla is a Gla domain of
FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX;
AP1 is the N-terminal sequence portion of the activator peptide
domain of FIX that includes a first native cleavage sequence of the
AP domain; AP2 is the C-terminal sequence portion of the activator
peptide domain of FIX that includes a second native cleavage
sequence of the AP domain; PRO is a protease domain of FIX; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence; u is either 0 or
1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; z is
either 0 or 1 with the proviso that u+v+w+x+z.gtoreq.1; and XTEN is
an extended recombinant polypeptide.
[0020] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula VI:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(XTEN).sub.w-(Pro)-(S).sub-
.x--(XTEN).sub.y VI
wherein independently for each occurrence, Gla is a Gla domain of
FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of
FVII; PRO is a protease domain of FVII; S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence; u is either 0 or 1; v is
either 0 or 1; x is either 0 or 1; y is either 0 or 1 with the
proviso that u+v+w+y.gtoreq.1; and XTEN is an extended recombinant
polypeptide.
[0021] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula VII:
(Gla)-(XTENN).sub.t-(EGF1)-(XTEN).sub.u-(EGF2)-(AP1).sub.v--(XTEN).sub.w-
-(AP2).sub.x-(Pro)-(S).sub.y--(XTEN).sub.z VII
wherein independently for each occurrence, Gla is a Gla domain of
FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of
FVII; PRO is a protease domain of FVII; AP1 is the N-terminal
sequence portion of the activator peptide domain of FIX that
includes the native cleavage sequence; AP2 is the C-terminal
sequence portion of the activator peptide domain of FIX that
includes the native cleavage sequence; S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence; t is either 0 or 1; u is
either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either
0 or 1; z is either 0 or 1 with the proviso that t+u+w+z.gtoreq.1;
and XTEN is an extended recombinant polypeptide. In the embodiment,
the CFXTEN composition can include the entirety of the FIX
activator peptide domain sequence or one or both cleavage sequences
from the activator peptide domain of factor IX, e.g., a sequence of
at least about 3 to about 12 amino acids that flank the R145-A146
cleavage site and the sequence of at least about 1 to about 5 amino
acids that flank the R180-V181 cleavage site, as described more
fully above. The invention also contemplates substitution of any of
the other cleavage sequences of Table 7 for the AP cleavage
sequences.
[0022] The CFXTEN compositions of the embodiments described herein
can be evaluated for retention of activity (including after
cleavage of any incorporated XTEN-releasing cleavage sites) using
any appropriate in vitro assay disclosed herein (e.g., the assays
of Table 40 or the assays described in the Examples), to determine
the suitability of the configuration for use as a therapeutic agent
in the treatment of a coagulation-factor related disease, disorder
or condition. In one embodiment, the CFXTEN exhibits at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% of the activity compared to the native CF not linked to
XTEN. In another embodiment, the CF component released from the
CFXTEN by enzymatic cleavage of the incorporated cleavage sequence
linking the CF and XTEN components exhibits at least about 60%, or
at least about 70%, or at least about 80%, or at least about 90% of
the activity compared to the native CF not linked to XTEN.
[0023] The XTEN of the CFXTEN compositions have at least about 200,
or at least about 400, or at least about 800, or at least about
900, or at least about 1000, or at least about 2000, up to about
3000 amino acids residues. The XTEN of the CFXTEN fusion protein
compositions is characterized in that they have one or more of the
following characteristics: (a) at least a first XTEN comprises at
least about 200 contiguous amino acids that exhibits at least about
90%, or about 95%, or about 96%, or about 97%, or about 98%, or
about 99% identity to a comparable length of an amino acid sequence
selected from a sequence shown in Table 4; (b) the XTEN sequence
lacks a predicted T-cell epitope when analyzed by TEPITOPE
algorithm, wherein the TEPITOPE algorithm prediction for epitopes
within the XTEN sequence is based on a score of -5, or -6, or -7,
or -8, or -9 or greater; (c) the XTEN has a subsequence score of
less than 10, or less than 9, or less than 8, or less than 7, or
less than 6, or less than 5, or even less; (d) the sum of
asparagine and glutamine residues is less than 10% of the total
amino acid sequence of the XTEN; (e) the sum of methionine and
tryptophan residues is less than 2% of the total amino acid
sequence of the XTEN; (f) the XTEN has greater than 90% random coil
formation, or about 95%, or about 96%, or about 97%, or about 98%,
or about 99% random coil formation as determined by GOR algorithm;
(g) the XTEN sequence has less than 2% alpha helices and 2%
beta-sheets as determined by the Chou-Fasman algorithm; and (h) the
sum of glycine (G), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P) residues constitutes more than about
90%, or about 95%, or about 96%, or about 97%, or about 98%, or
about 99% of the total amino acid residues of the XTEN.
[0024] In another embodiment, the invention provides CFXTEN fusion
proteins, wherein the XTEN is characterized in that the sum of
asparagine and glutamine residues is less than 10% of the total
amino acid sequence of the XTEN, the sum of methionine and
tryptophan residues is less than 2% of the total amino acid
sequence of the XTEN, the XTEN sequence has less than 5% amino acid
residues with a positive charge, the XTEN sequence has greater than
90% random coil formation, or about 95%, or about 96%, or about
97%, or about 98%, or about 99% random coil formation as determined
by GOR algorithm; and the XTEN sequence has less than 2% alpha
helices and 2% beta-sheets as determined by the Chou-Fasman
algorithm. In some embodiments, no one type of amino acid
constitutes more than 30% of the XTEN sequence of the CFXTEN.
[0025] In another embodiment, the invention provides CFXTEN fusion
proteins, wherein the XTEN is characterized in that at least about
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99% of the XTEN sequence
consists of non-overlapping sequence motifs wherein each of the
sequence motifs has about 9 to about 14 amino acid residues and
wherein the sequence of any two contiguous amino acid residues does
not occur more than twice in each of the sequence motifs consist of
four to six types of amino acids selected from glycine (G), alanine
(A), serine (S), threonine (T), glutamate (E) and proline (P). In
one embodiment, the XTEN is characterized in that at least about
80%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99% of the XTEN sequence
consists of non-overlapping sequence motifs wherein the motifs are
selected from Table 3.
[0026] In some embodiments, the XTEN has a sequence in which no
three contiguous amino acids are identical unless the amino acid is
serine, in which case no more than three contiguous amino acids are
serine residues. In other embodiment, the XTEN component of the
CFXTEN has a subsequence score of less than 10, or less than 9, or
less than 8, or less than 7, or less than 6, or less than 5, or
less. In the embodiments of this paragraph, the XTEN is
characterized as "substantially non-repetitive."
[0027] In some embodiments, the invention provides CFXTEN
comprising at least a second XTEN, wherein the XTEN sequence
exhibits at least about 80%, or at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%
sequence identity compared to a sequence from Table 4, Table 9,
Table 10, Table 11, Table 12, or Table 13.
[0028] In some embodiments, CFXTEN fusion proteins exhibits
enhanced pharmacokinetic properties compared to CF not linked to
XTEN, wherein the enhanced properties include but are not limited
to longer terminal half-life, larger area under the curve,
increased time in which the blood concentration remains within the
therapeutic window, increased time between consecutive doses
results in blood concentrations within the therapeutic window, and
decreased dose in moles over time that can be administered compared
to a CF not linked to XTEN, yet still result in a blood
concentration within the therapeutic window for that composition.
In some embodiments, the terminal half-life of the CFXTEN fusion
protein administered to a subject is increased at least about
three-fold, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
eight-fold, or at least about ten-fold, or at least about 20-fold,
or at least about 40-fold, or at least about 60-fold, or at least
about 100-fold, or even higher as compared to CF not linked to XTEN
and administered to a subject at a comparable dose. In other
embodiments, the terminal half-life of the CFXTEN fusion protein
administered to a subject is at least about 12 h, or at least about
24 h, or at least about 48 h, or at least about 72 h, or at least
about 96 h, or at least about 120 h, or at least about 144 h, or at
least about 21 days or greater. In other embodiments, the enhanced
pharmacokinetic property is reflected by the fact that the blood
concentrations that remain within the therapeutic window for the
CFXTEN fusion protein for a given period are at least about two
fold, or at least about three-fold, or at least about four-fold, or
at least about five-fold, or at least about six-fold, or at least
about eight-fold, or at least about ten-fold longer, or at least
about 20-fold, or at least about 40-fold, or at least about
60-fold, or at least about 100-fold compared to CF not linked to
XTEN and administered to a subject at a comparable dose. The
increase in half-life and time spent within the therapeutic window
permits less frequent dosing and decreased amounts of the fusion
protein (in moles equivalent) that are administered to a subject,
compared to the corresponding CF not linked to XTEN. In one
embodiment, administration of a CFXTEN to a subject using a
therapeutically-effective dose regimen results in a gain in time of
at least two-fold, or at least three-fold, or at least four-fold,
or at least five-fold, or at least six-fold, or at least
eight-fold, or at least 10-fold, or at least about 20-fold, or at
least about 40-fold, or at least about 60-fold, or at least about
100-fold or higher between at least two consecutive C.sub.max peaks
and/or C.sub.min troughs for blood levels of the fusion protein
compared to the corresponding CF not linked to the XTEN and
administered using a comparable dose regimen to a subject.
[0029] In some embodiments, the XTEN enhances thermostability of CF
when linked to the XTEN wherein the thermostability is ascertained
by measuring the retention of biological activity after exposure to
a temperature of about 37.degree. C. for at least about 7 days of
the biologically active protein in comparison to the biologically
active protein not linked to the XTEN. In one embodiment of the
foregoing, the retention of biological activity increases by at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 100%, or about 150%,
at least about 200%, at least about 300%, or about 500% longer
compared to the CF not linked to the XTEN.
[0030] In some embodiments, the isolated CFXTEN fusion protein is
configures to have reduced binding affinity for a clearance
receptor as compared to the corresponding CF not linked to the
XTEN. In one embodiment, the CFXTEN fusion protein exhibits binding
affinity for a clearance receptor of the CF in the range of about
0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or
about 2% to about 10% of the binding affinity of the corresponding
CF not linked to the XTEN. In another embodiment, a CFXTEN fusion
protein with reduced affinity can have reduced active clearance and
a corresponding increase in half-life of at least about 3-fold, or
at least about 5-fold, or at least about 6-fold, or at least about
7-fold, or at least about 8-fold, or at least about 9-fold, or at
least about 10-fold, or at least about 12-fold, or at least about
15-fold, or at least about 17-fold, or at least about 20-fold, or
at least about 30-fold, or at least about 50-fold, or at least
about 100-fold longer compared to the corresponding CF that is not
linked to the XTEN.
[0031] In some embodiments, the invention provides CFXTEN fusion
proteins wherein the CFXTEN exhibits increased solubility of at
least three-fold, or at least about four-fold, or at least about
five-fold, or at least about six-fold, or at least about
seven-fold, or at least about eight-fold, or at least about
nine-fold, or at least about ten-fold, or at least about 15-fold,
or at least a 20-fold, or at least 40-fold, or at least 60-fold at
physiologic conditions compared to the CF not linked to XTEN.
[0032] In some embodiments, CFXTEN fusion proteins exhibit an
increased apparent molecular weight as determined by size exclusion
chromatography, compared to the actual molecular weight. In some
embodiments the CF comprising a FIX and at least a first XTEN
exhibits an apparent molecular weight of at least about 400 kD, or
at least about 500 kD, or at least about 700 kD, or at least about
1000 kD, or at least about 1400 kD, or at least about 1600 kD, or
at least about 1800 kD, or at least about 2000 kD, while the actual
molecular weight of each FIX component of the fusion protein is
about 50 kD and the molecular weight of the fusion protein ranges
from about 70 to about 125 kDa. In other embodiments, the CF
comprising a FVII and at least a first XTEN exhibits an apparent
molecular weight of at least about 400 kD, or at least about 500
kD, or at least about 700 kD, or at least about 1000 kD, or at
least about 1400 kD, or at least about 1600 kD, or at least about
1800 kD, or at least about 2000 kD, while the actual molecular
weight of each FIX component of the fusion protein is about 50 kD
and the molecular weight of the fusion protein ranges from about 70
to about 125 kDa. Accordingly, the CFXTEN fusion proteins can have
an apparent molecular weight that is about 6-fold greater, or about
8-fold greater, or about 10-fold greater, or about 12-fold greater,
or about 15-fold greater than the actual molecular weight of the
fusion protein. In some cases, the isolated CFXTEN fusion protein
of any of the embodiments disclosed herein exhibit an apparent
molecular weight factor under physiologic conditions that is
greater than about 4, or about 5, or about 6, or about 7, or about
8, or about 10, or greater than about 15.
[0033] In some embodiments, administration of a therapeutically
effective dose of a fusion protein of one of formulae I-VII to a
subject in need thereof can result in a gain in time of at least
two-fold, or at least three-fold, or at least four-fold, or at
least five-fold or more spent within a therapeutic window for the
fusion protein compared to the corresponding CF not linked to the
XTEN of and administered at a comparable dose to a subject. In
other cases, administration of a therapeutically effective dose of
a fusion protein of an embodiment of formulas I-VII to a subject in
need thereof can result in a gain in time between consecutive doses
necessary to maintain a therapeutically effective dose regimen of
at least 48 h, or at least 72 h, or at least about 96 h, or at
least about 120 h, or at least about 7 days, or at least about 14
days, or at least about 21 days between consecutive doses compared
to a CF not linked to XTEN and administered at a comparable
dose.
[0034] The fusion proteins of the disclosed compositions can be
designed to have different configurations, N- to C-terminus, of a
CF and XTEN and optional spacer sequences, including but not
limited to XTEN-CF, CF-XTEN, XTEN-S--CF, CF--S-XTEN, XTEN-CF-XTEN,
CF--CF-XTEN, XTEN-CF--CF, CF--S--CF-XTEN, XTEN-CF--S--CF, and
multimers thereof. The choice of configuration can, as disclosed
herein, confer particular pharmacokinetic, physico/chemical, or
pharmacologic properties including, in the case of an incorporated
cleavage sequence, the release of the CF with a concomitant
increase in activity.
[0035] In some embodiments, the CFXTEN fusion protein is
characterized in that: (i) it has a longer half-life when
administered to a subject compared to the corresponding coagulation
factor not linked to the XTEN administered to a subject under an
otherwise equivalent dose; (ii) when a smaller molar amount of the
fusion protein is administered to a subject in comparison to the
corresponding coagulation factor that lacks the XTEN administered
to a subject under an otherwise equivalent dose regimen, the fusion
protein achieves a comparable area under the curve (AUC) as the
corresponding coagulation factor not linked to the XTEN; (iii) when
a smaller molar amount of the fusion protein is administered to a
subject in comparison to the corresponding coagulation factor that
lacks the XTEN administered to a subject under an otherwise
equivalent dose regimen, the fusion protein achieves a comparable
therapeutic effect as the corresponding coagulation factor not
linked to the XTEN; (iv) when the fusion protein is administered to
a subject less frequently in comparison to the corresponding
coagulation factor not linked to the XTEN administered to a subject
using an otherwise equivalent molar amount, the fusion protein
achieves a comparable area under the curve (AUC) as the
corresponding coagulation factor not linked to the XTEN; (v) when
the fusion protein is administered to a subject less frequently in
comparison to the corresponding coagulation factor not linked to
the XTEN administered to a subject using an otherwise equivalent
molar amount, the fusion protein achieves a comparable therapeutic
effect as the corresponding coagulation factor not linked to the
XTEN; (vi) when an accumulatively smaller molar amount of the
fusion protein is administered to a subject in comparison to the
corresponding coagulation factor not linked to the XTEN
administered to a subject under an otherwise equivalent dose
period, the fusion protein achieves comparable area under the curve
(AUC) as the corresponding coagulation factor not linked to the
XTEN; or (vii) when an accumulatively smaller molar amount of the
fusion protein is administered to a subject in comparison to the
corresponding coagulation factor not linked to the XTEN
administered to a subject under an otherwise equivalent dose
period, the fusion protein achieves comparable therapeutic effect
as the corresponding coagulation factor not linked to the XTEN.
[0036] The invention provides a method of producing a fusion
protein comprising a factor VII or factor IX or a factor VII-factor
IX hybrid coagulation factor fused to one or more extended
recombinant polypeptides (XTEN), comprising: (a) providing host
cell comprising a recombinant polynucleotide molecule encoding the
fusion protein (b) culturing the host cell under conditions
permitting the expression of the fusion protein; and (c) recovering
the fusion protein from the culture. In one embodiment of the
method, the coagulation factor of the fusion protein has at least
90% sequence identity compared to a sequence selected from Table 1
or Table 2. In another embodiment of the method, the one or more
XTEN of the expressed fusion protein has at least about 90%, or
about 91%, or about 92%, or about 93%, or about 94%, or about 95%,
or about 96%, or about 97%, or about 98%, or about 99% to about
100% sequence identity compared to a sequence selected from Table
4. In another embodiment of the method, the host cell is a
eukaryotic cell. In another embodiment of the method, the host cell
is CHO cell. In another embodiment of the method the isolated
fusion protein is recovered from the host cell cytoplasm in
substantially soluble form.
[0037] The invention provides isolated nucleic acids comprising a
polynucleotide sequence selected from (a) a polynucleotide encoding
the fusion protein of any of the foregoing embodiments, or (b) the
complement of the polynucleotide of (a). In one embodiment, the
invention provides an isolated nucleic acid comprising a
polynucleotide sequence that has at least 80% sequence identity, or
about 85%, or at least about 90%, or about 91%, or about 92%, or
about 93%, or about 94%, or about 95%, or about 96%, or about 97%,
or about 98%, or about 99% to about 100% sequence identity compared
to (a) a polynucleotide sequence of comparable length selected from
Table 41 and Table 42; or (b) the complement of the polynucleotide
of (a). The invention provides expression vectors comprising the
nucleic acid of any of the embodiments hereinabove described in
this paragraph. In one embodiment, the expression vector of the
foregoing further comprises a recombinant regulatory sequence
operably linked to the polynucleotide sequence. In another
embodiment, the polynucleotide sequence of the expression vectors
of the foregoing is fused in frame to a polynucleotide encoding a
secretion signal sequence, which can be a CF native signal
sequence. The invention provides a host cell that comprises an
expression vector of any of the embodiments hereinabove described
in this paragraph. In one embodiment, the host cell is a eukaryotic
cell. In another embodiment, the host cell is a CHO cell. In
another embodiment, the host cell is HEK cell.
[0038] In one embodiment, the invention provides pharmaceutical
compositions comprising the fusion protein of any of the foregoing
embodiments and a pharmaceutically acceptable carrier. In another
embodiment, the invention provides kits, comprising packaging
material and at least a first container comprising the
pharmaceutical composition of the foregoing embodiment and a label
identifying the pharmaceutical composition and storage and handling
conditions, and a sheet of instructions for the reconstitution
and/or administration of the pharmaceutical compositions to a
subject.
[0039] The invention provides a method of treating a coagulopathy
or a coagulation factor-related disease, disorder or condition in a
subject, comprising administering to the subject a therapeutically
effective amount of a CFXTEN fusion protein of any of the foregoing
embodiments. In one embodiment of the method, the
coagulation-factor related condition is selected from bleeding
disorders (e.g., defective platelet function, thrombocytopenia or
von Willebrand's disease), coagulopathies (any disorder of blood
coagulation, including coagulation factor deficiencies), hemophilia
B (aka Christmas disease), factor IX-related bleeding disorders,
factor VII deficiency, hemophilia A, vascular injury, uncontrolled
bleeding in subjects not suffering from hemophilia, bleeding from
trauma or surgery, bleeding due to anticoagulant therapy, and
bleeding due to liver disease. In one embodiment of the method of
treatment, the coagulopathy is hemophilia A. In one embodiment of
the method of treatment, the coagulopathy is hemophilia B. In
another embodiment of the method of treatment, the coagulopathy is
factor VII deficiency. In another embodiment of the method of
treatment, the CFXTEN is administered to a subject to control a
bleeding episode. In another embodiment of the method of treatment,
a CFXTEN comprising a factor VII-factor IX sequence hybrid is
administered to a subject to control a bleeding episode, wherein
the CFXTEN is activated by a pro-coagulant protease of the
intrinsic coaguation cascade (e.g., activated factor XI). In
another embodiment, the present invention provides a method of
treating a clotting factor deficiency in a subject, comprising:
administering to said subject a composition comprising a
therapeutically effective amount of the factor VII provided
herein.
[0040] In some embodiments, the composition can be administered
subcutaneously, intramuscularly, or intravenously. In one
embodiment, the composition is administered at a therapeutically
effective amount, wherein the administration results in a gain in
time spent within a therapeutic window for the fusion protein
compared to the corresponding CF of the fusion protein not linked
to the XTEN and administered at a comparable dose to a subject. The
gain in time spent within the therapeutic window can at least
three-fold longer than the corresponding CF not linked to the XTEN,
or alternatively, at least four-fold, or five-fold, or six-fold, or
seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at
least 20-fold, or at least about 30-fold, or at least about
50-fold, or at least about 100-fold longer than the corresponding
CF not linked to XTEN. In some embodiments of the method of
treatment, (i) a smaller molar amount of (e.g. of about two-fold
less, or about three-fold less, or about four-fold less, or about
five-fold less, or about six-fold less, or about eight-fold less,
or about 100 fold-less or greater) the fusion protein is
administered in comparison to the corresponding coagulation factor
not linked to the XTEN under an otherwise same dose regimen, and
the fusion protein achieves a comparable area under the curve
and/or a comparable therapeutic effect as the corresponding
coagulation factor not linked to the XTEN; (ii) the fusion protein
is administered less frequently (e.g., every two days, about every
seven days, about every 14 days, about every 21 days, or about,
monthly) in comparison to the corresponding coagulation factor not
linked to the XTEN under an otherwise same dose amount, and the
fusion protein achieves a comparable area under the curve and/or a
comparable therapeutic effect as the corresponding coagulation
factor not linked to the XTEN; or (iii) an accumulative smaller
molar amount (e.g. about 5%, or about 10%, or about 20%, or about
40%, or about 50%, or about 60%, or about 70%, or about 80%, or
about 90% less) of the fusion protein is administered in comparison
to the corresponding coagulation factor not linked to the XTEN
under the otherwise same dose regimen the fusion protein achieves a
comparable area under the curve and/or a comparable therapeutic
effect as the corresponding coagulation factor not linked to the
XTEN. The accumulative smaller molar amount is measured for a
period of at least about one week, or about 14 days, or about 21
days, or about one month. In some embodiments of the method of
treatment, the therapeutic effect is a measured parameter selected
from blood concentrations of coagulation factor, prothrombin (PT)
assay, activated partial prothrombin (aPTT) assay, bleeding time
assay, whole blood clotting time (WBCT), and
thrombelastography.
[0041] In another embodiment, invention provides a method of
treating a disease, disorder or condition, comprising administering
the pharmaceutical composition described above to a subject using
multiple consecutive doses of the pharmaceutical composition
administered using a therapeutically effective dose regimen. In one
embodiment of the foregoing, the therapeutically effective dose
regimen can result in a gain in time of at least three-fold, or
alternatively, at least four-fold, or five-fold, or six-fold, or
seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at
least 20-fold, or at least about 30-fold, or at least about
50-fold, or at least about 100-fold longer time between at least
two consecutive C.sub.max peaks and/or C.sub.min troughs for blood
levels of the fusion protein compared to the corresponding CF of
the fusion protein not linked to the fusion protein and
administered at a comparable dose regimen to a subject. In another
embodiment of the foregoing, the administration of the fusion
protein results in improvement in at least one measured parameter
of a coagulation factor-related disease using less frequent dosing
or a lower total dosage in moles of the fusion protein of the
pharmaceutical composition compared to the corresponding
biologically active protein component(s) not linked to the fusion
protein and administered to a subject d using a therapeutically
effective regimen to a subject.
[0042] The invention further provides use of the compositions
comprising the fusion protein of any of the foregoing embodiments
in the preparation of a medicament for treating a disease, disorder
or condition in a subject in need thereof. In one embodiment of the
foregoing, the disease, disorder or condition is selected from
group consisting of bleeding disorders, coagulopathies, hemophilia
B (aka Christmas disease), factor IX-related bleeding disorders,
factor VII deficiency, vascular injury, bleeding from trauma or
surgery, bleeding due to anticoagulant therapy, and liver disease.
Any of the disclosed embodiments can be practiced alone or in
combination depending on the interested application.
INCORPORATION BY REFERENCE
[0043] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The features and advantages of the invention may be further
explained by reference to the following detailed description and
accompanying drawings that sets forth illustrative embodiments.
[0045] FIG. 1 shows a schematic representation of exemplary CFXTEN
(FIX-XTEN) fusion proteins.
[0046] FIG. 1A shows the domain architecture of native FIX, with
the gamma-carboxyglutamate domain, the EGF1 and EGF2 domains, the
activation peptide, and the protease domain, with a linked XTEN at
the C-terminus. Arrows indicate the cleavage sites for the
activation peptide domain. FIG. 1B shows a FIX molecule with an
XTEN polypeptide attached to the C-terminus via a cleavage
sequence, and indicates a site for proteolytic cleavage to release
the XTEN (arrows indicate the cleavage sites for the activation
peptide domain and the release point for the XTEN).
[0047] FIG. 2 illustrates several examples of CXTEN configurations
of FIX-XTEN and associated protease cleavage sites. FIG. 2A shows
an FIX-XTEN with two proteolytic cleavage sites (arrows) within the
activation peptide of FIX, and a C-terminus XTEN without a cleavage
site linkage. FIG. 2B is similar to the configuration of FIG. 2A,
but the C-terminus XTEN is linked via a cleavage sequence, with the
arrow indicating the release point. FIG. 2C shows three
configurations of FIX-XTEN, with the XTEN integrated between the
various domains of FIX. FIG. 2D shows an FIX-XTEN with the XTEN
portion inserted into the activation peptide between the native
cleavage sites, which would release the XTEN upon the proteolytic
activation of FIX. FIG. 2E illustrates FIX-XTEN that contain
multiple XTEN sequences inserted between different domains with the
addition of a releasable XTEN at the C-terminus. FIG. 2F
illustrates FIX-XTEN where the XTEN has been inserted within loop
domains of FIX.
[0048] FIG. 3 is a schematic of the coagulation cascade, showing
both the extrinsic and intrinsic pathways.
[0049] FIG. 4 shows several examples of CXTEN configurations of
FVII-XTEN. FIG. 4A shows a FVII-XTEN that has not been activated.
FIG. 4B shows a FVII-XTEN in which the peptide has been cleaved,
resulting in an activated FVIIa-XTEN; FIG. 4C illustrates a
FVII-XTEN composition with a cleavage sequence for releasable XTEN
in which the FVII component has not been activated, containing a
cleavage site for the activation protease (AP) and a second
cleavage site for the release protease (RP). FIG. 4D shows a
composition of activated FVIIa-XTEN containing a cleavage site for
the release protease.
[0050] FIG. 5 illustrates a strategy for FVII-XTEN design approach
using internal XTEN. FIGS. 5A-D show exemplary sites for XTEN
insertion between boundaries of the FVII domains with inactive FVII
on the left and an activated form of FVII on the right (A:
Insertion of XTEN between Gla and EGF1 domain, B: Insertion of XTEN
between EGF1 and EGF2. C: Insertion of XTEN at C-terminus of
activation peptide, D: Insertion of XTEN at N-terminus of
activation peptide). FIG. 5E shows examples of FVII-XTEN in which
the XTEN is located within external loops within individual domains
fusion proteins, with inactive FVII on the left and FVIIa on the
right. The activation peptide in FVII is shown as a thin line
versus XTEN that is shown as a fat line.
[0051] FIG. 6 illustrates essentially the same constructs as FIG.
5, but with an XTEN linked at the C-terminus of each construct.
[0052] FIG. 7 is a schematic that shows some of the various
locations in which XTEN can be inserted internal to the sequences
of the coagulation factors FVII or FIX.
[0053] FIG. 8 is a schematic of the key components of the clotting
system. FIG. 7A: Normal clotting system with the intrinsic and
extrinsic cascade components. FIG. 7B illustrates a variation in
which an inactive/low active form of FVII-XTEN (FVII*) is intended
to bypass the FIX and FVIII components of the intrinsic system when
activated endogenously after administration.
[0054] FIG. 9 is a graph of the distribution of cell cluster size
(gray bars) and FVII ELISA titers in ng/ml (black bars) by ELISA of
clones from primary screening of pBC0014 CHO-K1 transformants (not
all clones were labeled underneath the bars due to insufficient
space) (see Example 25 for experimental details). Clones were
sorted according to ELISA titer low to high (left to right).
[0055] FIG. 10 is a graph of cell counts (white bars) and FVII
titers in ng/ml (black bars) of the top pBC0014 clones (see Example
25 for experimental details). Clones were sorted according to ELISA
titer, low to high (left to right).
[0056] FIG. 11 is a graph of the ratio of FVII titer over cell
count of the top pBC0014 clones (see Example 25 for experimental
details). Clones were sorted according to the ratio, low to high
(left to right).
[0057] FIG. 12 is a Western blot of top pBC0014 clones according to
ELISA, clotting, ELISA/cell count and clotting/cell count ratios
(see Example 25 for experimental details). Clone 6G1 expressed a
truncated product and was not evaluated further.
[0058] FIG. 13 is a Western blot of the top pBC0016 clones
according to ELISA, clotting, ELISA/cell count and clotting/cell
count ratios (see Example 25 for experimental details).
[0059] FIG. 14 is a Western blot of the top pBC0018 clones
according to ELISA, clotting, ELISA/cell count and clotting/cell
count ratios (see Example 25 for experimental details). Clone 3B2
expressed a truncated product and was not evaluated further.
[0060] FIG. 15 shows purification of FVII-AE864 by anti-GLA
affinity chromatography (see Example 26 for experimental details).
SDS-PAGE analysis demonstrating the purification of FVII-AE864 from
concentrated supernatant and the >90% purity of the EDTA eluted
fractions.
[0061] FIG. 16 shows activation of FVII-XTEN fusions to FVIIa-XTEN
fusions by FXa treatment (see Example 26 for experimental details).
SDS-PAGE analysis demonstrates the appearance of a light chain band
under reducing conditions after FXa treatment, but not in the
untreated sample. Additionally, there is a downwards shift in the
upper band indicating the loss of the light chain.
[0062] FIG. 17 shows an SDS-PAGE demonstrating auto-activation of
FVII-XTEN fusions to FVIIa-XTEN fusions (see Example 26 for
experimental details). SDS-PAGE analysis demonstrating appearance
of a light chain band under reducing conditions after FXa treatment
and after incubation at 4.degree. C. at high concentration with
CaCl.sub.2. Additionally, there is a downwards shift in the upper
band indicating the loss of the light chain.
[0063] FIG. 18 shows SEC Analysis of FVII-AE864 and FVII-AE288 (see
Example 26 for experimental details). The SEC shows a monodispersed
population with minimal contamination and no aggregates at the void
volume of the column (-22 ml).
[0064] FIG. 19 shows the purification of FVII-AE864 by anion
exchange chromatography (see Example 26 for experimental details).
The chromatograms depict the elution profiles of the total protein
content and the FVII activity from a Macrocap Q column with the
bulk of the activity eluting later than the contaminant proteins,
creating a net 5-fold purification.
[0065] FIG. 20 shows purification of FVII-AE864 by hydrophobic
interaction chromatography (see Example 26 for experimental
details). The chromatograms depict the elution profiles of the
total protein content and the FVII activity from a toyopearl phenyl
column with the bulk of the activity eluting earlier than the
contaminant proteins, creating a net 2-fold purification
[0066] FIG. 21 shows two chromatography outputs demonstrating
removal of aggregated protein from monomeric FVII-AE864 with anion
exchange chromatography (see Example 26 for experimental details).
FIG. 21A is a chromatogram depicting the elution profile of
FVII-XTEN from a macrocap Q column with two peaks eluting after the
buffer related early peak. FIG. 21B shows SEC chromatograms of the
early and late macrocap Q peaks demonstrating the absence of
aggregates in the early peak.
[0067] FIG. 22 shows results of ELISA or aPTT assays, showing
FIX/cFXI/XTEN has enhanced activity compared to FIX-XTEN (see
Example 29 for experimental details). Transiently expressed FIX
constructs were assayed for antigen content by ELISA and for
activity by aPTT based assays. While the antigen content of
FIX-XTEN was similar to the FIX/cFXI/XTEN constructs the activity
was significantly increased. This increase is attributed to the
specific action of the FXI protease in the assays as the
FIX/cTEV/XTEN does not show a significantly different activity to
FIX-XTEN. Note the ELISA titer of the FIX sample is 197 ng/ml and
is off the scale of the graph.
[0068] FIG. 23 shows the pharmacokinetic profile after a single
dose administered subcutaneously to rats, with the derived
equivalent FVII concentration shown, as described in Example
30.
[0069] FIG. 24 shows the pharmacokinetic profile after a single
dose administered subcutaneously to rats, with the derived
equivalent FIX concentration shown, as described in Example 31.
[0070] FIG. 25 shows the pharmacokinetic profile (plasma
concentrations) in cynomolgus monkeys after single doses of
different compositions of GFP linked to unstructured polypeptides
of varying length, administered either subcutaneously or
intravenously, as described in Example 39. The compositions were
GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP.
Blood samples were analyzed at various times after injection and
the concentration of GFP in plasma was measured by ELISA using a
polyclonal antibody against GFP for capture and a biotinylated
preparation of the same polyclonal antibody for detection. Results
are presented as the plasma concentration versus time (h) after
dosing and show, in particular, a considerable increase in
half-life for the XTEN_AD836-GFP, the composition with the longest
sequence length of XTEN. The construct with the shortest sequence
length, the GFP-L288 had the shortest half-life.
[0071] FIG. 26 shows an SDS-PAGE gel of samples from a stability
study of the fusion protein of XTEN_AE864 fused to the N-terminus
of GFP (see Example 40). The GFP-XTEN was incubated in cynomolgus
plasma and rat kidney lysate for up to 7 days at 37.degree. C. In
addition, GFP-XTEN administered to cynomolgus monkeys was also
assessed. Samples were withdrawn at 0, 1 and 7 days and analyzed by
SDS PAGE followed by detection using Western analysis with
antibodies against GFP.
[0072] FIG. 27 shows three randomized libraries used for the third
and fourth codons in the N-terminal sequences of clones from
LCW546, LCW547 and LCW552 (see Example 14 for experimental
details). The libraries were designed with the third and fourth
residues modified such that all combinations of allowable XTEN
codons were present at these positions, as shown. In order to
include all the allowable XTEN codons for each library, nine pairs
of oligonucleotides encoding 12 amino acids with codon diversities
of third and fourth residues were designed, annealed and ligated
into the NdeI/BsaI restriction enzyme digested stuffer vector
pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed into E. coli
BL21Gold(DE3) competent cells to obtain colonies of the three
libraries LCW0569, LCW0570, and LCW0571.
[0073] FIG. 28 shows a histogram of a retest of the top 75 clones
after the optimization step, as described in Example 15, for GFP
fluorescence signal, relative to the benchmark CBD_AM875 construct.
The results indicated that several clones were now superior to the
benchmark clones.
[0074] FIG. 29 is a schematic of a combinatorial approach
undertaken for the union of codon optimization preferences for two
regions of the N-terminus 48 amino acids (see Example 16 for
experimental details). The approach created novel 48 mers at the
N-terminus of the XTEN protein for evaluation of the optimization
of expression that resulted in leader sequences that can be a
solution for the expression of XTEN proteins where the XTEN is
N-terminal to the CF.
[0075] FIG. 30 shows an SDS-PAGE gel confirming the expression of
preferred clones obtained from the XTEN N-terminal codon
optimization experiments, in comparison to benchmark XTEN clones
comprising CBD leader sequences at the N-terminus of the construct
sequences, as described in Example 17.
[0076] FIG. 31 is a schematic flowchart of representative steps in
the assembly, production and the evaluation of a XTEN.
[0077] FIG. 32 is a schematic flowchart of representative steps in
the assembly of a CFXTEN polynucleotide construct encoding a fusion
protein. Individual oligonucleotides 501 are annealed into sequence
motifs 502 such as a 12 amino acid motif ("12-mer"), which is
subsequently ligated with an oligo containing BbsI, and KpnI
restriction sites 503. Additional sequence motifs from a library
are annealed to the 12-mer until the desired length of the XTEN
gene 504 is achieved. The XTEN gene is cloned into a stuffer
vector. In this case, the vector encodes an optional Flag sequence
506 followed by a stopper sequence that is flanked by BsaI, BbsI,
and KpnI sites 507 and an FVII gene 508, resulting in the gene 500
encoding an XTEN-FVII fusion protein.
[0078] FIG. 33 is a schematic flowchart of representative steps in
the assembly of a gene encoding fusion protein comprising a CF and
XTEN, its expression and recovery as a fusion protein, and its
evaluation as a candidate CFXTEN product.
[0079] FIG. 34 is a schematic representation of the design of
CFXTEN expression vectors with different processing strategies.
FIG. 34A shows an expression vector encoding XTEN fused to the 3'
end of the sequence encoding FVII. Note that no additional leader
sequences are required in this vector. FIG. 7B depicts an
expression vector encoding XTEN fused to the 5' end of the sequence
encoding FVII with a CBD leader sequence and a TEV protease site.
FIG. 7C depicts an expression vector as in FIG. 7B where the CBD
and TEV processing sites have been replaced with an optimized
N-terminal leader sequence (NTS). FIG. 7D depicts an expression
vector encoding an NTS sequence, an XTEN, a sequence encoding VFII,
and than a second sequence encoding an XTEN.
[0080] FIG. 35 shows results of a size exclusion chromatography
analysis of glucagon-XTEN construct samples measured against
protein standards of known molecular weight, with the graph output
as absorbance versus retention volume, as described in Example 37.
The glucagon-XTEN constructs are 1) glucagon-Y288; 2)
glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results
indicate an increase in apparent molecular weight with increasing
length of XTEN moiety.
[0081] FIG. 36 shows sequence alignments between portions of native
FIX, native FVII, and FVII-FIX sequence hybrids with different
portions of the AP domain incorporated in the portion of the
molecule spanning the EGF2 and Pro domains. The legend provides
construct names. Gaps in an individual sequence (dashes) represents
stretches of non-homology to FIX but are otherwise continuous,
linked sequences. The underlined amino acids are FIX-derived
sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0082] Before the embodiments of the invention are described, it is
to be understood that such embodiments are provided by way of
example only, and that various alternatives to the embodiments of
the invention described herein may be employed in practicing the
invention. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention.
[0083] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. Numerous
variations, changes, and substitutions will now occur to those
skilled in the art without departing from the invention.
DEFINITIONS
[0084] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0085] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0086] The terms "polypeptide", "peptide", and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified, for example, by disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component.
[0087] As used herein the term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including but
not limited to both the D or L optical isomers, and amino acid
analogs and peptidomimetics. Standard single or three letter codes
are used to designate amino acids.
[0088] The term "natural L-amino acid" means the L optical isomer
forms of glycine (G), proline (P), alanine (A), valine (V), leucine
(L), isoleucine (I), methionine (M), cysteine (C), phenylalanine
(F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K),
arginine (R), glutamine (Q), asparagine (N), glutamic acid (E),
aspartic acid (D), serine (S), and threonine (T).
[0089] The term "non-naturally occurring," as applied to sequences
and as used herein, means polypeptide or polynucleotide sequences
that do not have a counterpart to, are not complementary to, or do
not have a high degree of homology with a wild-type or
naturally-occurring sequence found in a mammal. For example, a
non-naturally occurring polypeptide or fragment may share no more
than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid
sequence identity as compared to a natural sequence when suitably
aligned.
[0090] The terms "hydrophilic" and "hydrophobic" refer to the
degree of affinity that a substance has with water. A hydrophilic
substance has a strong affinity for water, tending to dissolve in,
mix with, or be wetted by water, while a hydrophobic substance
substantially lacks affinity for water, tending to repel and not
absorb water and tending not to dissolve in or mix with or be
wetted by water. Amino acids can be characterized based on their
hydrophobicity. A number of scales have been developed. An example
is a scale developed by Levitt, M, et al., J Mol Biol (1976)
104:59, which is listed in Hopp, T P, et al., Proc Natl Acad Sci
USA (1981) 78:3824. Examples of "hydrophilic amino acids" are
arginine, lysine, threonine, alanine, asparagine, and glutamine. Of
particular interest are the hydrophilic amino acids aspartate,
glutamate, and serine, and glycine. Examples of "hydrophobic amino
acids" are tryptophan, tyrosine, phenylalanine, methionine,
leucine, isoleucine, and valine.
[0091] A "fragment" is a truncated form of a native biologically
active protein that retains at least a portion of the therapeutic
and/or biological activity. A "variant" is a protein with sequence
homology to the native biologically active protein that retains at
least a portion of the therapeutic and/or biological activity of
the biologically active protein. For example, a variant protein may
share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
amino acid sequence identity compared with the reference
biologically active protein. As used herein, the term "biologically
active protein moiety" includes proteins modified deliberately, as
for example, by site directed mutagenesis, insertions, or
accidentally through mutations.
[0092] As used herein, "internal XTEN" refers to XTEN sequences
that have been inserted into the sequence of the coagulation
factor. Internal XTENs can be constructed by insertion of an XTEN
sequence into the sequence of a coagulation factor such as FIX or
FVII, either by insertion between two adjacent amino acids or
domains of the coagulation factor or wherein XTEN replaces a
partial, internal sequence of the coagulation factor.
[0093] As used herein, "terminal XTEN" refers to XTEN sequences
that have been fused to or in the N- or C-terminus of the
coagulation factor or to a proteolytic cleavage sequence at the N-
or C-terminus of the coagulation factor. Terminal XTENs can be
fused to the native termini of the coagulation factor.
Alternatively, terminal XTENs can replace a terminal sequence of
the coagulation factor.
[0094] The term "XTEN release site" refers to a sequence in CFXTEN
fusion proteins that can be recognized and cleaved by a mammalian
protease, effecting release of an XTEN or a portion of an XTEN from
the CFXTEN fusion protein. As used herein, "mammalian protease"
means a protease that normally exists in the body fluids, cells or
tissues of a mammal. XTEN release sites can be engineered to be
cleaved by various mammalian proteases (a.k.a. "XTEN release
proteases") such as FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa
(thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any
protease that is present during a clotting event.
[0095] "Activity" as applied to form(s) of a CFXTEN polypeptide
provided herein, refers to retention of a biological activity of
the native coagulation factor, wherein "biological activity" refers
to an in vitro or in vivo biological function or effect, including
but not limited to either receptor or ligand binding, enzymatic
activity, or an effect on coagulation generally known in the art
for the coagulation factor.
[0096] A "therapeutic effect" as applied to form(s) of a CFXTEN
polypeptide provided herein, refers to a physiologic effect,
including but not limited to the curing, mitigation, reversal,
amelioration or prevention of disease or conditions in humans or
other animals, or to otherwise enhance physical or mental wellbeing
of humans or animals. A "therapeutically effective amount" means an
amount of compound effective to prevent, alleviate, reverse or
ameliorate symptoms of disease or a condition (e.g., a bleeding
episode) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within
the capability of those skilled in the art, especially in light of
the detailed disclosure provided herein.
[0097] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for the subject vectors. Host
cells include progeny of a single host cell. The progeny may not
necessarily be completely identical (in morphology or in genomic of
total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a vector of this invention.
[0098] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. As
is apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody, or
fragments thereof, does not require "isolation" to distinguish it
from its naturally occurring counterpart. In addition, a
"concentrated", "separated" or "diluted" polynucleotide, peptide,
polypeptide, protein, antibody, or fragments thereof, is
distinguishable from its naturally occurring counterpart in that
the concentration or number of molecules per volume is generally
greater than that of its naturally occurring counterpart. In
general, a polypeptide made by recombinant means and expressed in a
host cell is considered to be "isolated."
[0099] An "isolated" polynucleotide or polypeptide-encoding nucleic
acid or other polypeptide-encoding nucleic acid is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the polypeptide-encoding
nucleic acid. An isolated polypeptide-encoding nucleic acid
molecule is other than in the form or setting in which it is found
in nature. Isolated polypeptide-encoding nucleic acid molecules
therefore are distinguished from the specific polypeptide-encoding
nucleic acid molecule as it exists in natural cells. However, an
isolated polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal or extra-chromosomal location
different from that of natural cells.
[0100] A "chimeric" protein contains at least one fusion
polypeptide comprising regions in a different position in the
sequence than that which occurs in nature. The regions may normally
exist in separate proteins and are brought together in the fusion
polypeptide; or they may normally exist in the same protein but are
placed in a new arrangement in the fusion polypeptide. A chimeric
protein may be created, for example, by chemical synthesis, or by
creating and translating a polynucleotide in which the peptide
regions are encoded in the desired relationship.
[0101] "Conjugated", "linked," "fused," and "fusion" are used
interchangeably herein. These terms refer to the joining together
of two or more chemical elements or components, by whatever means
including chemical conjugation or recombinant means. For example, a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence. Generally, "operably
linked" means that the DNA sequences being linked are contiguous,
and in reading phase or in-frame. An "in-frame fusion" refers to
the joining of two or more open reading frames (ORFs) to form a
continuous longer ORF, in a manner that maintains the correct
reading frame of the original ORFs. Thus, the resulting recombinant
fusion protein is a single protein containing two or more segments
that correspond to polypeptides encoded by the original ORFs (which
segments are not normally so joined in nature).
[0102] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminus direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide. A "partial sequence" is a linear sequence of part
of a polypeptide that is known to comprise additional residues in
one or both directions.
[0103] "Heterologous" means derived from a genotypically distinct
entity from the rest of the entity to which it is being compared.
For example, a glycine rich sequence removed from its native coding
sequence and operatively linked to a coding sequence other than the
native sequence is a heterologous glycine rich sequence. The term
"heterologous" as applied to a polynucleotide, a polypeptide, means
that the polynucleotide or polypeptide is derived from a
genotypically distinct entity from that of the rest of the entity
to which it is being compared.
[0104] The terms "polynucleotides", "nucleic acids", "nucleotides"
and "oligonucleotides" are used interchangeably. They refer to a
polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof.
Polynucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: coding or non-coding
regions of a gene or gene fragment, loci (locus) defined from
linkage analysis, exons, introns, messenger RNA (mRNA), transfer
RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and
primers. A polynucleotide may comprise modified nucleotides, such
as methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component.
[0105] The term "complement of a polynucleotide" denotes a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence, such that
it could hybridize with a reference sequence with complete
fidelity.
[0106] "Recombinant" as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of in vitro
cloning, restriction and/or ligation steps, and other procedures
that result in a construct that can potentially be expressed in a
host cell.
[0107] The terms "gene" and "gene fragment" are used
interchangeably herein. They refer to a polynucleotide containing
at least one open reading frame that is capable of encoding a
particular protein after being transcribed and translated. A gene
or gene fragment may be genomic or cDNA, as long as the
polynucleotide contains at least one open reading frame, which may
cover the entire coding region or a segment thereof. A "fusion
gene" is a gene composed of at least two heterologous
polynucleotides that are linked together.
[0108] "Homology" or "homologous" refers to sequence similarity or
interchangeability between two or more polynucleotide sequences or
two or more polypeptide sequences. When using a program such as
BestFit to determine sequence identity, similarity or homology
between two different amino acid sequences, the default settings
may be used, or an appropriate scoring matrix, such as blosum45 or
blosum80, may be selected to optimize identity, similarity or
homology scores. Preferably, polynucleotides that are homologous
are those which hybridize under stringent conditions as defined
herein and have at least 70%, preferably at least 80%, more
preferably at least 90%, more preferably 95%, more preferably 97%,
more preferably 98%, and even more preferably 99% sequence identity
compared to those sequences.
[0109] "Ligation" refers to the process of forming phosphodiester
bonds between two nucleic acid fragments or genes, linking them
together. To ligate the DNA fragments or genes together, the ends
of the DNA must be compatible with each other. In some cases, the
ends will be directly compatible after endonuclease digestion.
However, it may be necessary to first convert the staggered ends
commonly produced after endonuclease digestion to blunt ends to
make them compatible for ligation.
[0110] The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a
polynucleotide will hybridize to its target sequence, to a
detectably greater degree than other sequences (e.g., at least
2-fold over background). Generally, stringency of hybridization is
expressed, in part, with reference to the temperature and salt
concentration under which the wash step is carried out. Typically,
stringent conditions will be those in which the salt concentration
is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short
polynucleotides (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long polynucleotides (e.g., greater than 50
nucleotides)--for example, "stringent conditions" can include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and three washes for 15 min each in 0.1.times.SSC/1% SDS at
60.degree. C. to 65.degree. C. Alternatively, temperatures of about
65.degree. C., 60.degree. C., 55.degree. C., or 42.degree. C. may
be used. SSC concentration may be varied from about 0.1 to
2.times.SSC, with SDS being present at about 0.1%. Such wash
temperatures are typically selected to be about 5.degree. C. to
20.degree. C. lower than the thermal melting point for the specific
sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An
equation for calculating Tm and conditions for nucleic acid
hybridization are well known and can be found in Sambrook, J. et
al. (1989) Molecular Cloning: A Laboratory Manual, 2.sup.nded.,
vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.; specifically
see volume 2 and chapter 9. Typically, blocking reagents are used
to block non-specific hybridization. Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at
about 100-200 .mu.g/ml. Organic solvent, such as formamide at a
concentration of about 35-50% v/v, may also be used under
particular circumstances, such as for RNA:DNA hybridizations.
Useful variations on these wash conditions will be readily apparent
to those of ordinary skill in the art.
[0111] The terms "percent identity" and "% identity," as applied to
polynucleotide sequences, refer to the percentage of residue
matches between at least two polynucleotide sequences aligned using
a standardized algorithm. Such an algorithm may insert, in a
standardized and reproducible way, gaps in the sequences being
compared in order to optimize alignment between two sequences, and
therefore achieve a more meaningful comparison of the two
sequences. Percent identity may be measured over the length of an
entire defined polynucleotide sequence, or may be measured over a
shorter length, for example, over the length of a fragment taken
from a larger, defined polynucleotide sequence, for instance, a
fragment of at least 45, at least 60, at least 90, at least 120, at
least 150, at least 210 or at least 450 contiguous residues. Such
lengths are exemplary only, and it is understood that any fragment
length supported by the sequences shown herein, in the tables,
figures or Sequence Listing, may be used to describe a length over
which percentage identity may be measured.
[0112] "Percent (%) sequence identity," with respect to the
polypeptide sequences identified herein, is defined as the
percentage of amino acid residues in a query sequence that are
identical with the amino acid residues of a second, reference
polypeptide sequence or a portion thereof, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. Percent identity may be measured over the length of
an entire defined polypeptide sequence, or may be measured over a
shorter length, for example, over the length of a fragment taken
from a larger, defined polypeptide sequence, for instance, a
fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least 150 contiguous residues. Such
lengths are exemplary only, and it is understood that any fragment
length supported by the sequences shown herein, in the tables,
figures or Sequence Listing, may be used to describe a length over
which percentage identity may be measured.
[0113] The term "non-repetitiveness" as used herein in the context
of a polypeptide refers to a lack or limited degree of internal
homology in a peptide or polypeptide sequence. The term
"substantially non-repetitive" can mean, for example, that there
are few or no instances of four contiguous amino acids in the
sequence that are identical amino acid types or that the
polypeptide has a subsequence score (defined infra) of 10 or less
or that there isn't a pattern in the order, from N- to C-terminus,
of the sequence motifs that constitute the polypeptide sequence.
The term "repetitiveness" as used herein in the context of a
polypeptide refers to the degree of internal homology in a peptide
or polypeptide sequence. In contrast, a "repetitive" sequence may
contain multiple identical copies of short amino acid sequences.
For instance, a polypeptide sequence of interest may be divided
into n-mer sequences and the number of identical sequences can be
counted. Highly repetitive sequences contain a large fraction of
identical sequences while non-repetitive sequences contain few
identical sequences. In the context of a polypeptide, a sequence
can contain multiple copies of shorter sequences of defined or
variable length, or motifs, in which the motifs themselves have
non-repetitive sequences, rendering the full-length polypeptide
substantially non-repetitive. The length of polypeptide within
which the non-repetitiveness is measured can vary from 3 amino
acids to about 200 amino acids, about from 6 to about 50 amino
acids, or from about 9 to about 14 amino acids. "Repetitiveness"
used in the context of polynucleotide sequences refers to the
degree of internal homology in the sequence such as, for example,
the frequency of identical nucleotide sequences of a given length.
Repetitiveness can, for example, be measured by analyzing the
frequency of identical sequences.
[0114] A "vector" is a nucleic acid molecule, preferably
self-replicating in an appropriate host, which transfers an
inserted nucleic acid molecule into and/or between host cells. The
term includes vectors that function primarily for insertion of DNA
or RNA into a cell, replication of vectors that function primarily
for the replication of DNA or RNA, and expression vectors that
function for transcription and/or translation of the DNA or RNA.
Also included are vectors that provide more than one of the above
functions. An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide(s). An "expression system" usually
connotes a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0115] "Serum degradation resistance," as applied to a polypeptide,
refers to the ability of the polypeptides to withstand degradation
in blood or components thereof, which typically involves proteases
in the serum or plasma. The serum degradation resistance can be
measured by combining the protein with human (or mouse, rat,
monkey, as appropriate) serum or plasma, typically for a range of
days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about
37.degree. C. The samples for these time points can be run on a
Western blot assay and the protein is detected with an antibody.
The antibody can be to a tag in the protein. If the protein shows a
single band on the western, where the protein's size is identical
to that of the injected protein, then no degradation has occurred.
In this exemplary method, the time point where 50% of the protein
is degraded, as judged by Western blots or equivalent techniques,
is the serum degradation half-life or "serum half-life" of the
protein.
[0116] The term "t.sub.1/2" as used herein means the terminal
half-life calculated as ln(2)/K.sub.el. K.sub.elis the terminal
elimination rate constant calculated by linear regression of the
terminal linear portion of the log concentration vs. time curve.
Half-life typically refers to the time required for half the
quantity of an administered substance deposited in a living
organism to be metabolized or eliminated by normal biological
processes. The terms "t.sub.1/2", "terminal half-life",
"elimination half-life" and "circulating half-life" are used
interchangeably herein.
[0117] "Active clearance" means the mechanisms by which CF is
removed from the circulation other than by filtration or
coagulation, and which includes removal from the circulation
mediated by cells, receptors, metabolism, or degradation of the
CF.
[0118] "Apparent molecular weight factor" and "apparent molecular
weight" are related terms referring to a measure of the relative
increase or decrease in apparent molecular weight exhibited by a
particular amino acid sequence. The apparent molecular weight is
determined using size exclusion chromatography (SEC) and similar
methods compared to globular protein standards and is measured in
"apparent kD" units. The apparent molecular weight factor is the
ratio between the apparent molecular weight and the actual
molecular weight; the latter predicted by adding, based on amino
acid composition, the calculated molecular weight of each type of
amino acid in the composition or by estimation from comparison to
molecular weight standards in an SDS electrophoresis gel.
[0119] The terms "hydrodynamic radius" or "Stokes radius" is the
effective radius (R.sub.hin nm) of a molecule in a solution
measured by assuming that it is a body moving through the solution
and resisted by the solution's viscosity. In the embodiments of the
invention, the hydrodynamic radius measurements of the XTEN fusion
proteins correlate with the `apparent molecular weight factor`,
which is a more intuitive measure. The "hydrodynamic radius" of a
protein affects its rate of diffusion in aqueous solution as well
as its ability to migrate in gels of macromolecules. The
hydrodynamic radius of a protein is determined by its molecular
weight as well as by its structure, including shape and
compactness. Methods for determining the hydrodynamic radius are
well known in the art, such as by the use of size exclusion
chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and
7,294,513. Most proteins have globular structure, which is the most
compact three-dimensional structure a protein can have with the
smallest hydrodynamic radius. Some proteins adopt a random and
open, unstructured, or `linear` conformation and as a result have a
much larger hydrodynamic radius compared to typical globular
proteins of similar molecular weight.
[0120] "Physiological conditions" refers to a set of conditions in
a living host as well as in vitro conditions, including
temperature, salt concentration, pH, that mimic those conditions of
a living subject. A host of physiologically relevant conditions for
use in in vitro assays have been established. Generally, a
physiological buffer contains a physiological concentration of salt
and is adjusted to a neutral pH ranging from about 6.5 to about
7.8, and preferably from about 7.0 to about 7.5. A variety of
physiological buffers are listed in Sambrook et al. (1989).
Physiologically relevant temperature ranges from about 25.degree.
C. to about 38.degree. C., and preferably from about 35.degree. C.
to about 37.degree. C.
[0121] A "reactive group" is a chemical structure that can be
coupled to a second reactive group. Examples for reactive groups
are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl
groups, aldehyde groups, azide groups. Some reactive groups can be
activated to facilitate coupling with a second reactive group.
Non-limiting examples for activation are the reaction of a carboxyl
group with carbodiimide, the conversion of a carboxyl group into an
activated ester, or the conversion of a carboxyl group into an
azide function.
[0122] "Controlled release agent", "slow release agent", "depot
formulation" and "sustained release agent" are used interchangeably
to refer to an agent capable of extending the duration of release
of a polypeptide of the invention relative to the duration of
release when the polypeptide is administered in the absence of
agent. Different embodiments of the present invention may have
different release rates, resulting in different therapeutic
amounts.
[0123] The terms "antigen", "target antigen" and "immunogen" are
used interchangeably herein to refer to the structure or binding
determinant that an antibody fragment or an antibody fragment-based
therapeutic binds to or has specificity against.
[0124] The term "payload" as used herein refers to a protein or
peptide sequence that has biological or therapeutic activity; the
counterpart to the pharmacophore of small molecules. Examples of
payloads include, but are not limited to, cytokines, enzymes,
hormones and blood and growth factors. Payloads can further
comprise genetically fused or chemically conjugated moieties such
as chemotherapeutic agents, antiviral compounds, toxins, or
contrast agents. These conjugated moieties can be joined to the
rest of the polypeptide via a linker that may be cleavable or
non-cleavable.
[0125] The term "antagonist", as used herein, includes any molecule
that partially or fully blocks, inhibits, or neutralizes a
biological activity of a native polypeptide disclosed herein.
Methods for identifying antagonists of a polypeptide may comprise
contacting a native polypeptide with a candidate antagonist
molecule and measuring a detectable change in one or more
biological activities normally associated with the native
polypeptide. In the context of the present invention, antagonists
may include proteins, nucleic acids, carbohydrates, antibodies or
any other molecules that decrease the effect of a biologically
active protein.
[0126] The term "agonist" is used in the broadest sense and
includes any molecule that mimics a biological activity of a native
polypeptide disclosed herein. Suitable agonist molecules
specifically include agonist antibodies or antibody fragments,
fragments or amino acid sequence variants of native polypeptides,
peptides, small organic molecules, etc. Methods for identifying
agonists of a native polypeptide may comprise contacting a native
polypeptide with a candidate agonist molecule and measuring a
detectable change in one or more biological activities normally
associated with the native polypeptide.
[0127] "Activity" for the purposes herein refers to an action or
effect of a component of a fusion protein consistent with that of
the corresponding native biologically active protein, wherein
"biological activity" refers to an in vitro or in vivo biological
function or effect, including but not limited to receptor binding,
antagonist activity, agonist activity, or a cellular or physiologic
response.
[0128] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" is used interchangeably herein. These terms refer
to an approach for obtaining beneficial or desired results
including but not limited to a therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the subject, notwithstanding that the subject may still
be afflicted with the underlying disorder. For prophylactic
benefit, the compositions may be administered to a subject at risk
of developing a particular disease, or to a subject reporting one
or more of the physiological symptoms of a disease, even though a
diagnosis of this disease may not have been made.
[0129] A "therapeutic effect", as used herein, refers to a
physiologic effect, including but not limited to the cure,
mitigation, amelioration, or prevention of disease in humans or
other animals, or to otherwise enhance physical or mental wellbeing
of humans or animals, caused by a fusion polypeptide of the
invention other than the ability to induce the production of an
antibody against an antigenic epitope possessed by the biologically
active protein. Determination of a therapeutically effective amount
is well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein.
[0130] The terms "therapeutically effective amount" and
"therapeutically effective dose", as used herein, refer to an
amount of a biologically active protein, either alone or as a part
of a fusion protein composition, that is capable of having any
detectable, beneficial effect on any symptom, aspect, measured
parameter or characteristics of a disease state or condition when
administered in one or repeated doses to a subject. Such effect
need not be absolute to be beneficial.
[0131] The term "therapeutically effective dose regimen", as used
herein, refers to a schedule for consecutively administered
multiple doses (i.e., at least two or more) of a biologically
active protein, either alone or as a part of a fusion protein
composition, wherein the doses are given in therapeutically
effective amounts to result in sustained beneficial effect on any
symptom, aspect, measured parameter or characteristics of a disease
state or condition.
I). General Techniques
[0132] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, J. et al., "Molecular Cloning: A
Laboratory Manual," 3.sup.rd edition, Cold Spring Harbor Laboratory
Press, 2001; "Current protocols in molecular biology", F. M.
Ausubel, et al. eds., 1987; the series "Methods in Enzymology,"
Academic Press, San Diego, Calif.; "PCR 2: a practical approach",
M. J. MacPherson, B. D. Hames and G. R. Taylor eds., Oxford
University Press, 1995; "Antibodies, a laboratory manual" Harlow,
E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; "Goodman
& Gilman's The Pharmacological Basis of Therapeutics,"
11.sup.thEdition, McGraw-Hill, 2005; and Freshney, R. I., "Culture
of Animal Cells: A Manual of Basic Technique," 4.sup.th edition,
John Wiley & Sons, Somerset, N.J., 2000, the contents of which
are incorporated in their entirety herein by reference.
II). Coagulation Factors
[0133] The present invention relates in part to fusion protein
compositions comprising coagulation factors (CF). As used herein,
"coagulation factor" or "CF" refers to factor IX (FIX), factor VII
(FVII), sequence combinations of FVII and FIX, or mimetics,
sequence variants and truncated versions thereof.
(a) Factor IX
[0134] "Factor IX" or "FIX" means a coagulation factor protein and
species and sequence variants thereof, and includes, but is not
limited to, the 461 single-chain amino acid sequence of human FIX
precursor polypeptide ("prepro") and the 415 single-chain amino
acid sequence of mature human FIX. FIX includes any form of factor
IX molecule with the typical characteristics of blood coagulation
factor IX. As used herein "factor IX" and "FIX" are intended to
encompass polypeptides that comprise the domains Gla (region
containing .gamma.-carboxyglutamic acid residues), EGF1 and EGF2
(region containing sequences homologous to human epidermal growth
factor), activation peptide (formed by residues R136-R180 of the
mature FIX), and the C-terminal protease domain ("Pro"), or
synonyms of these domains known in the art, or can be a truncated
fragment or a sequence variant that retains at least a portion of
the biological activity of the native protein. FIX or sequence
variants have been cloned, as described in U.S. Pat. Nos.
4,770,999, 7,700,734, and cDNA coding for human factor IX has been
isolated, characterized, and cloned into expression vectors (see,
for example, Choo et al., Nature 299:178-180 (1982); Fair et al.,
Blood 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad.
Sci., U.S.A. 79:6461-6464 (1982)).
[0135] Human factor IX (FIX) is encoded by a single-copy gene
residing on the X-chromosome at q27.1. The human FIX mRNA is
composed of 205 bases for the 5' untranslated region, 1383 bases
for the prepro factor IX, a stop codon and 1392 bases for the 3'
untranslated region. The FIX polypeptide is 55 kDa, synthesized as
a prepropolypetide chain composed of three regions: a signal
peptide of 28 amino acids, a propeptide of 18 amino acids, which is
required for gamma-carboxylation of glutamic acid residues, and a
mature factor IX of 415 amino acids. The propeptide is an 18-amino
acid residue sequence N-terminal to the gamma-carboxyglutamate
domain. The propeptide binds vitamin K-dependent gamma carboxylase
and then is cleaved from the precursor polypeptide of FIX by an
endogenous protease, most likely PACE (paired basic amino acid
cleaving enzyme), also known as furin or PCSK3. Without the gamma
carboxylation, the Gla domain is unable to bind calcium to assume
the correct conformation necessary to anchor the protein to
negatively charged phospholipid surfaces, thereby rendering factor
IX nonfunctional. Even if it is carboxylated, the Gla domain also
depends on cleavage of the propeptide for proper function, since
retained propeptide interferes with conformational changes of the
Gla domain necessary for optimal binding to calcium and
phospholipid. In humans, the resulting mature factor IX is secreted
by liver cells into the blood stream as an inactive zymogen, a
single chain protein of 415 amino acid residues that contains
approximately 17% carbohydrate by weight (Schmidt, A. E., et al.
(2003) Trends Cardiovasc Med, 13: 39). The mature factor IX is
composed of several domains that in an N- to C-terminus
configuration are: a Gla domain, an EGF1 domain, an EGF2 domain, an
activation peptide (AP) domain, and a protease (or catalytic)
domain. FIX contains two activation peptides formed by R145-A146
and R180-V181, respectively. Following activation, the single-chain
FIX becomes a 2-chain molecule, in which the two chains are linked
by a disulfide bond attaching the enzyme to the Gla domain. CFs can
be engineered by replacing their activation peptides resulting in
altered activation specificity. In mammals, mature FIX must be
activated by activated factor XI to yield factor IXa. The protease
domain provides, upon activation of FIX to FIXa, the catalytic
activity of FIX. Activated factor VIII (FVIIIa) is the specific
cofactor for the full expression of FIXa activity.
[0136] Proteins involved in clotting include factor I, factor II,
factor III, factor IV, factor V, factor VI, factor VII, factor
VIII, factor IX, factor X, factor XI, factor XII, factor XIII,
Protein C, and tissue factor ("clotting proteins"). The majority of
the clotting proteins is present in zymogen form that when
activated exhibits a pro-coagulant protease activity to activate
other clotting proteins, contributing to the intrinsic or extrinsic
coagulation pathway and clot formation. In the intrinsic pathway of
the coagulation cascade, FIX associates with a complex of activated
factor VIII, factor X, calcium, and phospholipid. In the complex,
FIX is activated by factor XIa. The activation of factor IX is
achieved by a two-step removal of the activation peptide (Ala
146-Arg 180) from the molecule (Bajaj et al., Human factor IX and
factor IXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is
made at the Arg 145-Ala 146 site by either factor XIa or factor
VIIa/tissue factor. The second and rate limiting cleavage is made
at Arg 180-Val 181. The activation removes 35 residues. Activated
human factor IX exists as a heterodimer of the C-terminal heavy
chain (28 kDa) and an N-terminal light chain (18 kDa), which are
held together by one disulfide bridge attaching the enzyme to the
Gla domain. Factor IXa in turn activates factor X in concert with
activated factor VIII. Alternatively, factors IX and X can both be
activated by factor VIIa complexed with lipidated Tissue Factor,
generated via the extrinsic pathway. Factor Xa then participates in
the final common pathway whereby prothrombin is converted to
thrombin, and thrombin in turn converts fibrinogen to fibrin to
form the clot.
[0137] Defects in the coagulation process can lead to bleeding
disorders in which the time taken for clot formation is prolonged.
Such defects can be congenital or acquired. For example, hemophilia
A and B are inherited diseases characterized by deficiencies in
factor VIII (FVIII) and FIX, respectively. Replacement therapy with
these proteins, generally prepared as recombinant proteins, may be
used in the therapeutic intervention of hemophilia B (Christmas
Disease) and factor IX-related bleeding disorders. Factor IX can be
used in the treatment of both conditions. In some cases, however,
patients develop antibodies against the administered proteins that
reduce or negate the efficacy of the treatment.
[0138] The invention contemplates inclusion of FIX sequences in the
CFXTEN compositions that have homology to FIX sequences, sequence
fragments that are natural, such as from humans, non-human
primates, mammals (including domestic animals), and non-natural
sequence variants which retain at least a portion of the biologic
activity or biological function of FIX and/or that are useful for
preventing, treating, mediating, or ameliorating a coagulation
factor-related disease, deficiency, disorder or condition (e.g.,
bleeding episodes related to trauma, surgery, of deficiency of a
coagulation factor). Sequences with homology to human FIX can be
found by standard homology searching techniques, such as NCBI
BLAST.
[0139] In one embodiment, the FIX incorporated into the subject
compositions is a recombinant polypeptide with a sequence
corresponding to a protein found in nature. In another embodiment,
the FIX is a sequence variant, fragment, homolog, or a mimetics of
a natural sequence that retains at least a portion of the
biological activity of the corresponding native FIX. Table 1
provides a non-limiting list of amino acid sequences of FIX that
are encompassed by the CFXTEN fusion proteins of the invention. Any
of the FIX sequences or homologous derivatives to be incorporated
into the fusion protein compositions can be constructed by
shuffling individual mutations between the amino acid sequences of
Table 1 and evaluated for activity. Those that retain at least a
portion of the biological activity of the native FIX are useful for
the fusion protein compositions of this invention. FIX that can be
incorporated into a CFXTEN fusion protein includes a protein that
has at least about 80% sequence identity, or alternatively 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an
amino acid sequence selected from Table 1.
TABLE-US-00001 TABLE 1 FIX amino acid and nucleic acid sequences
Name Amino Acid Sequence FIX precursor
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN
polypeptide
LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV
PFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDA
KPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTE
QKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGY
VSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQG
DSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT FIX Homo
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN sapiens
GGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYR
LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 4
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN from
Patent LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
US WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV
20080214462
PFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDA
KPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTE
QKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYV
SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGD
SGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 6
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN from
Patent LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
US WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV
20080214462
PFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDA
KPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTE
QKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYV
SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIFNNMFCAGFHEGGRDSCQGD
SGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 8
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN from
Patent LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
US WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEG 20080214462
YRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQ
SFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKIT
VVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVLNSYVTPICIA
DKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDATCLRSTKFTIFNNMFC
AGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEGCAMKGKYGIYTKVSRYVNW IKEKTKLT
Sequence 2
MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN from
Patent LERECMEEKCSFEEPREVFENTEKITEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
U.S. 7,125,841
WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV
PFPCGRVSVSQTSKLTRAEAVFPDVDYVNPTEAETILDNITQGTQSFNDFTRVVGGEDA
KPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTE
QKRNVIRAIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSG
YVSGWARVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQ
GDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 1
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 2
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 3
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 4
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 5
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 6
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE KTKLT
Sequence 8
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLN from
Patent GGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYR
US LAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFN
20080167219
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA
GEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKE
YTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAG
FHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE
KTKLT
(b) Factor VII.
[0140] "Factor VII" or "FVII" means a coagulation factor protein
and species and sequence variants thereof, and includes, but is not
limited to, both the inactive and activated forms (unless indicated
to the contrary) of the 406 single-chain amino acid sequence of
human FVII, and the 444 amino acid sequence of the precursor
protein. As used herein, factor VII and FVII encompass polypeptides
that comprise the domains Gla (region containing
.gamma.-carboxyglutamic acid residues), EGF1 and EGF2 (region
containing sequences homologous to human epidermal growth factor),
an activation peptide domain that spans the sequence between the
EGF2 and Pro domains, and a catalytic or peptidase S1 domain ("Pro"
region containing the serine protease catalytic triad), or synonyms
of these domains known in the art, or can be a truncated fragment
or a sequence variant that retains at least a portion of the
biological activity of the native protein. Factor VII (FVII), a
vitamin K-dependent plasma protein produced by the liver, initially
circulates in the blood as a zymogen. The main role of factor VII
is to initiate the process of coagulation in conjunction with
tissue factor (TF). Upon vessel injury, tissue factor is exposed to
the blood and circulating factor VII. Once bound to TF, FVII is
activated to become the activated form of factor VII (FVIIa) by
different proteases, among which are thrombin (factor IIa), factor
Xa, IXa, XIIa, and the FVIIa-TF complex itself. The FVII zymogen is
activated by proteolytic cleavage at a single site,
Arg.sup.152-Ile.sup.153, resulting in a two-chain protease linked
by a single disulphide bond (FVIIa). FVIIa binds its cofactor,
tissue factor (TF), to form a complex which can activate factor X
(FX) to FXa, thereby initiating a coagulation cascade that results
in fibrin formation and hemostasis. The complete nucleotide and
amino acid sequences for human factor VII are known, and human FVII
or sequence variants have been cloned, as described in U.S. Pat.
Nos. 4,784,950, 5,833,982, 6,911,323, and 7,026,524.
[0141] Current therapeutic uses of factor VII exist but can be
problematic in the treatment of individuals exhibiting a deficiency
in factor VII, factor VIII, or factor IX, and individuals with Von
Willebrand's disease with FVIIa formulations. More specifically,
individuals receiving factors VIII and IX in replacement therapy
frequently develop antibodies to these proteins. Continuing
treatment is exceedingly difficult because of the presence of these
antibodies. Patients experiencing this problem are normally treated
with an activated prothrombin complex known to consist of a mixture
of active and inactive clotting enzymes, including factor VIIa.
FVII also is utilized in connection with treatment of uncontrolled
bleedings, such as trauma, and it is believed that factor VIIa is
capable of activating factor X to factor Xa without binding to
tissue factor, and this activation reaction is believed to occur
primarily on activated blood platelets (Hedner et al. Blood
Coagulation & Fibrinolysis, 2000; 11; 107-111).
[0142] Sequence variants of factor VII, whether exhibiting
substantially the same or better bioactivity than wild-type factor
VII, or, alternatively, exhibiting substantially modified or
reduced bioactivity relative to wild-type factor VII, include,
polypeptides having an amino acid sequence that differs from the
sequence of wild-type factor VII by insertion, deletion, or
substitution of one or more amino acids. Such FVII variants are
known in the art, including those described in U.S. Pat. Nos.
6,960,657, 7,176,288, 7,414,022, 7,700,733, 20060205036A1,
20080318276A1, and 20090011992A1, which are incorporated herein by
reference.
[0143] Recombinant FVIIa has been approved for the treatment of
hemophilia A or B patients that have inhibitors to FVIII or FIX,
and also is used to stop bleeding episodes or prevent bleeding
associated with trauma and/or surgery. Recombinant FVIIa also has
been approved for the treatment of patients with congenital FVII
deficiency, and is increasingly being utilized in off-label uses,
such as the treatment of bleeding associated with other congenital
or acquired bleeding disorders, trauma, and surgery in hemophilic
and non-hemophilic patients.
[0144] The invention contemplates inclusion in the CFXTEN
compositions sequences with homology to FVII sequences, sequence
fragments, mimetics and non-natural sequence variants which retain
at least a portion of the biologic activity or biological function
of FVIIa that are useful for preventing, treating, mediating, or
ameliorating a CF-related disease, deficiency, disorder or
condition. In addition, because of the comparatively long-half life
of CFXTEN comprising FVII, compositions comprising the inactive
form of FVII that can be activated by mammalian endogenous
proteases (described more fully below) or undergo autoactivation
represents a means to treat subjects with certain forms of chronic
coagulopathies with what is essentially a "prodrug" form of FVII.
Table 2 provides a list of sequences of FVII that are encompassed
by the CFXTEN fusion proteins of the invention. FVII sequences or
homologous derivatives constructed by shuffling individual
mutations between species or families that retain at least a
portion of the biological activity of the native CF are useful for
the fusion proteins of this invention. FVII that can be
incorporated into a CFXTEN fusion protein include a protein that
exhibits at least about 80% sequence identity, or alternatively
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to
a sequence selected from Table 2.
TABLE-US-00002 TABLE 2 Factor VII amino acid sequences Name Amino
Acid Sequence FVII
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE
precursor
QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN
polypeptide
CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP
ILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI
KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH
VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR
KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVG
HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP Human FVII
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGS
(mature)
CKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYS
LLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLC
GGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGT
TNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALEL
MVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG
TWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKSLTRNGPLKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKSMTRVVGGKVCPKGECPWQVLLLVNGAQLC
GGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGT
TNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALEL
MVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG
TWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKCGQRLRKSKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKIKPRIVGGKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKKLTRAETVKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCPKGECPWQVLLLVNGAQLC
GGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGT
TNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALEL
MVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG
TWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKIQIRSVAKKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII variant
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGS
CKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYS
LLADGVSCTPTVEYPCGKIPILEKRNASKIEPRSPSQKVCPKGECPWQVLLLVNGAQLCG
GTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELM
VLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP
III). Coagulation Factor Fusion Protein Compositions
[0145] The present invention provides fusion protein compositions
comprising coagulation factors (CF). One way to increase the
circulation half-life of a therapeutic protein is to reduce the
renal clearance of the protein. This may be achieved by conjugating
the protein to a polymer that is capable of conferring an increased
molecular size (or hydrodynamic radius) to the protein, and hence,
reduces renal clearance. Thus, one object of the present invention
is to provide improved FIX or FVII (or FVIIa) molecules with a
longer circulation, or terminal half-life (thereby decreasing the
number of necessary administrations) and that retain at least a
portion of the activity of the native coagulation factors, thereby
to treat coagulation deficiencies and uncontrolled bleedings more
efficiently. In one aspect, the invention provides isolated
monomeric fusion proteins of CF comprising the full-length sequence
or sequence variants of a CF, such as FIX or FVII, covalently
linked to extended recombinant polypeptides ("XTEN" or "XTENs"). As
described more fully below, the fusion proteins optionally include
spacer sequences that further comprise cleavage sequences to
release the CF from the fusion protein when acted on by a
protease.
[0146] In one aspect, the invention provides an isolated fusion
protein comprising at least a first biologically active coagulation
factor protein covalently linked to one or more extended
recombinant polypeptides ("XTEN"), resulting in a fusion protein
composition (hereinafter "CFXTEN"). The term "CFXTEN", as used
herein, is meant to encompass fusion polypeptides that comprise one
or more payload regions each comprising a biologically active CF
that mediates one or more biological or therapeutic activities
associated with a coagulation factor and at least one other region
comprising at least a first XTEN polypeptide that serves as a
carrier. In one embodiment, the coagulation factor is FIX or a
sequence variant of FIX, as disclosed above (including sequences
with homology to the sequences of Table 1). In another embodiment,
the coagulation factor is FVII, which can include the activated
form of FVII, or a sequence variant of FVII, as disclosed above
(including sequences with homology with the sequences of Table 2).
In the case of CFXTEN compositions of the invention comprising
FVII, activation of the FVII component may be carried out by
exposure to activated factor X, by auto-activation, or according to
procedures known in the art, such as those disclosed by Osterud, et
al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.
4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983);
or Kisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983).
Alternatively, factor VII can be activated by passing it through an
ion-exchange chromatography column (see, e.g., Bjoern et al.
Research Disclosure (1986) 269:564-565), such as Mono Q (Pharmacia
fine Chemicals) or similar chromatography resins.
[0147] The CF of the subject compositions, particularly those
disclosed in Tables 1 and 2, together with their corresponding
nucleic acid and amino acid sequences, are well known in the art
and descriptions and sequences are available in public databases
such as Chemical Abstracts Services Databases (e.g., the CAS
Registry), GenBank, The Universal Protein Resource (UniProt) and
subscription provided databases such as GenSeq (e.g., Derwent).
Polynucleotide sequences may be a wild type polynucleotide sequence
encoding a given CF (e.g., either full length or mature), or in
some instances the sequence may be a variant of the wild type
polynucleotide sequence (e.g., a polynucleotide which encodes the
wild type biologically active protein, wherein the DNA sequence of
the polynucleotide has been optimized, for example, for expression
in a particular species; or a polynucleotide encoding a variant of
the wild type protein, such as a site directed mutant or an allelic
variant. It is well within the ability of the skilled artisan to
use a wild-type or consensus cDNA sequence or a codon-optimized
variant of a CF to create CFXTEN constructs contemplated by the
invention using methods known in the art and/or in conjunction with
the guidance and methods provided herein, and described more fully
in the Examples.
[0148] The CF for inclusion in the CFXTEN of the invention include
coagulation factors or sequence variants that are useful, when
administered to a subject, for mediating or preventing or
ameliorating a disease, disorder or condition associated with
bleeding disorders, coagulation factor deficiencies or defects in a
coagulation factor. Of particular interest are CFXTEN fusion
protein compositions for which an increase in a pharmacokinetic
parameter, increased solubility, increased stability, or some other
enhanced pharmaceutical property compared to native CF is sought,
or for which increasing the terminal half-life would improve
efficacy, safety, or result in reduced dosing frequency and/or
improve patient compliance. Thus, the CFXTEN fusion protein
compositions are prepared with various objectives in mind,
including improving the therapeutic efficacy of the bioactive CF
by, for example, increasing the in vivo exposure or the length that
the CFXTEN remains within the therapeutic window when administered
to a subject, compared to a CF not linked to XTEN.
[0149] In one embodiment, the CF incorporated into the subject
compositions can be a recombinant polypeptide with a sequence
corresponding to a protein found in nature. In another embodiment,
the CF is a sequence variant, fragment, homolog, or mimetic of a
natural sequence that retain at least a portion of the biological
activity of the native CF. In non-limiting examples, a CF is a
sequence that exhibits at least about 80% sequence identity, or
alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or
100% sequence identity compared to a protein sequence selected from
Table 1 or from Table 2. In one embodiment, a CFXTEN fusion protein
comprises a single CF molecule linked to a single XTEN (e.g., an
XTEN as described more fully below). In another embodiment, the
CFXTEN comprises a first CF and a second molecule of the same CF,
resulting in a fusion protein comprising the two CF linked to one
or more XTEN in an N- to C-terminus configuration selected from
Table 6. In another embodiment, the CFXTEN fusion protein comprises
a single CF molecule linked to a first and a second XTEN, in which
the CF is a sequence that exhibits at least about 80% sequence
identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about
99%, or 100% sequence identity compared to a protein sequence
selected from Table 1 or from Table 2, and the first and/or the
second XTEN are sequences that exhibits at least about 80% sequence
identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about
99%, or 100% sequence identity compared to a sequence selected from
Table 4.
[0150] The subject CFXTEN of the present invention exhibits an
enhancement of one or more pharmacokinetic parameters compared to
the native CF. The CFXTEN with enhanced pharmacokinetic parameters
permits less frequent dosing or an enhanced pharmacologic effect,
including but not limited to maintaining the biologically active
CFXTEN within the therapeutic window between the minimum effective
dose or blood concentration (C.sub.min) and the maximum tolerated
dose or blood concentration (C.sub.max) for a longer period of time
compared to the CF not linked to XTEN. In such cases, the linking
of the CF to a fusion protein comprising a select XTEN sequence(s)
can result in an improvement in these properties, making them more
useful as therapeutic or preventive agents compared to CF not
linked to XTEN. In some embodiments, the subject CFXTEN of the
present invention has a cleavage sequence incorporated between the
CF and the XTEN and the biologic activity of the CF component is
enhanced by the release of the CF from the fusion protein by
cleavage of the cleavage sequence by an endogenous protease, as
described below.
IV). Xtended Recombinant Polypeptides
[0151] In one aspect, the invention provides XTEN polypeptide
compositions that are useful as a fusion protein partner to which
CF is linked, resulting in a CFXTEN fusion protein. XTEN are
generally extended length polypeptides with non-naturally
occurring, substantially non-repetitive sequences that are composed
mainly of small hydrophilic amino acids, with the sequence having a
low degree or no secondary or tertiary structure under physiologic
conditions.
[0152] XTENs have utility as a fusion protein partners in that they
serve as a "carrier," conferring certain desirable pharmacokinetic,
physicochemical and pharmaceutical properties when linked to a CF
protein to a create a fusion protein. Such desirable properties
include but are not limited to enhanced pharmacokinetic parameters
and solubility characteristics of the compositions, amongst other
properties described herein. Such fusion protein compositions have
utility to treat certain coagulation factor-related diseases,
disorders or conditions, as described herein. As used herein,
"XTEN" specifically excludes whole antibodies or antibody fragments
(e.g. single-chain antibodies and Fc fragments).
[0153] In some embodiments, the XTEN is a long polypeptide having
greater than about 100 to about 3000 amino acid residues when used
as a carrier or greater than 400 to about 3000 residues
cumulatively when more than one XTEN unit is used in a single
fusion protein. In other embodiments, when XTEN is used as a linker
between fusion protein components or where an increase in half-life
of the fusion protein is not needed but where an increase in
solubility or some other physico/chemical property for the CF
fusion partner component is desired, an XTEN sequence shorter than
100 amino acid residues, such as about 96, or about 84, or about
72, or about 60, or about 48, or about 36 amino acid residues are
incorporated into a fusion protein composition with the CF to
effect the property.
[0154] The selection criteria for the XTEN to be linked to the
biologically active proteins used to create the inventive fusion
proteins compositions generally relate to attributes of
physical/chemical properties and conformational structure of the
XTEN that is, in turn, used to confer enhanced pharmaceutical and
pharmacokinetic properties to the fusion proteins compositions. The
XTEN of the present invention exhibits one or more of the following
advantageous properties: conformational flexibility, enhanced
aqueous solubility, high degree of protease resistance, low
immunogenicity, low binding to mammalian receptors, and increased
hydrodynamic (or Stokes) radii; properties that make them
particularly useful as fusion protein partners. Non-limiting
examples of the properties of the fusion proteins comprising CF
that are enhanced by XTEN include increases in the overall
solubility and/or metabolic stability, reduced susceptibility to
proteolysis, reduced immunogenicity, reduced rate of absorption
when administered subcutaneously or intramuscularly, and enhanced
pharmacokinetic properties such as longer terminal half-life and
increased area under the curve (AUC), slower absorption after
subcutaneous or intramuscular injection (compared to CF not linked
to XTEN and administered by a similar route) such that the
C.sub.maxis lower, which, in turn, results in reductions in adverse
effects of the CF that, collectively, results in an increased
period of time that a fusion protein of a CFXTEN composition
administered to a subject retains therapeutic activity.
[0155] A variety of methods and assays are known in the art for
determining the physical/chemical properties of proteins such as
the compositions comprising the inventive XTEN. Such properties
include but are not limited to secondary or tertiary structure,
solubility, protein aggregation, melting properties, contamination
and water content. Such methods include analytical centrifugation,
EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase,
light scattering, capillary electrophoresis, circular dichroism,
differential scanning calorimetry, fluorescence, HPLC-ion exchange,
HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry,
and UV/Visible spectroscopy. Additional methods are disclosed in
Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.
[0156] In one embodiment, XTEN is designed to behave like denatured
peptide sequence under physiological conditions, despite the
extended length of the polymer. "Denatured" describes the state of
a peptide in solution that is characterized by a large
conformational freedom of the peptide backbone. Most peptides and
proteins adopt a denatured conformation in the presence of high
concentrations of denaturants or at elevated temperature. Peptides
in denatured conformation have, for example, characteristic
circular dichroism (CD) spectra and are characterized by a lack of
long-range interactions as determined by NMR. "Denatured
conformation" and "unstructured conformation" are used synonymously
herein. In some embodiments, the invention provides XTEN sequences
that, under physiologic conditions, resemble denatured sequences
that are largely devoid in secondary structure. In other cases, the
XTEN sequences are substantially devoid of secondary structure
under physiologic conditions. "Largely devoid," as used in this
context, means that less than 50% of the XTEN amino acid residues
of the XTEN sequence contribute to secondary structure as measured
or determined by the means described herein. "Substantially
devoid," as used in this context, means that at least about 60%, or
about 70%, or about 80%, or about 90%, or about 95%, or at least
about 99% of the XTEN amino acid residues of the XTEN sequence do
not contribute to secondary structure, as measured or determined by
the methods described herein.
[0157] A variety of methods have been established in the art to
discern the presence or absence of secondary and tertiary
structures in a given polypeptide. In particular, secondary
structure can be measured spectrophotometrically, e.g., by circular
dichroism spectroscopy in the "far-UV" spectral region (190-250
nm). Secondary structure elements, such as alpha-helix and
beta-sheet, each give rise to a characteristic shape and magnitude
of CD spectra. Secondary structure can also be predicted for a
polypeptide sequence via certain computer programs or algorithms,
such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al.
(1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson
("GOR") algorithm (Garnier J, Gibrat J F, Robson B. (1996), GOR
method for predicting protein secondary structure from amino acid
sequence. Methods Enzymol 266:540-553), as described in US Patent
Application Publication No. 20030228309A1. For a given sequence,
the algorithms can predict whether there exists some or no
secondary structure at all, expressed as the total and/or
percentage of residues of the sequence that form, for example,
alpha-helices or beta-sheets or the percentage of residues of the
sequence predicted to result in random coil formation (which lacks
secondary structure).
[0158] In some embodiments, the XTEN sequences used in the subject
fusion protein compositions can have an alpha-helix percentage
ranging from 0% to less than about 5% as determined by the
Chou-Fasman algorithm. In other cases, the XTEN sequences of the
fusion protein compositions have a beta-sheet percentage ranging
from 0% to less than about 5% as determined by the Chou-Fasman
algorithm. In some embodiments, the XTEN sequences of the fusion
protein compositions have an alpha-helix percentage ranging from 0%
to less than about 5% and a beta-sheet percentage ranging from 0%
to less than about 5% as determined by the Chou-Fasman algorithm.
In some embodiments, the XTEN sequences of the fusion protein
compositions have an alpha-helix percentage less than about 2% and
a beta-sheet percentage less than about 2%. In other cases, the
XTEN sequences of the fusion protein compositions have a high
degree of random coil percentage, as determined by the GOR
algorithm. In some embodiments, an XTEN sequence have at least
about 80%, more preferably at least about 90%, more preferably at
least about 91%, more preferably at least about 92%, more
preferably at least about 93%, more preferably at least about 94%,
more preferably at least about 95%, more preferably at least about
96%, more preferably at least about 97%, more preferably at least
about 98%, and most preferably at least about 99% random coil, as
determined by the GOR algorithm.
1. Non-Repetitive Sequences
[0159] In some embodiments, XTEN sequences of the compositions are
substantially non-repetitive. In general, repetitive amino acid
sequences have a tendency to aggregate or form higher order
structures, as exemplified by natural repetitive sequences such as
collagens and leucine zippers. These repetitive amino acids may
also tend to form contacts resulting in crystalline or
pseudocrystalline structures. In contrast, the low tendency of
non-repetitive sequences to aggregate enables the design of
long-sequence XTENs with a relatively low frequency of charged
amino acids that would otherwise be likely to aggregate if the
sequences were repetitive. Typically, the CFXTEN fusion proteins
comprise XTEN sequences of greater than about 100 to about 3000
amino acid residues wherein the sequences are substantially
non-repetitive. In one embodiment, the XTEN sequences have greater
than about 100 to about 3000 amino acid residues in which no three
contiguous amino acids in the sequence are identical amino acid
types unless the amino acid is serine, in which case no more than
three contiguous amino acids are serine residues. In the foregoing
embodiment, the XTEN sequence is "substantially
non-repetitive."
[0160] The degree of repetitiveness of a polypeptide or a gene can
be measured by computer programs or algorithms or by other means
known in the art. Repetitiveness in a polypeptide sequence can, for
example, be assessed by determining the number of times shorter
sequences of a given length occur within the polypeptide. For
example, a polypeptide of 200 amino acid residues has 192
overlapping 9-amino acid sequences (or 9-mer "frames") and 198
3-mer frames, but the number of unique 9-mer or 3-mer sequences
will depend on the amount of repetitiveness within the sequence. A
score is generated (hereinafter "subsequence score") that is
reflective of the degree of repetitiveness of the subsequences in
the overall polypeptide sequence. In the context of the present
invention, "subsequence score" means the sum of occurrences of each
unique 3-mer frame across a 200 consecutive amino acid sequence of
the polypeptide divided by the absolute number of unique 3-mer
subsequences within the 200 amino acid sequence. Examples of such
subsequence scores derived from the first 200 amino acids of
repetitive and non-repetitive polypeptides are presented in Example
44. In some embodiments, the present invention provides CFXTEN each
comprising one or more XTEN in which the XTEN has a subsequence
score less than 12, more preferably less than 10, more preferably
less than 9, more preferably less than 8, more preferably less than
7, more preferably less than 6, and most preferably less than 5. In
the embodiments hereinabove described in this paragraph, an XTEN
with a subsequence score less than about 10 (i.e., 9, 8, 7, etc.)
is "substantially non-repetitive."
[0161] The non-repetitive characteristic of XTEN imparts a CF
fusion proteins a greater degree of solubility and less tendency to
aggregate compared to polypeptides having repetitive sequences.
These properties facilitate the formulation of XTEN-comprising
pharmaceutical preparations containing extremely high drug
concentrations, in some cases exceeding 100 mg/ml.
[0162] Furthermore, the XTEN polypeptide sequences of the
embodiments are designed to have a low degree of internal
repetitiveness in order to reduce or substantially eliminate
immunogenicity when administered to a mammal. Polypeptide sequences
composed of short, repeated motifs largely limited to three amino
acids, such as glycine, serine and glutamate, may result in
relatively high antibody titers when administered to a mammal
despite the absence of predicted T-cell epitopes in these
sequences. This may be caused by the repetitive nature of
polypeptides, as it has been shown that immunogens with repeated
epitopes, including protein aggregates, cross-linked immunogens,
and repetitive carbohydrates are highly immunogenic and can, for
example, result in the cross-linking of B-cell receptors causing
B-cell activation. (Johansson, J., et al. (2007) Vaccine,
25:1676-82; Yankai, Z., et al. (2006) Biochem Biophys Res Commun,
345:1365-71; Hsu, C. T., et al. (2000) Cancer Res, 60:3701-5);
Bachmann M F, et al. Eur J. Immunol. (1995) 25(12):3445-3451).
2. Exemplary Sequence Motifs
[0163] The present invention encompasses XTEN used as fusion
partners that comprise multiple units of shorter sequences, or
motifs, in which the amino acid sequences of the motifs are
non-repetitive. The non-repetitive criterion can be met despite the
use of a "building block" approach using a library of sequence
motifs that are multimerized to create the XTEN sequences. Thus,
while an XTEN sequence may consist of multiple units of as few as
four different types of sequence motifs, because the motifs
themselves generally consist of non-repetitive amino acid
sequences, the overall XTEN sequence is rendered substantially
non-repetitive.
[0164] In one embodiment, XTEN have a non-repetitive sequence of
greater than about 100 to about 3000 amino acid residues wherein at
least about 80%, or at least about 85%, or at least about 90%, or
at least about 95%, or at least about 97%, or about 100% of the
XTEN sequence consists of non-overlapping sequence motifs, wherein
each of the motifs has about 9 to 36 amino acid residues. In other
embodiments, at least about 80%, or at least about 85%, or at least
about 90%, or at least about 95%, or at least about 97%, or about
100% of the XTEN sequence consists of non-overlapping sequence
motifs wherein each of the motifs has 9 to 14 amino acid residues.
In still other embodiments, at least about 80%, or at least about
85%, or at least about 90%, or at least about 95%, or at least
about 97%, or about 100% of the XTEN sequence component consists of
non-overlapping sequence motifs wherein each of the motifs has 12
amino acid residues. In these embodiments, it is preferred that the
sequence motifs be composed mainly of small hydrophilic amino
acids, such that the overall sequence has an unstructured, flexible
characteristic. Examples of amino acids that are included in XTEN
are, e.g., arginine, lysine, threonine, alanine, asparagine,
glutamine, aspartate, glutamate, serine, and glycine. As a result
of testing variables such as codon optimization, assembly
polynucleotides encoding sequence motifs, expression of protein,
charge distribution and solubility of expressed protein, and
secondary and tertiary structure, it was discovered that XTEN
compositions with enhanced characteristics mainly include glycine
(G), alanine (A), serine (S), threonine (T), glutamate (E) and
proline (P) residues wherein the sequences are designed to be
substantially non-repetitive. In one embodiment, XTEN sequences
have predominately four to six types of amino acids selected from
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
or proline (P) that are arranged in a substantially non-repetitive
sequence that is greater than about 100 to about 3000 amino acid
residues, preferably greater than 400 to about 3000 residues in
length. In some embodiments, XTEN have sequences of greater than
about 100 to about 3000 amino acid residues wherein at least about
80% of the sequence consists of non-overlapping sequence motifs
wherein each of the motifs has 9 to 36 amino acid residues wherein
each of the motifs consists of 4 to 6 types of amino acids selected
from glycine (G), alanine (A), serine (S), threonine (T), glutamate
(E) and proline (P), and wherein the content of any one amino acid
type in the full-length XTEN does not exceed 30%. In other
embodiments, at least about 90% of the XTEN sequence consists of
non-overlapping sequence motifs wherein each of the motifs has 9 to
36 amino acid residues wherein the motifs consist of 4 to 6 types
of amino acids selected from glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P), and wherein the
content of any one amino acid type in the full-length XTEN does not
exceed 30%. In other embodiments, at least about 90% of the XTEN
sequence consists of non-overlapping sequence motifs wherein each
of the motifs has 12 amino acid residues consisting of 4 to 6 types
of amino acids selected from glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P), and wherein the
content of any one amino acid type in the full-length XTEN does not
exceed 30%. In yet other embodiments, at least about 90%, or about
91%, or about 92%, or about 93%, or about 94%, or about 95%, or
about 96%, or about 97%, or about 98%, or about 99%, to about 100%
of the XTEN sequence consists of non-overlapping sequence motifs
wherein each of the motifs has 12 amino acid residues consisting of
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
and proline (P), and wherein the content of any one amino acid type
in the full-length XTEN does not exceed 30%.
[0165] In still other embodiments, XTENs comprise non-repetitive
sequences of greater than about 100 to about 3000 amino acid
residues wherein at least about 80%, or at least about 90%, or
about 91%, or about 92%, or about 93%, or about 94%, or about 95%,
or about 96%, or about 97%, or about 98%, or about 99% of the
sequence consists of non-overlapping sequence motifs of 9 to 14
amino acid residues wherein the motifs consist of 4 to 6 types of
amino acids selected from glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P), and wherein the
sequence of any two contiguous amino acid residues in any one motif
is not repeated more than twice in the sequence motif. In other
embodiments, at least about 90%, or about 91%, or about 92%, or
about 93%, or about 94%, or about 95%, or about 96%, or about 97%,
or about 98%, or about 99% of an XTEN sequence consists of
non-overlapping sequence motifs of 12 amino acid residues wherein
the motifs consist of 4 to 6 types of amino acids selected from
glycine (G), alanine (A), serine (S), threonine (T), glutamate (E)
and proline (P), and wherein the sequence of any two contiguous
amino acid residues in any one sequence motif is not repeated more
than twice in the sequence motif. In other embodiments, at least
about 90%, or about 91%, or about 92%, or about 93%, or about 94%,
or about 95%, or about 96%, or about 97%, or about 98%, or about
99% of an XTEN sequence consists of non-overlapping sequence motifs
of 12 amino acid residues wherein the motifs consist of glycine
(G), alanine (A), serine (S), threonine (T), glutamate (E) and
proline (P), and wherein the sequence of any two contiguous amino
acid residues in any one sequence motif is not repeated more than
twice in the sequence motif. In yet other embodiments, XTENs
consist of 12 amino acid sequence motifs wherein the amino acids
are selected from glycine (G), alanine (A), serine (S), threonine
(T), glutamate (E) and proline (P), and wherein the sequence of any
two contiguous amino acid residues in any one sequence motif is not
repeated more than twice in the sequence motif, and wherein the
content of any one amino acid type in the full-length XTEN does not
exceed 30%. In the foregoing embodiments hereinabove described in
this paragraph, the XTEN sequences is substantially
non-repetitive.
[0166] In some embodiments, the invention provides compositions
comprising non-repetitive XTEN sequence(s) of greater than about
100 to about 3000 amino acid residues wherein at least about 80%,
or at least about 90%, or about 91%, or about 92%, or about 93%, or
about 94%, or about 95%, or about 96%, or about 97%, or about 98%,
or about 99% to about 100% of the sequence consists of multiple
units of two or more non-overlapping sequence motifs selected from
the amino acid sequences of Table 3. In some embodiments, the XTEN
comprises non-overlapping sequence motifs in which about 80%, or at
least about 90%, or about 91%, or about 92%, or about 93%, or about
94%, or about 95%, or about 96%, or about 97%, or about 98%, or
about 99% to about 100% of the sequence consists of two or more
non-overlapping sequences selected from a single motif family of
Table 3, resulting in a "family" sequence in which the overall
sequence remains substantially non-repetitive. Accordingly, in
these embodiments, an XTEN sequence comprises multiple units of
non-overlapping sequence motifs of the AD motif family, or the AE
motif family, or the AF motif family, or the AG motif family, or
the AM motif family, or the AQ motif family, or the BC family, or
the BD family of sequences of Table 3. In other embodiments, the
XTEN comprises motif sequences from two or more of the motif
families of Table 3.
TABLE-US-00003 TABLE 3 XTEN Sequence Motifs of 12 Amino Acids and
Motif Families Motif Family* MOTIF SEQUENCE AD GESPGGSSGSES AD
GSEGSSGPGESS AD GSSESGSSEGGP AD GSGGEPSESGSS AE, AM GSPAGSPTSTEE
AE, AM, AQ GSEPATSGSETP AE, AM, AQ GTSESATPESGP AE, AM, AQ
GTSTEPSEGSAP AF, AM GSTSESPSGTAP AF, AM GTSTPESGSASP AF, AM
GTSPSGESSTAP AF, AM GSTSSTAESPGP AG, AM GTPGSGTASSSP AG, AM
GSSTPSGATGSP AG, AM GSSPSASTGTGP AG, AM GASPGTSSTGSP AQ
GEPAGSPTSTSE AQ GTGEPSSTPASE AQ GSGPSTESAPTE AQ GSETPSGPSETA AQ
GPSETSTSEPGA AQ GSPSEPTEGTSA BC GSGASEPTSTEP BC GSEPATSGTEPS BC
GTSEPSTSEPGA BC GTSTEPSEPGSA BD GSTAGSETSTEA BD GSETATSGSETA BD
GTSESATSESGA BD GTSTEASEGSAS *Denotes individual motif sequences
that, when used together in various permutations, results in a
"family sequence"
[0167] In other embodiments, the CFXTEN composition comprises a
non-repetitive XTEN sequence of greater than about 100 to about
3000 amino acid residues, wherein at least about 80%, or at least
about 90%, or about 91%, or about 92%, or about 93%, or about 94%,
or about 95%, or about 96%, or about 97%, or about 98%, or about
99% to about 100% of the sequence consists of non-overlapping 36
amino acid sequence motifs selected from one or more of the
polypeptide sequences of Tables 9-12.
[0168] In those embodiments wherein the XTEN component of the
CFXTEN fusion protein has less than 100% of its amino acids
consisting of four to six amino acid selected from glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P), or less than 100% of the sequence consisting of the sequence
motifs of Table 3, or less than 100% sequence identity compared
with an XTEN from Table 3, the other amino acid residues are
selected from any other of the 14 natural L-amino acids, but are
preferentially selected from hydrophilic amino acids such that the
XTEN sequence contains at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98%, or at least about 99% hydrophilic
amino acids. The XTEN amino acids that are not glycine (G), alanine
(A), serine (S), threonine (T), glutamate (E) and proline (P) are
interspersed throughout the XTEN sequence, are located within or
between the sequence motifs, or are concentrated in one or more
short stretches of the XTEN sequence. In such cases where the XTEN
component of the CFXTEN comprises amino acids other than glycine
(G), alanine (A), serine (S), threonine (T), glutamate (E) and
proline (P), it is preferred that the amino acids not be
hydrophobic residues and should not substantially confer secondary
structure of the XTEN component. Hydrophobic residues that are less
favored in construction of XTEN include tryptophan, phenylalanine,
tyrosine, leucine, isoleucine, valine, and methionine.
Additionally, one can design the XTEN sequences to contain few
(e.g. less than 5%) or none of the following amino acids: cysteine
(to avoid disulfide formation and oxidation), methionine (to avoid
oxidation), asparagine and glutamine (to avoid desamidation). Thus,
in some embodiments, the XTEN component of the CFXTEN fusion
protein comprising other amino acids in addition to glycine (G),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P) would have a sequence with less than 5% of the residues
contributing to alpha-helices and beta-sheets as measured by the
Chou-Fasman algorithm and have at least 90%, or at least about 95%
or more random coil formation as measured by the GOR algorithm.
3. Length of Sequence
[0169] In another aspect of the present invention, the invention
encompasses CFXTEN compositions comprising carriers of XTEN
polypeptides with extended length sequences. The present invention
makes use of the discovery that increasing the length of the
non-repetitive, unstructured polypeptides enhances the unstructured
nature of the XTENs and correspondingly enhances the biological and
pharmacokinetic properties of fusion proteins comprising the XTEN
carrier. As described more fully in the Examples, proportional
increases in the length of the XTEN, even if created by a fixed
repeat order of single family sequence motifs (e.g., the four AE
motifs of Table 3), result in a sequence with a higher percentage
of random coil formation, as determined by GOR algorithm, compared
to shorter XTEN lengths. In general, increasing the length of the
unstructured polypeptide fusion partner, as described in the
Examples, results in a fusion protein with a disproportionate
increase in terminal half-life compared to fusion proteins with
unstructured polypeptide partners with shorter sequence
lengths.
[0170] Non-limiting examples of XTEN contemplated for inclusion in
the CFXTEN of the invention are presented in Table 4, below. In one
embodiment, the invention provides CFXTEN compositions wherein the
XTEN sequence length of the fusion protein(s) is greater than about
100 to about 3000 amino acid residues, and in some cases is greater
than 400 to about 3000 amino acid residues, wherein the XTEN
confers enhanced pharmacokinetic properties on the CFXTEN in
comparison to CF not linked to XTEN. In some embodiments, the XTEN
sequences of the CFXTEN compositions of the present invention can
be about 100, or about 144, or about 288, or about 401, or about
500, or about 600, or about 700, or about 800, or about 900, or
about 1000, or about 1500, or about 2000, or about 2500 or up to
about 3000 amino acid residues in length. In other cases, the XTEN
sequences can be about 100 to 150, about 150 to 250, about 250 to
400, 401 to about 500, about 500 to 900, about 900 to 1500, about
1500 to 2000, or about 2000 to about 3000 amino acid residues in
length. In one embodiment, the CFXTEN can comprise an XTEN sequence
wherein the sequence exhibits at least about 80% sequence identity,
or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity compared to a XTEN selected from Table 4. In some
embodiments, the XTEN sequence is designed for optimized expression
as the N-terminal component of the CFXTEN by inclusion of encoding
nucleotides for an optimized N-terminal leader sequence (NTS) in
the XTEN portion of the gene encoding the fusion protein. In one
embodiment, the N-terminal XTEN sequence of the expressed CFXTEN
has at least 90% sequence identity compared to the sequence of AE48
or AM48, AE624, or AE912 or AM923. In another embodiment, the XTEN
has the N-terminal residues described in Examples 14-17.
[0171] In other embodiments, the CFXTEN fusion protein comprises a
first and a second XTEN sequence, wherein the cumulative total of
the residues in the XTEN sequences is greater than about 400 to
about 3000 amino acid residues and the XTEN can be identical or
they can be different in sequence. In embodiments of the foregoing,
the CFXTEN fusion protein comprises a first and a second XTEN
sequence wherein the sequences each exhibit at least about 80%
sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity compared to at least a first or additionally
a second XTEN selected from Table 4. Examples where more than one
XTEN is used in a CFXTEN composition include, but are not limited
to constructs with an XTEN linked to both the N- and C-termini of
at least one CF.
[0172] As described more fully below, the invention provides
methods in which the CFXTEN is designed by selecting the length of
the XTEN to confer a target half-life on a fusion protein
administered to a subject. In general, XTEN lengths longer that
about cumulative 400 residues incorporated into the CFXTEN
compositions result in longer half-life compared to shorter
cumulative lengths; e.g., shorter than about 280 residues. However,
in another embodiment, CFXTEN fusion proteins are designed to
comprise XTEN with a longer sequence length that is selected to
additionally confer slower rates of systemic absorption after
subcutaneous or intramuscular administration to a subject. In such
embodiments, the C.sub.maxis reduced in comparison to a comparable
dose of a CF not linked to XTEN, thereby contributing to the
ability to keep the CFXTEN within the therapeutic window for the
composition. Thus, the XTEN confers the property of a depot to the
administered CFXTEN, in addition to the other physical/chemical
properties described herein.
TABLE-US-00004 TABLE 4 XTEN Polypeptides XTEN Name Amino Acid
Sequence AE48 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS AM48
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEP
ATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSG
SETPGTSTEPSEGSAP AF144
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSS
TAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESST
APGTSPSGESSTAP AE288
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAP AF504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSXP
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGT
SSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG
SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AF540
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST
APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGS
TSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTA
ESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASP
GSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGT
APGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGT
STPESGSASPGSTSESPSGTAP AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSE
SGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSS
EGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGS
SGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSG
GEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGS
SGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGS
SGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGES
PGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPS
ESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS
AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
AF576
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST
APGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGS
TSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTA
ESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASP
GSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGT
APGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGT
STPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AD836
GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGESP
GGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSS
GSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGP
GSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSG
GEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGS
SEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEG
GPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGS
EGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGE
PSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSG
SEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPG
GSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSE
GGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSES
GSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESGSG
GEPSESGSS AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP
GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAP AF864
GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTP
ESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESST
APGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT
STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTA
ESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAP
GTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPS
GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSG
TAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPG
TSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPE
SGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSAS
PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTS
ESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESS
TAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPG
SSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AG864
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSP
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTS
STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS
PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGAS
PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
STGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS
TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGT
SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST
GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG
SSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT
STEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSES
ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESS
TAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPG
SEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGS
EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGT
SSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AE912
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEEGT
SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS
PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS
EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA
TPESGPGTSTEPSEGSAP AM923
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAPGS
EPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESP
SGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
EGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGS
PGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSP
AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSS
PGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGST
SSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSE
GSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAP
GTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST
EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT
STEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAP
GSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPA
TSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
TEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPG
TSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGS
APGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPS
GATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESST
APGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS
SPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSG
ESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGT
APGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSES
PSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS
SPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP BC 864
GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEP
ATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSE
PGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPS
GSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEP
ATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSG
TEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEP
GTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEP
ATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSG
TEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPS
GTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTST
EPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSE
PGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPS
GSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEP
ATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA BD864
GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETAGS
ETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTE
ASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATS
ESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETST
EAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEA
GSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTS
ESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETA
TSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASE
GSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSE
TAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGA
GSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGST
AGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETA
TSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATS
GSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA Y288
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGSGEG
SEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSE
GSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEG
SEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGE
GEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE Y576
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGE
GSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEG
SEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEG
EGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGEGGSEGSEGEGGSEGSEGEGGEGS
GEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEGSE
GSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGE
GSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSE
GEGGGEGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEG
GSEGEGSEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEG
GEGSGEGEGGGEGSEGEGSEGSGEGEGSGEGSE
4. XTEN Segments
[0173] In one embodiment, the invention provides an isolated CFXTEN
fusion protein wherein the cumulative length of the XTEN component
is greater than about 100 to about 3000 amino acid residues
containing at least one polypeptide sequence segment selected from
Tables 4, 9, 10, 11, 12, and 13 and wherein at least about 90%, or
at least about 91%, or at least about 92%, or at least about 93%,
or at least about 94%, or at least about 95%, or at least about
96%, or at least about 97%, or at least about 98% or more of the
remainder of the XTEN sequence contains hydrophilic amino acids and
less than about 2% of the remainder of the XTEN consists of
hydrophobic or aromatic amino acids or cysteine. In some
embodiments, the XTEN contains multiple segments wherein the
segments are identical or different. In another embodiment, the
invention provides an isolated CFXTEN fusion protein wherein the
cumulative length of the XTEN component is greater than about 100
to about 3000 amino acid residues and comprises at least one
sequence segment of at least about 100 to about 923, or at least
about 100 to about 875, or at least about 100 to about 576, or at
least about 100 to about 288, or at least about 100 to about 144
amino acid residues wherein the sequence segment(s) consists of at
least three different types of amino acids and the sum of glycine
(G), alanine (A), serine (S), threonine (T), glutamate (E) and
proline (P) residues in the sequence segment(s) constitutes at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about
98%, or at least about 99% of the total amino acid sequence of the
sequence segment and at least about 90%, or at least about 91%, or
at least about 92%, or at least about 93%, or at least about 94%,
or at least about 95%, or at least about 96%, or at least about
97%, or at least about 98% of the remainder of the XTEN sequence(s)
consist of hydrophilic amino acids and less than about 2% of the
remainder of the XTEN sequence(s) consists of hydrophobic or
aromatic amino acids, or cysteine. In another embodiment, the
invention provides an isolated CFXTEN fusion protein wherein the
cumulative length of the XTEN component is greater than about 100
to about 3000 amino acid residues and comprises at least one
sequence segment of at least about 200 to about 923, or at least
about 200 to about 875, or at least about 200 to about 576, or at
least about 200 to about 288 amino acid residues wherein the
sequence segment(s) the sum of glycine (G), alanine (A), serine
(S), threonine (T), glutamate (E) and proline (P) residues in the
sequence segment(s) constitutes at least about 90%, or at least
about 91%, or at least about 92%, or at least about 93%, or at
least about 94%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99% of
the total amino acid sequence of the sequence segment and wherein
the subsequence score of the segment is less than 12, more
preferably less than 10, more preferably less than 9, more
preferably less than 8, more preferably less than 7, more
preferably less than 6, and most preferably less than 5, and at
least about 90%, or at least about 91%, or at least about 92%, or
at least about 93%, or at least about 94%, or at least about 95%,
or at least about 96%, or at least about 97%, or at least about 98%
of the remainder of the XTEN sequence(s) consist of hydrophilic
amino acids and less than about 2% of the remainder of the XTEN
sequence(s) consists of hydrophobic, aromatic or cysteine amino
acids.
5. N-terminal XTEN Expression-Enhancing Sequences
[0174] In some embodiments, the invention provides a short-length
XTEN sequence incorporated as the N-terminal portion of the CFXTEN
fusion protein. It has been discovered that the expression of the
fusion protein is enhanced in a host cell transformed with a
suitable expression vector comprising an optimized N-terminal
leader polynucleotide sequence (that encodes the N-terminal XTEN)
incorporated into the polynucleotide encoding the binding fusion
protein. As described in Examples 14-17, a host cell transformed
with such an expression vector comprising an optimized N-terminal
leader sequence (NTS) in the binding fusion protein gene results in
greatly-enhanced expression of the fusion protein compared to the
expression of a corresponding fusion protein from a polynucleotide
not comprising the NTS, and obviates the need for incorporation of
a non-XTEN leader sequence used to enhance expression. In one
embodiment, the invention provides CFXTEN fusion proteins
comprising an NTS wherein the expression of the binding fusion
protein from the encoding gene in a host cell is enhanced about
50%, or about 75%, or about 100%, or about 150%, or about 200%, or
about 400% compared to expression of a CFXTEN fusion protein not
comprising the N-terminal XTEN sequence (where the encoding gene
lacks the NTS).
[0175] In one embodiment, the N-terminal XTEN polypeptide of the
CFXTEN comprises a sequence that exhibits at least about 80%, more
preferably at least about 90%, more preferably at least about 91%,
more preferably at least about 92%, more preferably at least about
93%, more preferably at least about 94%, more preferably at least
about 95%, more preferably at least about 96%, more preferably at
least about 97%, more preferably at least about 98%, more
preferably at least 99%, or exhibits 100% sequence identity
compared to the amino acid sequence of AE48 or AM48, the respective
amino acid sequences of which are as follows:
TABLE-US-00005 AE48: MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTS
STGS AM48: MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSG ATGS
[0176] In another embodiment, the short-length N-terminal XTEN is
linked to an XTEN of longer length to form the N-terminal region of
the CFXTEN fusion protein, wherein the polynucleotide sequence
encoding the short-length N-terminal XTEN confers the property of
enhanced expression in the host cell, and wherein the long length
of the expressed XTEN contributes to the enhanced properties of the
XTEN carrier in the fusion protein, as described above. In the
foregoing, the short-length XTEN is linked to any of the XTEN
disclosed herein (e.g., an XTEN of Table 3) and the resulting XTEN,
in turn, is linked to the N-terminal of any of the CF disclosed
herein (e.g., a CF of Table 1 or Table 2) as a component of the
fusion protein. Alternatively, polynucleotides encoding the
short-length XTEN (or its complement) is linked to polynucleotides
encoding any of the XTEN (or its complement) disclosed herein and
the resulting gene encoding the N-terminal XTEN, in turn, is linked
to the 5' end of polynucleotides encoding any of the CF (or to the
3' end of its complement) disclosed herein. In some embodiments,
the N-terminal XTEN polypeptide with long length exhibits at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 96%, or at least about 97%,
or at least about 98%, or at least 99%, or exhibits 100% sequence
identity compared to an amino acid sequence selected from the group
consisting of the sequences AE624, AE912, and AM923.
[0177] In any of the foregoing N-terminal XTEN embodiments
described above, the N-terminal XTEN can have from about one to
about six additional amino acid residues, preferably selected from
GESTPA, to accommodate the restriction endonuclease restriction
sites that is employed to join the nucleotides encoding the
N-terminal XTEN to the gene encoding the targeting moiety of the
fusion protein. The methods for the generation of the N-terminal
sequences and incorporation into the fusion proteins of the
invention are described more fully in the Examples.
6. Net Charge
[0178] In other embodiments, the XTEN polypeptides have an
unstructured characteristic imparted by incorporation of amino acid
residues with a net charge and/or reducing the proportion of
hydrophobic amino acids in the XTEN sequence. The overall net
charge and net charge density is controlled by modifying the
content of charged amino acids in the XTEN sequences. In some
embodiments, the net charge density of the XTEN of the compositions
may be above +0.1 or below -0.1 charges/residue. By "net charge
density" of a protein or peptide herein is meant the net charge
divided by the total number of amino acids in the protein or
protide. In other embodiments, the net charge density of a XTEN can
be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%,
about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about
12%, about 13%, about 14%, about 15%, about 16%, about 17%, about
18%, about 19%, or about 20% or more.
[0179] Since most tissues and surfaces in a human or animal have a
net negative charge, in some embodiments, the XTEN sequences are
designed to have a net negative charge to minimize non-specific
interactions between the XTEN containing compositions and various
surfaces such as blood vessels, healthy tissues, or various
receptors. Not to be bound by a particular theory, the XTEN can
adopt open conformations due to electrostatic repulsion between
individual amino acids of the XTEN polypeptide that individually
carry a net negative charge and that are distributed across the
sequence of the XTEN polypeptide. Such a distribution of net
negative charge in the extended sequence lengths of XTEN can lead
to an unstructured conformation that, in turn, can result in an
effective increase in hydrodynamic radius. In preferred
embodiments, the negative charge is conferred by incorporation of
glutamic acid residues. Accordingly, in one embodiment the
invention provides XTEN in which the XTEN sequences contain about
8, 10, 15, 20, 25, or even about 30% glutamic acid. Generally, the
glutamic residues is spaced uniformly across the XTEN sequence. In
some cases, the XTEN can contain about 10-80, or about 15-60, or
about 20-50 glutamic residues per 20 KDa of XTEN that can result in
an XTEN with charged residues that would have very similar pKa,
which can increase the charge homogeneity of the product and
sharpen its isoelectric point, enhance the physicochemical
properties of the resulting CFXTEN fusion protein for, and hence,
simplifying purification procedures.
[0180] The XTEN of the compositions of the present invention
generally have no or a low content of positively charged amino
acids. In some embodiments, the XTEN may have less than about 10%
amino acid residues with a positive charge, or less than about 7%,
or less than about 5%, or less than about 2%, or less than about 1%
amino acid residues with a positive charge. However, the invention
contemplates constructs where a limited number of amino acids with
a positive charge, such as lysine, are incorporated into XTEN to
permit conjugation between the epsilon amine of the lysine and a
reactive group on a peptide, a linker bridge, or a reactive group
on a drug or small molecule to be conjugated to the XTEN backbone.
In one embodiment of the foregoing, the XTEN has between about 1 to
about 100 lysine residues, or about 1 to about 70 lysine residues,
or about 1 to about 50 lysine residues, or about 1 to about 30
lysine residues, or about 1 to about 20 lysine residues, or about 1
to about 10 lysine residues, or about 1 to about 5 lysine residues,
or alternatively only a single lysine residue. Using the foregoing
lysine-containing XTEN, fusion proteins are constructed that
comprises XTEN, a coagulation factor, plus a chemotherapeutic agent
useful in the treatment of growth-related diseases or disorders,
wherein the maximum number of molecules of the agent incorporated
into the XTEN component is determined by the numbers of lysines or
other amino acids with reactive side chains (e.g., cysteine)
incorporated into the XTEN.
[0181] In some embodiments, the XTEN sequence comprises charged
residues separated by other residues such as serine or glycine,
which leads to better expression or purification behavior. Based on
the net charge, some XTENs have an isoelectric point (pI) of 1.0,
1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In
preferred embodiments, the XTEN will have an isoelectric point
between 1.5 and 4.5. In these embodiments, the XTEN incorporated
into the CFXTEN fusion protein compositions of the present
invention carry a net negative charge under physiologic conditions
that contribute to the unstructured conformation and reduced
binding of the XTEN component to mammalian proteins and
tissues.
[0182] As hydrophobic amino acids impart structure to a
polypeptide, the invention provides that the content of hydrophobic
amino acids in the XTEN will typically be less than 5%, or less
than 2%, or less than 1% hydrophobic amino acid content. In one
embodiment, the amino acid content of methionine and tryptophan in
the XTEN component of a CFXTEN fusion protein is typically less
than 5%, or less than 2%, and most preferably less than 1%. In
another embodiment, the XTEN will have a sequence that has less
than 10% amino acid residues with a positive charge, or less than
about 7%, or less that about 5%, or less than about 2% amino acid
residues with a positive charge, the sum of methionine and
tryptophan residues will be less than 2%, and the sum of asparagine
and glutamine residues will be less than 10% of the total XTEN
sequence.
7. Low Immunogenicity
[0183] In another aspect, the invention provides compositions in
which the XTEN sequences have a low degree of immunogenicity or are
substantially non-immunogenic. Several factors can contribute to
the low immunogenicity of XTEN, e.g., the non-repetitive sequence,
the unstructured conformation, the high degree of solubility, the
low degree or lack of self-aggregation, the low degree or lack of
proteolytic sites within the sequence, and the low degree or lack
of epitopes in the XTEN sequence.
[0184] Conformational epitopes are formed by regions of the protein
surface that are composed of multiple discontinuous amino acid
sequences of the protein antigen. The precise folding of the
protein brings these sequences into a well-defined, stable spatial
configurations, or epitopes, that can be recognized as "foreign" by
the host humoral immune system, resulting in the production of
antibodies to the protein or the activation of a cell-mediated
immune response. In the latter case, the immune response to a
protein in an individual is heavily influenced by T-cell epitope
recognition that is a function of the peptide binding specificity
of that individual's HLA-DR allotype. Engagement of a MHC Class II
peptide complex by a cognate T-cell receptor on the surface of the
T-cell, together with the cross-binding of certain other
co-receptors such as the CD4 molecule, can induce an activated
state within the T-cell. Activation leads to the release of
cytokines further activating other lymphocytes such as B cells to
produce antibodies or activating T killer cells as a full cellular
immune response.
[0185] The ability of a peptide to bind a given MHC Class II
molecule for presentation on the surface of an APC (antigen
presenting cell) is dependent on a number of factors; most notably
its primary sequence. In one embodiment, a lower degree of
immunogenicity is achieved by designing XTEN sequences that resist
antigen processing in antigen presenting cells, and/or choosing
sequences that do not bind MHC receptors well. The invention
provides CFXTEN fusion proteins with substantially non-repetitive
XTEN polypeptides designed to reduce binding with MHC II receptors,
as well as avoiding formation of epitopes for T-cell receptor or
antibody binding, resulting in a low degree of immunogenicity.
Avoidance of immunogenicity can attribute to, at least in part, a
result of the conformational flexibility of XTEN sequences; i.e.,
the lack of secondary structure due to the selection and order of
amino acid residues. For example, of particular interest are
sequences having a low tendency to adapt compactly folded
conformations in aqueous solution or under physiologic conditions
that could result in conformational epitopes. The administration of
fusion proteins comprising XTEN, using conventional therapeutic
practices and dosing, would generally not result in the formation
of neutralizing antibodies to the XTEN sequence, and also reduce
the immunogenicity of the CF fusion partner in the CFXTEN
compositions.
[0186] In one embodiment, the XTEN sequences utilized in the
subject fusion proteins can be substantially free of epitopes
recognized by human T cells. The elimination of such epitopes for
the purpose of generating less immunogenic proteins has been
disclosed previously; see for example WO 98/52976, WO 02/079232,
and WO 00/3317 which are incorporated by reference herein. Assays
for human T cell epitopes have been described (Stickler, M., et al.
(2003) J Immunol Methods, 281: 95-108). Of particular interest are
peptide sequences that can be oligomerized without generating T
cell epitopes or non-human sequences. This is achieved by testing
direct repeats of these sequences for the presence of T-cell
epitopes and for the occurrence of 6 to 15-mer and, in particular,
9-mer sequences that are not human, and then altering the design of
the XTEN sequence to eliminate or disrupt the epitope sequence. In
some embodiments, the XTEN sequences are substantially
non-immunogenic by the restriction of the numbers of epitopes of
the XTEN predicted to bind MHC receptors. With a reduction in the
numbers of epitopes capable of binding to MHC receptors, there is a
concomitant reduction in the potential for T cell activation as
well as T cell helper function, reduced B cell activation or
upregulation and reduced antibody production. The low degree of
predicted T-cell epitopes can be determined by epitope prediction
algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999)
Nat Biotechnol, 17: 555-61), as shown in Example 45. The TEPITOPE
score of a given peptide frame within a protein is the log of the
K.sub.d(dissociation constant, affinity, off-rate) of the binding
of that peptide frame to multiple of the most common human MHC
alleles, as disclosed in Sturniolo, T. et al. (1999) Nature
Biotechnology 17:555). The score ranges over at least 20 logs, from
about 10 to about -10 (corresponding to binding constraints of
10e.sup.10K.sub.dto 10e.sup.-1.degree. K.sub.d), and can be reduced
by avoiding hydrophobic amino acids that serve as anchor residues
during peptide display on MHC, such as M, I, L, V, F. In some
embodiments, an XTEN component incorporated into a CFXTEN does not
have a predicted T-cell epitope at a TEPITOPE score of about -5 or
greater, or -6 or greater, or -7 or greater, or -8 or greater, or
at a TEPITOPE score of -9 or greater. As used herein, a score of
"-9 or greater" would encompass TEPITOPE scores of 10 to -9,
inclusive, but would not encompass a score of -10, as -10 is less
than -9.
[0187] In another embodiment, the inventive XTEN sequences,
including those incorporated into the subject CFXTEN fusion
proteins, are rendered substantially non-immunogenic by the
restriction of known proteolytic sites from the sequence of the
XTEN, reducing the processing of XTEN into small peptides that can
bind to MHC II receptors. In another embodiment, the XTEN sequence
is rendered substantially non-immunogenic by the use a sequence
that is substantially devoid of secondary structure, conferring
resistance to many proteases due to the high entropy of the
structure. Accordingly, the reduced TEPITOPE score and elimination
of known proteolytic sites from the XTEN render the XTEN
compositions, including the XTEN of the CFXTEN fusion protein
compositions, substantially unable to be bound by mammalian
receptors, including those of the immune system. In one embodiment,
an XTEN of a CFXTEN fusion protein can have >100 nM K.sub.d
binding to a mammalian receptor, or greater than 500 nM K.sub.d, or
greater than 1 .mu.M K.sub.d towards a mammalian cell surface or
circulating polypeptide receptor.
[0188] Additionally, the non-repetitive sequence and corresponding
lack of epitopes of XTEN limit the ability of B cells to bind to or
be activated by XTEN. A repetitive sequence is recognized and can
form multivalent contacts with even a few B cells and, as a
consequence of the cross-linking of multiple T-cell independent
receptors, can stimulate B cell proliferation and antibody
production. In contrast, while a XTEN can make contacts with many
different B cells over its extended sequence, each individual B
cell may only make one or a small number of contacts with an
individual XTEN due to the lack of repetitiveness of the sequence.
Not being to be bound by any theory, XTENs typically have a much
lower tendency to stimulate proliferation of B cells and thus an
immune response. In one embodiment, the CFXTEN have reduced
immunogenicity as compared to the corresponding CF that is not
fused to an XTENT. In one embodiment, the administration of up to
three parenteral doses of a CFXTEN to a mammal result in detectable
anti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution
of 1:1000. In another embodiment, the administration of up to three
parenteral doses of a CFXTEN to a mammal result in detectable
anti-CF IgG at a serum dilution of 1:100 but not at a dilution of
1:1000. In another embodiment, the administration of up to three
parenteral doses of a CFXTEN to a mammal result in detectable
anti-XTEN IgG at a serum dilution of 1:100 but not at a dilution of
1:1000. In the foregoing embodiments, the mammal can be a mouse, a
rat, a rabbit, or a cynomolgus monkey.
[0189] An additional feature of XTENs with non-repetitive sequences
relative to sequences with a high degree of repetitiveness is
non-repetitive XTENs form weaker contacts with antibodies.
Antibodies are multivalent molecules. For instance, IgGs have two
identical binding sites and IgMs contain 10 identical binding
sites. Thus antibodies against repetitive sequences can form
multivalent contacts with such repetitive sequences with high
avidity, which can affect the potency and/or elimination of such
repetitive sequences. In contrast, antibodies against
non-repetitive XTENs may yield monovalent interactions, resulting
in less likelihood of immune clearance such that the CFXTEN
compositions can remain in circulation for an increased period of
time.
8. Increased Hydrodynamic Radius
[0190] In another aspect, the present invention provides XTEN in
which the XTEN polypeptides have a high hydrodynamic radius that
confers a corresponding increased apparent molecular weight to the
CFXTEN fusion protein incorporating the XTEN. As detailed in
Example 38, the linking of XTEN to CF sequences, such as FIX or
FVII sequences, results in CFXTEN compositions that can have
increased hydrodynamic radii, increased apparent molecular weight,
and increased apparent molecular weight factor compared to a CF not
linked to an XTEN. For example, in therapeutic applications in
which prolonged half-life is desired, compositions in which a XTEN
with a high hydrodynamic radius is incorporated into a fusion
protein comprising CF can effectively enlarge the hydrodynamic
radius of the composition beyond the glomerular pore size of
approximately 3-5 nm (corresponding to an apparent molecular weight
of about 70 kDA, which is larger than both native FIX and FVII)
(Caliceti. 2003. Pharmacokinetic and biodistribution properties of
poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev
55:1261-1277), resulting in reduced renal clearance of circulating
proteins. The hydrodynamic radius of a protein is determined by its
molecular weight as well as by its structure, including shape or
compactness. Not to be bound by a particular theory, the XTEN can
adopt open conformations due to electrostatic repulsion between
individual charges of the peptide or the inherent flexibility
imparted by the particular amino acids in the sequence that lack
potential to confer secondary structure. The open, extended and
unstructured conformation of the XTEN polypeptide can have a
greater proportional hydrodynamic radius compared to polypeptides
of a comparable sequence length and/or molecular weight that have
secondary and/or tertiary structure, such as typical globular
proteins. Methods for determining the hydrodynamic radius are well
known in the art, such as by the use of size exclusion
chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and
7,294,513. As the results of Example 38 demonstrate, the addition
of increasing lengths of XTEN results in proportional increases in
the parameters of hydrodynamic radius, apparent molecular weight,
and apparent molecular weight factor, permitting the tailoring of
CFXTEN to desired characteristic cut-off apparent molecular weights
or hydrodynamic radii. Accordingly, in certain embodiments, the
CFXTEN fusion protein can be configured with an XTEN such that the
fusion protein can have a hydrodynamic radius of at least about 5
nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or
at least about 15 nm. In the foregoing embodiments, the large
hydrodynamic radius conferred by the XTEN in an CFXTEN fusion
protein can lead to reduced renal clearance of the resulting fusion
protein, leading to a corresponding increase in terminal half-life,
an increase in mean residence time, and/or a decrease in renal
clearance rate.
[0191] In another embodiment, an XTEN of a chosen length and
sequence (e.g., a sequence from Table 4 or a sequence variant
thereof) can be selectively incorporated into a CFXTEN to create a
fusion protein that have, under physiologic conditions, an apparent
molecular weight of at least about 500 kDa, or at least about 800
kDa, or at least about 1000 kDa, or at least about 1500 kDA, or at
least about 1800 kDa, or at least about 2000 kDa, or at least about
2300 kDa or more. In another embodiment, an XTEN of a chosen length
and sequence can be selectively linked to a CF to result in a
CFXTEN fusion protein that has, under physiologic conditions, an
apparent molecular weight factor of at least four, alternatively of
at least five, alternatively of at least six, alternatively of at
least eight, alternatively of at least 10, alternatively of at
least 15, or an apparent molecular weight factor of at least 20 or
greater. In another embodiment, the CFXTEN fusion protein has,
under physiologic conditions, an apparent molecular weight factor
that is about 4 to about 20, or is about 6 to about 15, or is about
8 to about 12, or is about 9 to about 10 relative to the actual
molecular weight of the fusion protein.
V). CFXTEN Variants, Structural Configurations and Properties
[0192] The CF of the subject compositions are not limited to
native, full-length FIX or FVII polypeptides, but also include
recombinant versions as well as biologically and/or
pharmacologically active forms with sequence variants, combinations
of FVII and FIX sequences, or fragments thereof. For example, it
will be appreciated that various amino acid deletions, insertions
and substitutions can be made in the CF to create variants without
departing from the spirit of the invention with respect to the
biological activity or pharmacologic properties of the CF. Examples
of conservative substitutions for amino acids in polypeptide
sequences are shown in Table 5. However, in embodiments of the
CFXTEN in which the sequence identity of the CF is less than 100%
compared to a specific sequence disclosed herein, the invention
contemplates substitution of any of the other 19 natural L-amino
acids for a given amino acid residue of the given CF (e.g., FIX or
FVII), which may be at any position within the sequence of the CF,
including adjacent amino acid residues. If any one substitution
results in an undesirable change in biological activity, then one
of the alternative amino acids can be employed and the construct
evaluated by the methods described herein, or using any of the
techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S. Pat. No. 5,364,934, the
contents of which is incorporated by reference in its entirety, or
using methods generally known in the art. In addition, variants can
include, for instance, polypeptides wherein one or more amino acid
residues are added or deleted at the N- or C-terminus of the
full-length native amino acid sequence of a CF that retains some if
not all of the biological activity of the native peptide; e.g., the
ability to activate another coagulation factor and/or participate
in the coagulation cascade, leading to fibrin formation and
hemostasis.
[0193] In one embodiment, a factor IX incorporated into a CFXTEN
fusion protein has a sequence that exhibits at least about 80%
sequence identity compared to a sequence from Table 1,
alternatively at least about 81%, or about 82%, or about 83%, or
about 84%, or about 85%, or about 86%, or about 87%, or about 88%,
or about 89%, or about 90%, or about 91%, or about 92%, or about
93%, or about 94%, or about 95%, or about 96%, or about 97%, or
about 98%, or about 99%, sequence identity as compared with a
sequence from Table 1.
[0194] In one embodiment, a factor VII incorporated into a CFXTEN
fusion protein has a sequence that exhibits at least about 80%
sequence identity compared to a sequence from Table 2,
alternatively at least about 81%, or about 82%, or about 83%, or
about 84%, or about 85%, or about 86%, or about 87%, or about 88%,
or about 89%, or about 90%, or about 91%, or about 92%, or about
93%, or about 94%, or about 95%, or about 96%, or about 97%, or
about 98%, or about 99%, sequence identity as compared with a
sequence from Table 2.
TABLE-US-00006 TABLE 5 Exemplary conservative amino acid
substitutions Original Residue Exemplary Substitutions Ala (A) val;
leu; ile Arg (R) lys; gln; asn Asn (N) gin; his; lys; arg Asp (D)
Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Pro His (H) asn:
gin: lys: arg xIle (I) leu; val; met; ala; phe: norleucine Leu (L)
norleucine: ile: val; met; ala: phe Lys (K) arg: gin: asn Met (M)
leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) Gly Ser (S) Thr
Thr (T) Ser Trp (W) Tyr Tyr(Y) Trp: phe: thr: ser Val (V) Ile; leu;
met; phe; ala; norleucine
[0195] 1. Internal XTEN Sequences
[0196] The present invention encompasses CFXTEN that comprise one
or more XTEN sequences located internal to the CF sequence. The one
or more internally-located XTEN can be a sequence length of 36 to
.gtoreq.1000 amino acid residues. In some embodiments, the CFXTEN
can have one or two or three or four or more XTEN sequences with at
least about 80% sequence identity, or alternatively 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity compared to one or more
XTEN selected from Tables 4, 9, 10, 11, 12 and 13 wherein the XTEN
sequences are located internal to the CF sequence. In one
embodiment of the foregoing, the CFXTEN with one or more internal
XTEN has an additional XTEN located at the N- or C-terminus of the
fusion protein with at least about 80% sequence identity, or
alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity compared to one or more XTEN selected from Table 4. In
another embodiment, the invention provided CFXTEN with internal
XTEN (as detailed below) that further comprises a C-terminus XTEN
linked to the CF by a cleavage sequence (e.g., a cleavage sequence
of Table 7) such that the XTEN can be released when acted on by a
protease. The linkage of XTEN by cleavage sequences is more fully
described below and in the Examples.
[0197] In some embodiments, as illustrated in FIG. 2 and described
more fully in the Examples, an XTEN can be located between the
domains of a FIX sequence; e.g., between the Gla and EGF1, or
between the EGF1 and EGF2, or between the EGF2 and the activation
peptide, or within the sequence of the activation peptide between
the R145-A 146 and R180-V181 activation peptide residues of the AP
(i.e., between any two amino acids of the sequence
TVFPDVDYVNSTEAETILDNITQSTQSFNDF), or between the EGF2 and the
activation peptide, or between the activation peptide and the
protease domain, or any combination of the foregoing. In other
embodiments, as illustrated in FIG. 2 and detailed more fully in
the Examples, the XTEN can be inserted within an existing loop
sequence within an individual domain of the FIX sequence so that 1)
the XTEN forms a looped structure outside the domain and doesn't
disrupt the normal architecture of the domain; and 2) the XTEN can
be released by cleavage of incorporated cleavage sites.
[0198] In another embodiment, the invention provides a CFXTEN
comprising a FVII that incorporates one or more XTEN located
between the domains of a FVII sequence; e.g., between the Gla and
EGF1, or between the EGF1 and EGF2, or between the EGF2 and the
activating peptide, or between the activating peptide and the
protease domain, or any combination of the foregoing. The XTEN can
be a sequence of 36 to >1000 amino acid residues including, but
not limited to a sequence that has at least about 80%, or at least
about 85%, or at least about 90%, or at least about 95% or more
sequence identity compared to a sequence from Table 4, 8, 9, 10,
11, 12, and 13. In one embodiment, as illustrated in FIGS. 5 and 6,
an XTEN is incorporated between the EGF2 domain and the single
lytic cleavage site at residues Arg.sup.152-Ile.sup.153. In other
embodiments, as illustrated in FIGS. 5 and 6 and detailed more
fully in the Examples, the XTEN can be inserted within an existing
loop sequence within an individual domain of the FVII sequence so
that 1) the XTEN forms a looped structure outside the domain and
doesn't disrupt the normal architecture of the domain; and 2) the
XTEN can be released by cleavage of incorporated cleavage
sites.
[0199] 2. Factor VII-FIX Hybrid Sequence Variants
[0200] The invention provides an isolated factor VII polypeptide
comprising at least one heterologous sequence that is cleavable by
a pro-coagulant protease that does not activate a wildtype factor
VII, wherein upon cleavage heterologous sequence, the factor VII
polypeptide is activated. For example, CFXTEN with factor
VII-factor IX hybrid sequence variants that incorporate into, or
replace a portion of the sequence, a factor VII construct portions
of the activating peptide domain (AP) sequence from factor IX,
resulting in hybrid compositions that can be activated as part of
the intrinsic system of the coagulation cascade. The CFXTEN that
incorporate such factor VII-factor IX sequence variants as the CF
component of the fusion protein permit administration to a subject
a composition in which the CF component is not activated, and can
be dosed at high amounts because it remains as an inert,
circulating depot that is largely resistant to inactivation by
protease inhibitors until activated by the triggering of the
intrinsic coagulation cascade or by auto-activation, the latter a
slow process. Non-limiting examples of FVII/FIX hybrid sequences
are illustrated in FIG. 36, showing those portions of the hybrid
amino acid sequences that have homology with those of native FIX
and FVII. In some embodiments, the CFXTEN comprise factor
VII-factor IX sequence variants that substitute portions or the
entirety of the FIX activating peptide sequence with one or both
FIX AP cleavage sites for FVII sequence to the N-terminal side of
the protease domain of FVII; i.e., either towards the N-terminus
beginning with the arginine at position 212 of the full-length
precursor polypeptide or the isoleucine at position 213. In one
embodiment, the factor VII-factor IX sequence CF incorporates the
full-length FIX AP domain plus at least about 2, or at least about
3, or at least about 4, or at least about 5, or at least about 6,
or at least about 7, or at least about 8, or at least about 9, or
at least about 10, or at least about 11, or at least about 12 amino
acids flanking adjacent amino acid residues on one or both sides of
the R145-A146 and R180-V181 cleavage sites of FIX (e.g., the
sequence RVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE in
the case of 12 flanking amino acids on the N-terminus side and 5
flanking amino acids on the C-terminus side). In another
embodiment, the CFXTEN comprises a factor VII-factor IX sequence
variant that incorporates a portion of the AP that includes a
sequence of at least about 2, or at least about 3, or at least
about 4, or at least about 5 that flank the R145-A146 AP cleavage
site (e.g., the sequence TSKLTRAETVFP in the case of 6 flanking
amino acids on either side of the cleavage site). In another
embodiment, the CFXTEN comprises a factor VII-factor IX sequence
variant that incorporates a portion of the AP that includes a
sequence of at least about 2, or at least about 3, or at least
about 4, or at least about 5 amino acids that flank one or both
sides of the R180-V181 AP cleavage site (e.g., the sequence and
DFTRV in the case of 4 amino acids on the N-terminal flank and
valine as the C-terminus of the cleavage site from FIX). In another
embodiment, the CFXTEN comprises the factor VII-factor IX sequence
variant of any of the foregoing embodiments of this paragraph that
further includes the same AP sequence as a linker between the
C-terminus of the FVII component and the XTEN component of the
fusion protein; e.g., an N- to C-terminus configuration of FVII
variant-AP sequence-XTEN, thereby permitting the release of the
factor VII-factor IX sequence variant component from the CFXTEN
fusion protein by the same intrinsic coagulation factor as per that
of the FVII to FVIIa transition. In another embodiment, the CFXTEN
comprises the factor VII-factor IX sequence variant of any of the
foregoing embodiments of this paragraph that further includes the
factor XI cleavage sequence KLTRAET as the linker between the FVII
variant sequence and the XTEN, thereby permitting the release of
the factor VII-factor IX sequence variant component from the CFXTEN
fusion protein by the initiation of the intrinsic coagulation
cascade. It is expected d that with the release of the XTEN from
the factor VII-factor IX sequence variant, the activated factor
VII-factor IX sequence variant would have a shorter half-life
compared to the intact CFXTEN, thereby increasing the margin of
safety and tolerability of the composition in a subject. In the
embodiments of the paragraph, the activated factor VII-factor IX
sequence variant molecule can have at least about 60%, or at least
about 70%, or at least about 80%, or at least about 90%, or at
least about 95% of the biological activity as native FVIIa, as
measured by any of the appropriate assays or parameters disclosed
herein (e.g., PT or bleeding time assays).
[0201] In yet another embodiment, the invention provides the factor
VII-factor IX sequence variants of the foregoing embodiments of
this paragraph without a linked XTEN, permitting their
administration to a subject as a circulating depot of the factor
VII-factor IX hybrid that can be activated by either the intrinsic
or extrinsic coagulation cascade. In one embodiment, the invention
provides a CFXTEN with a factor VII-factor IX sequence variant with
incorporated FIX-derived sequence with an overall sequence that
exhibits at least about 80% sequence identity, or at least about
85%, or at least about 90%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at
least about 99%, sequence identity compared to a sequence from
Table 43. In another embodiment, the invention provides a factor
VII-factor IX sequence variant with incorporated FIX-derived
cleavage sequence (without an XTEN) with a sequence that exhibits
at least about 80% sequence identity, or at least about 85%, or at
least about 90%, or at least about 95%, or at least about 96%, or
at least about 97%, or at least about 98%, or at least about 99%,
sequence identity as compared with a sequence from Table 43 without
an XTEN.
[0202] The CFXTEN comprising factor VII-factor IX sequence variants
can be evaluated for biological activity using assays or in vivo
parameters as described herein (e.g., in vitro coagulation assays
or a pharmacodynamic effect in a hemophilia model), and those
sequences that retain at least about 40%, or about 50%, or about
55%, or about 60%, or about 70%, or about 80%, or about 90%, or
about 95% or more activity compared to the corresponding native
FVII sequence is considered suitable for inclusion in the subject
CFXTEN. The CF found to retain a suitable level of activity can be
linked to one or more XTEN polypeptides described hereinabove. In
one embodiment, a CF found to retain a suitable level of activity
can be linked to one or more XTEN polypeptides having at least
about 80% sequence identity to a sequence from Table 4,
alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
about 100% sequence identity as compared with a sequence of Table
4, resulting in a chimeric fusion protein.
[0203] 3. CFXTEN Fusion Protein Configurations
[0204] The invention provides CFXTEN fusion protein compositions
with the CF and XTEN components linked in specific N- to C-terminus
configurations. In some embodiments, one or more CFs are linked to
one or more XTENs, either at the N-terminus or at the C-terminus,
with or without a spacer, to form a block copolymer, and the
sequential arrangement of the CFs and the XTENs in the CFXTEN
fusion protein are the same as the configuration known in the block
copolymer chemistry. When there is more than one CF, XTEN, or
spacer, each of the CF, the XTEN, or the spacer have the same or
different sequences, and the CFs and/or XTENs are linked either
continuously or alternately (regular or irregular). Thus, in all of
the formulae provided herein, when there is more than one CF, XTEN,
or spacer, each of the CF, XTEN, and spacer are the same or
different. In some embodiments, the CFXTEN is a monomeric fusion
protein with a CF linked to one XTEN polypeptide. In other
embodiments, the CFXTEN is a monomeric fusion protein with a CF
linked to two or more XTEN polypeptides. In still other
embodiments, the CFXTEN is a monomeric fusion protein with two or
more CF linked to one XTEN polypeptide. In still other embodiments,
the CFXTEN is a monomeric fusion protein with two or more CF linked
to two or more XTEN polypeptide. In still other embodiment, the
CFXTEN is a monomeric fusion protein with a single CF in which XTEN
is located within the CF sequence (e.g., within a FIX sequence such
as between one or more domains as illustrated in FIGS. 2 and 5).
Table 6 provides non-limiting examples of configurations that are
encompassed by the CFXTEN fusion proteins of the invention;
numerous other variations will be apparent to the ordinarily
skilled artisan, including the incorporation the spacer and
cleavage sequences disclosed herein or known in the art.
TABLE-US-00007 TABLE 6 CFXTEN configurations Components*
Configuration** Single CF; Single XTEN CF-XTEN XTEN-CF Single CF;
Multiple XTEN XTEN-CF-XTEN CF-XTEN-XTEN XTEN-XTEN-CF
XTEN-CF-XTEN-XTEN XTEN-XTEN-CF-XTEN XTEN-XTEN-CF-XTEN Multiple CF,
Single XTEN CF-XTEN-CF XTEN-CF-CF CF-CF-XTEN CF-XTEN-CF-CF Multiple
CF; Multiple XTEN CF-XTEN-CF-XTEN XTEN-CF-XTEN-CF
XTEN-XTEN-CF-XTEN-CF XTEN-XTEN-CF-CF CF-XTEN-XTEN-CF
CF-CF-XTEN-XTEN CF-CF-XTEN-XTEN-CF CF-XTEN-CF-XTEN-CF
*Characterized as single for 1 component or multiple for 2 or more
of that component **Reflects N- to C-terminus configuration of the
growth factor and XTEN components
[0205] The invention contemplates CFXTEN fusion proteins
compositions comprising, but not limited to single or multiple CF
selected from Table 1 or Table 2 (or fragments or sequence variants
thereof), single or multiple XTEN selected from Table 4 (or
sequence variants thereof) that are in a configuration shown in
Table 6. Non-limiting examples of sequences of fusion proteins
containing a single CF linked to a single XTEN are presented in
Table 41. In one embodiment, a CFXTEN composition would comprise a
fusion protein having at least about 80% sequence identity compared
to a CFXTEN from Table 41, alternatively at least about 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or about 100% sequence identity as compared to
a CFXTEN from Table 41. Generally, the resulting CFXTEN retains at
least a portion of the biological activity of the corresponding CF
not linked to the XTEN. In the foregoing fusion proteins
hereinabove described in this paragraph, the CFXTEN fusion protein
can further comprise a cleavage sequence from Table 7; the cleavage
sequence being located between the CF and the XTEN or between
adjacent CF (if more than one CF is included in the CFXTEN). In
some cases, the CFXTEN comprising the cleavage sequences will also
have one or more spacer sequence amino acids between the CF and the
cleavage sequence or the XTEN and the cleavage sequence to
facilitate access of the protease; the spacer amino acids
comprising any natural amino acid, including glycine and alanine as
preferred amino acids. Non-limiting examples of CFXTEN comprising
CF, XTEN, cleavage sequence(s) and spacer amino acids are presented
in Table 42. However, the invention also contemplates substitution
of any of the CF sequences of Tables 1 and 2 for a CF sequence of
Table 42, substitution of any XTEN sequence of Table 4 for an XTEN
sequence of Table 42, and substitution of any cleavage sequence of
Table 7 for a cleavage sequence of Table 42. In CFXTEN embodiments
having one or more cleavage sequences, the CF component either
becomes biologically active or has an increase in activity upon its
release from the XTEN by cleavage of the cleavage sequence(s),
described more fully below.
[0206] In one embodiment of the CFXTEN composition, the invention
provides a fusion protein of formula I:
(XTEN).sub.x--CF--(XTEN).sub.y I
wherein independently for each occurrence, CF is a coagulation
factor; x is either 0 or 1 and y is either 0 or 1 wherein
x+y.gtoreq.1; and XTEN is an extended recombinant polypeptide.
[0207] In another embodiment of the CFXTEN composition, the
invention provides a fusion protein of formula II:
(XTEN).sub.x--(CF)--(S).sub.y--(XTEN).sub.y II
wherein independently for each occurrence, CF is a coagulation
factor a; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence; x is
either 0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN
is an extended recombinant polypeptide.
[0208] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein, wherein the fusion
protein is of formula III:
(XTEN).sub.x--(S).sub.x--(CF)--(S).sub.y--(XTEN).sub.y III
wherein independently for each occurrence, CF is a coagulation
factor; S is a spacer sequence having between 1 to about 50 amino
acid residues that can optionally include a cleavage sequence; x is
either 0 or 1 and y is either 0 or 1 wherein x+y.gtoreq.1; and XTEN
is an extended recombinant polypeptide.
[0209] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula IV:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(XTEN).sub.w-(AP)--(XTEN).-
sub.x-(Pro)-(S).sub.y--(XTEN).sub.z IV
wherein independently for each occurrence, Gla is a Gla domain of
FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX;
AP is an activator peptide of FIX; PRO is a protease domain of FIX;
S is a spacer sequence having between 1 to about 50 amino acid
residues that can optionally include a cleavage sequence; u is
either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either
0 or 1, z is either 0 or 1, with the proviso that u+v+x+z.gtoreq.1;
and XTEN is an extended recombinant polypeptide.
[0210] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula V:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(AP1)-(XTEN).sub.w-(AP2)-(-
XTEN).sub.x-(Pro)-(S).sub.y--(XTEN).sub.z V
wherein independently for each occurrence, Gla is a Gla domain of
FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX;
AP1 is the N-terminal sequence portion of the activator peptide
domain of FIX that includes a first native cleavage sequence of the
AP domain; AP2 is the C-terminal sequence portion of the activator
peptide domain of FIX that includes a second native cleavage
sequence of the AP domain; PRO is a protease domain of FIX; S is a
spacer sequence having between 1 to about 50 amino acid residues
that can optionally include a cleavage sequence; u is either 0 or
1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either
0 or 1; z is either 0 or 1 with the proviso that
u+v+w+x+z.gtoreq.1; and XTEN is an extended recombinant
polypeptide.
[0211] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula VI:
(Gla)-(XTEN).sub.u-(EGF1)-(XTEN).sub.v-(EGF2)-(XTEN).sub.w-(Pro)-(S).sub-
.x--(XTEN).sub.y VI
wherein independently for each occurrence, Gla is a Gla domain of
FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of
FVII; PRO is a protease domain of FVII; S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence; u is either 0 or 1; v is
either 0 or 1; x is either 0 or 1; y is either 0 or 1; and XTEN is
an extended recombinant polypeptide.
[0212] In another embodiment of the CFXTEN composition, the
invention provides an isolated fusion protein of formula VII:
(Gla)-(XTEN).sub.t-(EGF1)-(XTEN).sub.u-(EGF2)-(AP1).sub.v--(XTEN).sub.w--
(AP2).sub.x-(Pro)-(S).sub.y--(XTEN).sub.z VII
wherein independently for each occurrence, Gla is a Gla domain of
FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of
FVII; PRO is a protease domain of FVII; AP1 is the N-terminal
sequence portion of the activator peptide domain of FIX that
includes the native cleavage sequence; AP2 is the C-terminal
sequence portion of the activator peptide domain of FIX that
includes the native cleavage sequence; S is a spacer sequence
having between 1 to about 50 amino acid residues that can
optionally include a cleavage sequence; t is either 0 or 1; u is
either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either
0 or 1; z is either 0 or 1; and XTEN is an extended recombinant
polypeptide. In the embodiment, the factor VII variant includes can
include one or both cleavage sequences from the activator peptide
domain of factor IX; e.g., a sequence of at least about 2, or at
least about 3, or at least about 4, or at least about 5 amino acids
that flank the R145-A146 cleavage site (e.g., the sequence
TSKLTRAETVFP in the case of 5 flanking amino acids) and the
sequence of at least about 2, or at least about 3, or at least
about 4, or at least about 5 amino acids that flank the R180-V181
cleavage site (e.g., the sequence FNDFTRVVGGED in the case of 5
flanking amino acids, as described more fully above. The invention
also contemplates substitution of any of the other cleavage
sequences of Table 7 for the AP sequences of the factor VII
variant.
[0213] The embodiments of formulae V and VI encompass CFXTEN
configurations of factor IX and factor VII, respectively, wherein
one or more XTEN of lengths ranging from about 36 amino acids to
.gtoreq.1000 amino acids (e.g., sequences selected from Tables 4,
and 9-13) are inserted and linked between adjoining domains of the
factor IX or the factor VII sequence, respectively. The invention
contemplates all possible permutations of insertions of XTEN
between the domains of either FIX or FVII with optional linking of
an additional XTEN to the C-terminus of the FIX or the FVII,
optionally via an additional cleavage sequence selected from Table
7, resulting in a CFXTEN composition; non-limiting examples of
which are portrayed in FIGS. 2, 5 and 6. In the foregoing
embodiments hereinabove described in this paragraph, the CFXTEN
fusion proteins can be evaluated for retention of biological
activity (including after cleavage of any incorporated
XTEN-releasing cleavage sites) using any appropriate in vitro assay
disclosed herein (e.g., the assays of Table 40 or the assays
described in the Examples), to determine the suitability of the
configuration for use as a therapeutic agent in the treatment of a
coagulation-factor related disease, disorder or condition.
[0214] In some embodiments, administration of a therapeutically
effective amount of a fusion protein of one of formulae to a
subject in need thereof results in an increase of at least two-fold
in the terminal half-life, or at least three-fold, or at least
four-fold, or at least five-fold, or at least 10-fold, or at least
20-fold, or at least 40-fold, or at least 100-fold increase in the
terminal half-life for the fusion protein compared to the
corresponding CF not linked to the XTEN and administered at a
comparable amount administered to a subject. In some embodiments,
administration of a therapeutically effective amount of a fusion
protein of one of formulae I-VII to a subject in need thereof
results in a gain in time of at least two-fold, or at least
three-fold, or at least four-fold, or at least five-fold, or at
least 10-fold, or at least 20-fold, or at least 40-fold, or at
least 100-fold or more spent within a therapeutic window for the
fusion protein compared to the corresponding CF not linked to the
XTEN and administered at a comparable amount administered to a
subject. In other embodiments, administration of a therapeutically
effective dose of a fusion protein of one of formulae I-VII to a
subject in need thereof can result in a gain in time between
consecutive doses necessary to maintain a therapeutically effective
blood level of the fusion protein of at least 48 h, or at least 72
h, or at least about 96 h, or at least about 120 h, or at least
about 7 days, or at least about 14 days, or at least about 21 days
between consecutive doses compared to a CF not linked to XTEN and
administered at a comparable dose.
[0215] Any spacer sequence group optionally is introduced to a
subject fusion protein encompassed by the invention. The spacer is
provided to enhance expression of the fusion protein from a host
cell or to decrease steric hindrance such that the CF component may
assume its desired tertiary structure and/or interact appropriately
with its target substrate. For spacers and methods of identifying
desirable spacers, see, for example, George, et al. (2003) Protein
Engineering 15:871-879, specifically incorporated by reference
herein. In one embodiment, the spacer comprises one or more peptide
sequences that are between 1-50 amino acid residues in length, or
about 1-25 residues, or about 1-10 residues in length. Spacer
sequences, exclusive of cleavage sites, can comprise any of the 20
natural L amino acids, and will preferably comprise hydrophilic
amino acids that are sterically unhindered that can include, but
not be limited to, glycine (G), alanine (A), serine (S), threonine
(T), glutamate (E) and proline (P). In some cases, the spacer can
be polyglycines or polyalanines, or is predominately a mixture of
combinations of glycine and alanine residues. The spacer
polypeptide exclusive of a cleavage sequence is largely to
substantially devoid of secondary structure; e.g., less than about
10%, or less than about 5% as determined by the Chou-Fasman and/or
GOR algorithms. In one embodiment, a spacer sequence in a CFXTEN
fusion protein composition further contains one or more cleavage
sequences, which are identical or different, wherein the cleavage
sequence may be acted on by a protease to release the CF from the
fusion protein.
[0216] In some embodiments, the incorporation of the cleavage
sequence into the CFXTEN is designed to permit release of a CF that
becomes active or more active upon its release from the XTEN; e.g.,
the enzymatic activity of the CF component increases. In one
embodiment of the foregoing, the CF that becomes active after
release is a FIX or a sequence variant thereof. In another
embodiment of the foregoing, the CF that becomes active after
release is a FVII or a sequence variant thereof. The cleavage
sequences are located sufficiently close to the CF sequences,
generally within 18, or within 12, or within 6, or within 2 amino
acids of the CF sequence terminus, such that any remaining residues
attached to the CF after cleavage do not appreciably interfere with
the activity (e.g., such as binding to a ligand or substrate) of
the CF, yet provide sufficient access to the protease to be able to
effect cleavage of the cleavage sequence. In some embodiments, the
cleavage site is a sequence that can be cleaved by a protease
endogenous to the mammalian subject such that the CFXTEN can be
cleaved after administration to a subject. In such cases, the
CFXTEN can serve as a prodrug or a circulating depot for the CF. In
one embodiment, the CF that is released from the fusion protein by
cleavage of the cleavage sequence exhibits at least about a
two-fold, or at least about a three-fold, or at least about a
four-fold, or at least about a five-fold, or at least about a
six-fold, or at least about a eight-fold, or at least about a
ten-fold, or at least about a 20-fold increase in enzymatic
activity for its native substrate compared to the intact CFXTEN
fusion protein.
[0217] Examples of cleavage sites contemplated by the invention
include, but are not limited to, a polypeptide sequence cleavable
by a mammalian endogenous protease selected from FXIa, FXIIa,
kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, granzyme
B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases
such as TEV, enterokinase, PreScission.TM. protease (rhinovirus 3C
protease), and sortase A. Sequences known to be cleaved by the
foregoing proteases and others are known in the art. Exemplary
cleavage sequences and cut sites within the sequences are presented
in Table 7, as well as sequence variants thereof. For example,
thrombin (activated clotting factor II) acts on the sequence
LTPRSLLV [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], which is cut after the arginine at position 4 in the
sequence. Active FIIa is produced by cleavage of FII by FXa in the
presence of phospholipids and calcium and is down stream from
factor IX in the coagulation pathway. Once activated, its natural
role in coagulation is to cleave fibrinogen, which then in turn,
begins clot formation. FIIa activity is tightly controlled and only
occurs when coagulation is necessary for proper hemostasis.
However, as coagulation is an on-going process in mammals, by
incorporation of the LTPRSLLV sequence into the CFXTEN between and
linking the CF and the XTEN components, the XTEN is removed from
the adjoining CF concurrent with activation of either the extrinsic
or intrinsic coagulation pathways when coagulation is required
physiologically, thereby releasing CF over time. Similarly,
incorporation of other cleavage sequences into CFXTEN that are
acted upon by endogenous proteases, particularly those susceptible
to the activated clotting proteins listed in Table 7, would provide
for sustained release of CF that, in certain embodiments of the
CFXTEN, provide a higher degree of activity for the CF component
released from the intact form of the CFXTEN. In one embodiment, the
invention provides CFXTEN comprising one or more cleavage sequences
operably positioned to release the CF from the fusion protein upon
cleavage, wherein the one or more cleavage sequences has at least
about 86%, or at least about 92% or greater sequence identity to a
sequence selected from Table 7. In another embodiment, the CFXTEN
comprising a cleavage sequence would have at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%, or
at least about 96%, or at least about 97%, or at least about 98%,
or at least about 99% sequence identity compared to a sequence
selected from Table 42.
[0218] In some embodiments, only the two or three amino acids
flanking both sides of the cut site (four to six amino acids total)
are incorporated into the cleavage sequence that, in turn, is
incorporated into the CFXTEN of the embodiments. In other
embodiments, the known cleavage sequence have one or more deletions
or insertions or one or two or three amino acid substitutions for
any one or two or three amino acids in the known sequence, wherein
the deletions, insertions or substitutions result in reduced or
enhanced susceptibility but not an absence of susceptibility to the
protease, resulting in an ability to tailor the rate of release of
the CF from the XTEN. Exemplary substitutions are shown in Table
7.
TABLE-US-00008 TABLE 7 Protease Cleavage Sequences Protease Acting
Upon Exemplary Cleavage Sequence Sequence Minimal Cut Site* FXIa
KLTR.dwnarw.AET KD/FL/T/R.dwnarw.VA/ VE/GT/GV FXIa DFTR.dwnarw.VVG
KD/FL/T/R.dwnarw.VA/ VE/GT/GV FXIIa TMTR.dwnarw.VGG NA Kallikrein
SPFR.dwnarw.STGG -/-/FL/RY.dwnarw.SR/RT/-/- FVIIa LQVR.dwnarw.IVGG
NA FIXa PLGR.dwnarw.IVGG -/-/G/R.dwnarw.-/-/-/- FXa
IEGR.dwnarw.TVGG IA/E/GFP/R.dwnarw.STI/ VFS/-/G FIIa (thrombin)
LTPR.dwnarw.SLLV -/-/PLA/R.dwnarw.SAG/-/-/- Elastase-2
LGPV.dwnarw.SGVP -/-/-VIAT.dwnarw.-/-/-/- Granzyme-B
VAGD.dwnarw.SLEE V/-/-/D.dwnarw.-/-/-/- MMP-12 GPAG.dwnarw.LGGA
G/PA/-/G.dwnarw.L/-/G/- MMP-13 GPAG.dwnarw.LRGA
G/P/-/G.dwnarw.L/-/GA/- MMP-17 APLG.dwnarw.LRLR
-/PS/-/-.dwnarw.LQ/-/LT/- MMP-20 PALP.dwnarw.LVAQ NA TEV
ENLYFQ.dwnarw.G ENLYFQ.dwnarw.G/S Enterokinase DDDK.dwnarw.IVGG
DDDK.dwnarw.IVGG Protease 3C LEVLFQ.dwnarw.GP LEVLFQ.dwnarw.GP
(PreScission.TM.) Sortase A LPKT.dwnarw.GSES
L/P/KEAD/T.dwnarw.G/-/EKS/S .dwnarw.indicates cleavage site NA: not
applicable *the listing of multiple amino acids before, between, or
after a slash indicate alternative amino acids that can be
substituted at the position; "-" indicates that any amino acid may
be substituted for the corresponding amino acid indicated in the
middle column
[0219] (a) Pharmacokinetic Properties of CFXTEN
[0220] The invention provides CFXTEN fusion proteins with enhanced
pharmacokinetics compared to the CF not linked to XTEN. The
pharmacokinetic properties of a CF that can be enhanced by linking
a given XTEN to the CF include, but are not limited to, terminal
half-life, area under the curve (AUC), C.sub.max, volume of
distribution, and bioavailability; properties that provide enhanced
utility in the treatment of coagulation factor-related disorders,
diseases and related conditions. As a result of the enhanced
properties, the CFXTEN, when used at the dose and dose regimen
determined to be appropriate for the composition by the methods
described herein, can achieve a circulating concentration resulting
in a desired pharmacologic effect, yet stay within the safety range
for biologically active component of the composition for an
extended period of time compared to a comparable dose of the CF not
linked to XTEN. In such cases, the CFXTEN remains within the
therapeutic window for the fusion protein composition for the
extended period of time compared to a CF not liked to XTEN and
administered to a subject at a comparable dose. As used herein, a
"comparable dose" means a dose with an equivalent moles/kg for the
active CF pharmacophore (e.g., FIX or FVII) that is administered to
a subject in a comparable fashion. It will be understood in the art
that a "comparable dosage" of CFXTEN fusion protein would represent
a greater weight of agent but would have essentially the same
mole-equivalents of CF in the dose of the fusion protein
administered.
[0221] In some embodiments, the CFXTEN with enhanced
pharmacokinetic properties can be a sequence that has at least
about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity compared to a protein sequence
selected from any one of Tables 41, 42, or 43. In other
embodiments, the CFXTEN with enhanced pharmacokinetic properties
can comprise a CF sequence that has at least about 80% sequence
identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%
sequence identity compared to a sequence from Table 1 or from Table
2, linked to one or more XTEN that has at least about 80% sequence
identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%
sequence identity compared to a sequence from Table 4. For the
inventive compositions, CFXTEN with a longer terminal half-life are
generally preferred, so as to improve patient convenience, to
increase the interval between doses and to reduce the amount of
drug required to achieve a sustained effect. In the embodiments
hereinabove described in this paragraph the administration of the
fusion protein results in an improvement in at least one of the
parameters (disclosed herein as being useful for assessing the
subject diseases, conditions or disorders) using a lower unit dose
in moles of fusion protein compared to the corresponding CF
component not linked to the fusion protein and administered at a
comparable unit dose or dose regimen to a subject. In the foregoing
embodiments, the total dose in moles administered to achieve the
improvement is at least about three-fold lower, or at least about
four-fold, or at least about five-fold, or at least about six-fold,
or at least about eight-fold, or at least about 10-fold lower
compared to the corresponding CF component not linked to the fusion
protein.
[0222] As described more fully in the Examples pertaining to
pharmacokinetic characteristics of fusion proteins comprising XTEN,
it was observed that increasing the length of the XTEN sequence
confers a disproportionate increase in the terminal half-life of a
fusion protein comprising the XTEN. Accordingly, the invention
provides CFXTEN fusion proteins comprising XTEN wherein the XTEN is
selected to provide a targeted half-life for the CFXTEN composition
administered to a subject. In some embodiments, the invention
provides monomeric fusion proteins comprising XTEN wherein the XTEN
is selected to confer an increase in the terminal half-life for the
CFXTEN administered to a subject, compared to the corresponding CF
not linked to the fusion protein and administered at a comparable
dose, wherein the increase is at least about two-fold longer, or at
least about three-fold, or at least about four-fold, or at least
about five-fold, or at least about six-fold, or at least about
seven-fold, or at least about eight-fold, or at least about
nine-fold, or at least about ten-fold, or at least about 15-fold,
or at least a 20-fold, or at least a 40-fold, or at least a
80-fold, or at least a 100-fold or greater an increase in terminal
half-life compared to the CF not linked to the fusion protein.
Exogenously administered factor IX has been reported to have a
terminal half-life in humans of approximately 18-24 hours (Morfini,
M. Blood Transfus. (2008) 6(s2): s21-s25) and exogenously
administered factor VII is reported to have a terminal half-life of
approximately 4-6 hours (Klitgaard T, Br J Clin Pharmacol (2008)
65(1):3-11), whereas various CFXTEN compositions disclosed herein
that have been experimentally administered to animals, as described
in the Examples, have resulted in terminal half-life values
considerably longer. In one embodiment, the present invention
provides CFXTEN fusion proteins that exhibits an increase in ACU of
at least about 50%, or at least about 60%, or at least about 70%,
or at least about 80%, or at least about 90%, or at least about a
100%, or at least about 150%, or at least about 200%, or at least
about 300%, or at least about 500%, or at least about 1000%, or at
least about a 2000% compared to the corresponding CF not linked to
the XTEN and administered to a subject at a comparable dose. The
pharmacokinetic parameters of a CFXTEN can be determined by
standard methods involving dosing, the taking of blood samples at
times intervals, and the assaying of the protein using ELISA, HPLC,
radioassay, or other methods known in the art or as described
herein, followed by standard calculations of the data to derive the
half-life and other PK parameters.
[0223] The enhanced PK parameters allow for reduced dosing of the
CFXTEN compositions, compared to CF not linked to XTEN. In some
embodiments, a smaller molar amount of about two-fold less, or
about three-fold less, or about four-fold less, or about five-fold
less, or about six-fold less, or about eight-fold less, or about
10-fold less or greater of the fusion protein is administered in
comparison to the corresponding CF not linked to the XTEN under a
dose regimen needed to maintain hemostasis, and the fusion protein
achieves a comparable area under the curve as the corresponding
molar amount of the CF not linked to the XTEN. In other
embodiments, the fusion protein has a less frequent administration
regimen of about every two days, about every seven days, about
every 14 days, about every 21 days, or about monthly of the fusion
protein administered to a subject, compared to the daily
administration of an otherwise same dose amount of the
corresponding CF not linked to the XTEN, and the fusion protein
achieves a comparable area under the curve as the corresponding CF
not linked to the XTEN. In yet other embodiments, an accumulative
smaller molar amount of about 5%, or about 10%, or about 20%, or
about 40%, or about 50%, or about 60%, or about 70%, or about 80%,
or about 90% less of the fusion protein is administered to a
subject in comparison to the corresponding molar amount of the CF
not linked to the XTEN under a dose regimen needed to maintain
hemostasis, yet the fusion protein achieves at least a comparable
area under the curve as the corresponding CF not linked to the
XTEN. The accumulative smaller molar amount is measure for a period
of at least about one week, or about 14 days, or about 21 days, or
about one month.
[0224] The invention further provides CFXTEN comprising a CF
molecule separated from the XTEN sequence by one or more cleavage
sequences; e.g., a sequence from Table 7. In some embodiments, the
intact CFXTEN composition has less activity but a longer half-life
in its intact form compared to a corresponding CF not linked to the
XTEN, but is designed such that upon administration to a subject,
the CF component is gradually released from the fusion protein by
cleavage at the cleavage sequence(s) by endogenous proteases,
whereupon the CF component exhibits activity, i.e., the ability to
effectively bind to and activate its target coagulation protein
substrate. In non-limiting examples, the CFXTEN with a cleavage
sequence has about 80% sequence identity compared to a sequence
from Table 42, or about 85%, or about 90%, or about 95%, or about
97%, or about 98%, or about 99% sequence identity compared to a
sequence from Table 42. Accordingly, the CFXTEN of the foregoing
embodiments in this paragraph serve as prodrugs or a circulating
depot, resulting in a longer terminal half-life compared to CF not
linked to the XTEN. In such cases, a higher concentration of CFXTEN
can be administered to a subject, compared to the corresponding CF
not linked to XTEN because a smaller proportion of the circulating
composition is active.
[0225] (b) Pharmacology and Pharmaceutical Properties of CFXTEN
[0226] The present invention provides CFXTEN compositions
comprising CF covalently linked to XTEN that can have enhanced
properties compared to CF not linked to XTEN, as well as methods to
enhance the therapeutic and/or biologic activity or effect of the
respective two CF components of the compositions. In addition, the
invention provides CFXTEN compositions with enhanced properties
compared to those art-known fusion proteins containing albumin,
immunoglobulin polypeptide partners, polypeptides of shorter length
and/or polypeptide partners with repetitive sequences. In addition,
CFXTEN fusion proteins provide significant advantages over chemical
conjugates, such as pegylated constructs, notably the fact that
recombinant CFXTEN fusion proteins can be made in bacterial cell
expression systems, which can reduce time and cost at both the
research and development and manufacturing stages of a product, as
well as result in a more homogeneous, defined product with less
toxicity for both the product and metabolites of the CFXTEN
compared to pegylated conjugates.
[0227] As therapeutic agents, the CFXTEN possesses a number of
advantages over therapeutics not comprising XTEN, including one or
more of the following non-limiting exemplary enhanced properties:
increased solubility, increased thermal stability, reduced
immunogenicity, increased apparent molecular weight, reduced renal
clearance, reduced proteolysis, reduced metabolism, enhanced
therapeutic efficiency, a lower effective therapeutic dose,
increased bioavailability, increased time between dosages capable
of maintaining blood levels within the therapeutic window for the
CF, a "tailored" rate of absorption when administered
subcutaneously or intramuscularly, enhanced lyophilization
stability, enhanced serum/plasma stability, increased terminal
half-life, increased solubility in blood stream, decreased binding
by neutralizing antibodies, decreased active clearance, reduced
side effects, retention of substrate binding affinity, stability to
degradation, stability to freeze-thaw, stability to proteases,
stability to ubiquitination, ease of administration, compatibility
with other pharmaceutical excipients or carriers, persistence in
the subject, increased stability in storage (e.g., increased
shelf-life), reduced toxicity in an organism or environment and the
like. The net effect of the enhanced properties is that the use of
a CFXTEN composition can result in enhanced therapeutic and/or
biologic effect compared to a CF not linked to XTEN or result in
improved patient compliance when administered to a subject with a
coagulation factor-related disease or disorder.
[0228] Specific assays and methods for measuring the physical and
structural properties of expressed proteins are known in the art,
including methods for determining properties such as protein
aggregation, solubility, secondary and tertiary structure, melting
properties, contamination and water content, etc. Such methods
include analytical centrifugation, EPR, HPLC-ion exchange,
HPLC-size exclusion, HPLC-reverse phase, light scattering,
capillary electrophoresis, circular dichroism, differential
scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size
exclusion, IR, NMR, Raman spectroscopy, refractometry, and
UVNisible spectroscopy; several of which are applied to the
inventive CFXTEN as described in the Examples. Additional methods
are disclosed in Arnau et al, Prot Expr and Purif (2006) 48, 1-13.
Application of these methods to elucidate the enhanced properties
of the compositions of the invention is within the grasp of a
person skilled in the art.
[0229] In one embodiment, XTEN as a fusion partner increases the
solubility of the CF payload. Accordingly, where enhancement of the
pharmaceutical or physicochemical properties of the CF is
desirable, such as the degree of aqueous solubility or stability,
the length and/or the motif family composition of the XTEN
sequences incorporated into the fusion protein may each be selected
to confer a different degree of solubility and/or stability on the
respective fusion proteins such that the overall pharmaceutical
properties of the CFXTEN composition are enhanced. The CFXTEN
fusion proteins can be constructed and assayed, using methods
described herein, to confirm the physicochemical properties and the
XTEN adjusted, as needed, to result in the desired properties. In
one embodiment, the XTEN sequence of the CFXTEN is selected such
that the fusion protein has an aqueous solubility that is within at
least about 25% greater compared to a CF not linked to the fusion
protein, or at least about 30%, or at least about 40%, or at least
about 50%, or at least about 75%, or at least about 100%, or at
least about 200%, or at least about 300%, or at least about 400%,
or at least about 500%, or at least about 1000% greater than the
corresponding CF not linked to the fusion protein.
[0230] The invention provides methods to produce and recover
expressed CFXTEN from a host cell with enhanced solubility and ease
of recovery compared to CF not linked to XTEN. In some embodiments,
the method includes the steps of transforming a eukaryotic host
cell with a polynucleotide encoding a CFXTEN with one or more XTEN
components of cumulative sequence length greater than about 200, or
greater than about 400, or greater than about 600, or greater than
about 800 amino acid residues, expressing the CFXTEN fusion protein
in the host cell, and recovering the expressed fusion protein in
soluble form. In the embodiments hereinabove described in this
paragraph, the XTEN of the CFXTEN fusion proteins can have at least
about 80% sequence identity, or about 90%, or about 91%, or about
92%, or about 93%, or about 94%, or about 95%, or about 96%, or
about 97%, or about 98%, or about 99%, to about 100% sequence
identity compared to one or more XTEN selected from Table 4 and the
CF can have at least about 80% sequence identity, or about 90%, or
about 91%, or about 92%, or about 93%, or about 94%, or about 95%,
or about 96%, or about 97%, or about 98%, or about 99%, or 100%
sequence identity compared to a CF selected from Table 1 or Table 2
and the CFXTEN components can be in an N- to C-terminus
configuration selected from formulas I-VIII.
[0231] In one embodiment, the invention provides CFXTEN
compositions and methods to produce the compositions that can
maintain the CF component within a therapeutic window for a greater
period of time compared to comparable dosages of the corresponding
CF not linked to XTEN. It will be understood in the art that a
"comparable dosage" of CFXTEN fusion protein would represent a
greater weight of agent but would have the same approximate
mole-equivalents of CF in the dose of the fusion protein and/or
would have the same approximate molar concentration relative to the
CF. The method to produce the compositions that can maintain the CF
component within a therapeutic window includes the steps of
selecting the XTEN appropriate for conjugation to a CF to provide
the desired pharmacokinetic properties in view of a given dose and
dose regimen, administration of the CFXTEN to subjects in need
thereof, followed by assays to verify the pharmacokinetic
properties, the activity of the CFXTEN fusion protein, and the
safety of the administered composition. By the methods, CFXTEN
provided herein allow for increased efficacy of the administered
composition by maintaining the circulating concentrations of the CF
within the therapeutic window for an enhanced period of time. As
used herein, "therapeutic window" means that the amount of drug or
biologic as a blood or plasma concentration range, which provides
efficacy or a desired pharmacologic effect over time for the
disease or condition without unacceptable toxicity, i.e., the range
of the circulating blood concentrations between the minimal amount
to achieve any positive therapeutic effect and the maximum amount
which results in a response that is the response immediately before
toxicity to the subject (at a higher dose or concentration).
Additionally, therapeutic window generally encompasses an aspect of
time; the maximum and minimum concentration that results in a
desired pharmacologic effect over time that does not result in
unacceptable toxicity or adverse events. A dosed composition that
stays within the therapeutic window for the subject could also be
said to be within the "safety range."
[0232] The characteristics of CFXTEN compositions of the invention,
including functional characteristics or biologic and pharmacologic
activity and parameters that result, can be determined by any
suitable screening assay known in the art for measuring the desired
characteristic. The invention provides methods to assay the CFXTEN
fusion proteins of differing composition or configuration in order
to provide CFXTEN with the desired degree of biologic and/or
therapeutic activity, as well as safety profile. Specific in vivo
and ex vivo biological assays are used to assess the activity of
each configured CFXTEN and/or CF component to be incorporated into
CFXTEN, including but not limited to the assays of the Examples,
those assays of Table 40, as well as the following assays or other
such assays known in the art for assaying the properties and
effects of CF. Functional assays can be conducted that allow
determination of coagulation activity, such as prothrombin (PT) and
activated partial prothrombin (aPTT) assays (Belaaouaj A A et al.,
J. Biol. Chem. (2000) 275:27123-8; Diaz-Collier J A. Haemost (1994)
71:339-46), blood clotting time (WBCT), thrombelastography, or
bleeding time assays. Other possible assays may determine the
binding affinity of a CFXTEN for the target substrate of the
corresponding CF can be assayed using binding or competitive
binding assays, such as Biacore assays with chip-bound receptors or
binding proteins or ELISA assays, as described in U.S. Pat. No.
5,534,617, assays described in the Examples herein, radio-receptor
assays, or other assays known in the art. The foregoing assays can
also be used to assess CF sequence variants (assayed as single
components or as CFXTEN fusion proteins) and can be compared to the
native CF to determine whether they have the same degree of
biologic activity as the native CF, or some fraction thereof such
that they are suitable for inclusion in CFXTEN; e.g., at least
about 60%, or at least about 70%, or at least about 80%, or at
least about 90% of the activity compared to the native CF.
[0233] Dose optimization is important for all drugs, especially for
those with a narrow therapeutic window. For example, a standardized
single dose of CF for all patients presenting with a diverse
symptoms or abnormal clinical parameters may not always be
effective. A consideration of these factors is well within the
purview of the ordinarily skilled clinician for the purpose of
determining the therapeutically or pharmacologically effective
amount of the CFXTEN, versus that amount that would result in
unacceptable toxicity and place it outside of the safety range, or
insufficient potency such that clinical improvement is not
achieved.
[0234] In many cases, the therapeutic window for CF in subjects of
different ages or degree of disease have been established and are
available in published literature or are stated on the drug label
for approved products containing the CF. In other cases, the
therapeutic window can be established for new compositions,
including those CFXTEN of the disclosure. The methods for
establishing the therapeutic window for a given composition are
known to those of skill in the art (see, e.g., Goodman &
Gilman's The Pharmacological Basis of Therapeutics,
11.sup.thEdition, McGraw-Hill (2005)). For example, by using
dose-escalation studies in subjects with the target disease or
disorder to determine efficacy or a desirable pharmacologic effect,
appearance of adverse events, and determination of circulating
blood levels, the therapeutic window for a given subject or
population of subjects can be determined for a given drug or
biologic, or combinations of biologics or drugs. The dose
escalation studies can evaluate the activity of a CFXTEN through
metabolic studies in a subject or group of subjects that monitor
physiological or biochemical parameters, as known in the art or as
described herein for one or more parameters associated with the
metabolic disease or disorder, or clinical parameters associated
with a beneficial outcome for the particular indication, together
with observations and/or measured parameters to determine the no
effect dose, adverse events, maximum tolerated dose and the like,
together with measurement of pharmacokinetic parameters that
establish the determined or derived circulating blood levels. The
results can then be correlated with the dose administered and the
blood concentrations of the therapeutic that are coincident with
the foregoing determined parameters or effect levels. By these
methods, a range of doses and blood concentrations can be
correlated to the minimum effective dose as well as the maximum
dose and blood concentration at which a desired effect occurs and
above which toxicity occurs, thereby establishing the therapeutic
window for the dosed therapeutic. Blood concentrations of the
fusion protein (or as measured by the CF component) above the
maximum is considered outside the therapeutic window or safety
range. Thus, by the foregoing methods, a C.sub.min blood level is
established, below which the CFXTEN fusion protein would not have
the desired pharmacologic effect, and a C.sub.max blood level is
established that would represent the highest circulating
concentration before reaching a concentration that would elicit
unacceptable side effects, toxicity or adverse events, placing it
outside the safety range for the CFXTEN. With such concentrations
established, the frequency of dosing and the dosage can be further
refined by measurement of the C.sub.max and C.sub.min to provide
the appropriate dose and dose frequency to keep the fusion
protein(s) within the therapeutic window.
[0235] One of skill in the art can, by the means disclosed herein
or by other methods known in the art, confirm that the administered
CFXTEN remains in the therapeutic window for the desired interval
or requires adjustment in dose or length or sequence of XTEN.
Further, the determination of the appropriate dose and dose
frequency to keep the CFXTEN within the therapeutic window
establishes the therapeutically effective dose regimen; the
schedule for administration of multiple consecutive doses using a
therapeutically effective dose of the fusion protein to a subject
in need thereof resulting in consecutive C.sub.max peaks and/or
C.sub.min troughs that remain within the therapeutic window and
results in an improvement in at least one measured parameter
relevant for the target disease, disorder or condition. In some
cases, the CFXTEN administered at an appropriate dose to a subject
results in blood concentrations of the CFXTEN fusion protein that
remains within the therapeutic window for a period at least about
two-fold longer compared to the corresponding CF not linked to XTEN
and administered at a comparable dose; alternatively at least about
three-fold longer; alternatively at least about four-fold longer;
alternatively at least about five-fold longer; alternatively at
least about six-fold longer; alternatively at least about
seven-fold longer; alternatively at least about eight-fold longer;
alternatively at least about nine-fold longer or at least about
ten-fold longer or greater compared to the corresponding CF not
linked to XTEN and administered at a comparable dose. As used
herein, an "appropriate dose" means a dose of a drug or biologic
that, when administered to a subject, would result in a desirable
therapeutic or pharmacologic effect and/or a blood concentration
within the therapeutic window.
[0236] In one embodiment, the CFXTEN administered at a
therapeutically effective dose regimen results in a gain in time of
at least about three-fold longer; alternatively at least about
four-fold longer; alternatively at least about five-fold longer;
alternatively at least about six-fold longer; alternatively at
least about seven-fold longer; alternatively at least about
eight-fold longer; alternatively at least about nine-fold longer or
at least about ten-fold longer between at least two consecutive
C.sub.max peaks and/or C.sub.min troughs for blood levels of the
fusion protein compared to the corresponding biologically active
protein of the fusion protein not linked to the fusion protein and
administered at a comparable dose regimen to a subject. In another
embodiment, the CFXTEN administered at a therapeutically effective
dose regimen results in a comparable improvement in one, or two, or
three or more measured parameter using less frequent dosing or a
lower total dosage in moles of the fusion protein of the
pharmaceutical composition compared to the corresponding
biologically active protein component(s) not linked to the fusion
protein and administered to a subject using a therapeutically
effective dose regimen for the CF. The measured parameters include
any of the clinical, biochemical, or physiological parameters
disclosed herein, or others known in the art for assessing subjects
with coagulation factor-related disorders.
[0237] In some embodiments, the CFXTEN fusion proteins of the
invention retain at least about 0.05%, or about 0.1%, or about 1%,
or about 10%, or about 20%, or about 30%, or about 40%, or about
50%, or about 60%, or about 70%, or about 80%, or about 90%, or
about 95%, or about 98%, or about 99% percent of the biological
activity of the corresponding CF not linked to the fusion protein
with regard to an in vitro biologic activity or pharmacologic
effect known or associated with the use of the native CF in the
treatment and prevention of coagulation factor-related diseases,
disorders, and conditions. Non-limiting examples of parameters or
physiologic effects that can be assayed to assess the retained
activity of the CFXTEN fusion proteins include prothrombin time
(PT), activated partial thromboplastin time (aPTT), bleeding time,
whole blood clotting time (WBCT), and thrombelastography. In some
embodiments, the activity of the CF component is manifested by the
intact CFXTEN fusion protein, while in other cases the activity of
the CF component is primarily manifested upon cleavage and release
of the CF from the fusion protein by action of a protease that acts
on a cleavage sequence incorporated into the CFXTEN fusion protein,
embodiments of which are disclosed above. In the foregoing, the
CFXTEN is designed to reduce the binding affinity of the CF
component for the coagulation substrate when linked to the XTEN but
have restored or increased affinity when released from XTEN through
the cleavage of cleavage sequence(s) incorporated into the CFXTEN
sequence, as described more fully above. In one embodiment of the
foregoing, the invention provides an isolated fusion protein
comprising a FIX linked to XTEN by a cleavage sequence, wherein the
fusion protein is substantially inactive prior to cleavage and
wherein the FIX released from the fusion protein by proteolytic
cleavage at the cleavage sequence has biological activity that is
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 90%, or at least about 95% as active compared to
native FIX not linked to XTEN.
[0238] In other cases, the CFXTEN can be designed to reduce active
clearance of the CFXTEN to increase the terminal half-life of
CFXTEN administered to a subject, while still retaining biological
activity. The clearance mechanisms to remove CF from the
circulation have yet to be fully elucidated. Uptake, elimination,
and inactivation of CFs can occur in the circulatory system as well
as in the extravascular space. Coagulation factors are complex
proteins that interact with a large number of other proteins,
lipids, and receptors, and many of these interactions can
contribute to the elimination of CFs from the circulation. For
example, clearance mechanisms for FVII, a heterogeneously
glycosylated protein, may include clearance by the liver. The
effects of the gamma-carboxy glutamic acid (Gla) domain and the
sialic acid content of the protein on FVIIa clearance have been
investigated using a perfused liver model, with results suggesting
that carbohydrate receptors (e.g. the asialoglycoprotein receptor,
ASGPR) may play a role in FVIIa clearance. (Appa, R. S., et al.
Thromb Haemost. (2010, epub May 27) 104(2)). In addition, CF can be
lost through extravasation and rapid active clearance, which is
reflected in the generally poor bioavailability of intravenously
administered coagulation factors such as factor VIIa (see NovoSeven
package insert). It is believed that the CFXTEN of the present
invention has comparatively higher bioavailability achieved by
reduced active clearance and/or by reduced extravasation by
increasing the hydrodynamic radius, or apparent size, of the
molecule by the addition of unstructured XTEN to the coagulation
factor. In one embodiment, the invention provides CFXTEN that
reduce clearance of the fusion protein by linking one or more XTEN
to the CF component of the fusion protein, wherein the fusion
protein has an increase in apparent molecular weight factor of at
least about four-fold, or at least about five-fold, or at least
about six-fold, or at least about seven-fold, or at least about
eight-fold, or at least about ten-fold, or at least about
twelve-fold, or at least about fifteen-fold, and wherein the
terminal half-life of the CFXTEN when administered to a subject is
increased at least about two-fold, or at least about four-fold, or
at least about eight-fold, or at least about 10-fold, or at least
about 20-fold, or at least about 30-fold, or at least about
40-fold, or at least about 50-fold, or at least about 60-fold, or
at least about 70-fold, or at least about 80-fold or more compared
to the corresponding CF not linked to XTEN. In the foregoing
embodiment, wherein at least two XTEN molecules are incorporated
into the CFXTEN, the XTEN can be identical or they can be of a
different sequence composition (and net charge) or length.
Non-limiting examples of the foregoing embodiment with two XTEN
linked to a single FVII are illustrated in FIG. 6, and include the
constructs (expressed using the domains of FVII)
Gla-EGF1-EGF2-AE144-Protease-AE864 or
Gla-EGF1-AE288-EGF2-Protease-AE864 (wherein the AE XTEN components
have approximately a 17% net charge due to incorporated glutamic
acid), Gla-EGF1-EGF2-AG144-Protease-AG864 or
Gla-EGF1-AG144-EGF2-Protease-AE864 (wherein the AG XTEN components
have approximately no net charge). Not to be bound by a particular
theory, the XTEN of the CFXTEN compositions with the higher net
charge are expected, as described above, to have less non-specific
interactions with various negatively-charged surfaces such as blood
vessels, tissues, or various receptors, which would further
contribute to reduced active clearance. Conversely, the XTEN of the
CFXTEN compositions with a low (or no) net charge are expected to
have a higher degree of interaction with surfaces that, while
contributing to active clearance, can potentiate the activity of
the associated coagulation factor, given the known contribution of
cell (e.g., platelets) and vascular surfaces to the coagulation
process and the intensity of activation of coagulation factors
(Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London,
F., et al. Biochemistry (2000) 39(32):9850-9858). Thus, the
invention provides CFXTEN in which the degree of potency,
bioavailability, and half-life can be tailored by the selection and
placement of the type and length of the XTEN in the CFXTEN
compositions. Accordingly, the invention contemplates compositions
in which a CF from Table 1 or from Table 2 and XTEN from Table 4
are substituted for the respective components of the foregoing
examples, and are produced, for example, in a configuration from
Table 6 or from formulas I-VII such that the construct has reduced
clearance compared to an alternative configuration of the
respective components. In some embodiments, the foregoing method
for increasing the terminal half-life provides configured CFXTEN
that can result in an increase in the terminal half-life of at
least about 30%, or about 50%, or about 75%, or about 100%, or
about 150%, or about 200%, or about 300%, or about 400% or more
compared to the half-life of a CFXTEN in a second configuration
where active clearance is not reduced. The invention further takes
advantage of the fact that certain ligands wherein reduced binding
to a clearance receptor, either as a result of a decreased on-rate
or an increased off-rate, may be effected by the obstruction of
either the N- or C-terminus and using that terminus as the linkage
to another polypeptide of the composition, whether another molecule
of a CF, an XTEN, or a spacer sequence results in the reduced
binding. The choice of the particular configuration of the CFXTEN
fusion protein reduces the degree of binding to a clearance
receptor such that a reduced rate of active clearance is
achieved.
[0239] In cases where a reduction in active clearance is desired
but retention of at least a portion of the biological activity is
also desired, the CFXTEN is designed to retain sufficient biologic
activity for the intact molecule. Thus, in one embodiment, the
invention provides a CFXTEN configured such that the biologic
activity of the CFXTEN is in the range of about 0.01%-40%, or about
0.01%-30%, or about 0.01%-20%, or about 0.01%-10 of the biological
activity compared to the corresponding native coagulation factor.
The biological activity of the configured CFXTEN is thus reduced by
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 90%, or at least about 95%, or at least about
99%, or at least about 99.99% as compared to the biological
activity of the corresponding native coagulation factor not linked
to XTEN, determined under comparable conditions. In the foregoing
embodiments, the biological activity of the configured CFXTEN for
the target receptor is "substantially reduced" compared to a
corresponding native CF not linked to XTEN. Accordingly, the
present invention provides compositions and methods to produce
compositions with reduced biological activity but increased
half-life by configuring the CFXTEN, examples of which are provided
above, so as to be able to provide a desired in vivo biological
response yet avoid active clearance mechanisms. The increased
half-life permits higher dosages and reduced frequency of dosing
compared to CF not linked to XTEN or compared to CFXTEN
configurations wherein the fusion protein is subject to coagulation
factor clearance mechanisms.
VI). Uses of the Compositions of the Present Invention
[0240] In another aspect, the invention provides a method for
achieving a beneficial effect in bleeding disorders and/or in a
coagulation factor-related disease, disorder or condition mediated
by FIX or FVII. As used herein, "coagulation factor-related
diseases, disorders or conditions" is intended to include, but is
not limited to bleeding disorders (e.g., defective platelet
function, thrombocytopenia or von Willebrand's disease),
coagulopathies (any disorder of blood coagulation, including
coagulation factor deficiencies), hemophilia B (aka Christmas
disease), factor IX-related bleeding disorders, factor VII
deficiency, hemophilia A, vascular injury, uncontrolled bleeding in
subjects not suffering from hemophilia, bleeding from trauma or
surgery, bleeding due to anticoagulant therapy, and bleeding due to
liver disease or conditions that can be ameliorated or corrected by
administration of FIX or FVII to a subject. The present invention
addresses disadvantages and/or limitations of other methods of
treatment using factor IX or factor VII preparations that have a
relatively short terminal half-life and/or a narrow therapeutic
window.
[0241] In some embodiments, the invention provides methods for
treating a subject, such as a human, with a coagulation
factor-related disease, disorder or condition comprising the step
of administering to the subject a therapeutically- or
prophylactically-effective amount of an CFXTEN wherein said
administration results in the improvement of one or more
biochemical or physiological parameters or clinical endpoints
associated with the coagulation factor-related disease, disorder or
condition. In one embodiment of the foregoing, the CFXTEN comprises
a FVII. In another embodiment of the foregoing, the CFXTEN
comprises a FIX. The effective amount produces a beneficial effect
in helping to treat (e.g., cure or reduce the severity) or prevent
(e.g., reduce the likelihood of onset or severity) a coagulation
factor-related disease, disorder or condition. As used herein,
"treating" means administering a drug or a biologic (e.g., a
CFXTEN) to achieve an improvement in an existing disease, disorder
or condition or preventing the occurrence of a disease, disorder or
condition (including prophylaxis). A therapeutically-effective
amount of a CFXTEN fusion protein can be that amount of composition
that, when administered as a single or as repeated doses to a
subject, leads to improvements in or amelioration of the underlying
disease, disorder or condition, or improvements in signs or
symptoms or physiologic parameters associated with the underlying
disease, disorder or condition.
[0242] Hemostasis is regulated by multiple protein factors, and
such proteins, as well as analogues thereof, have found utility in
the treatment of coagulation factor-related diseases, disorders and
conditions. However, the use of commercially-available coagulation
factors has met with less than optimal success in the management of
subjects afflicted with such diseases, disorders and conditions. In
particular, dose optimization and frequency of dosing is important
for coagulation factors used in the treatment or prevention of
bleeding episodes in coagulation factor-related diseases,
disorders, or conditions, or uncontrolled bleeding in subjects not
suffering from hemophilia. The fact that coagulation factors have a
short half-life necessitates frequent dosing in order to achieve
clinical benefit, which results in difficulties in the management
of such patients.
[0243] The invention provides methods of treatment comprising
administering a CFXTEN composition to a subject suffering from or
at risk of developing a coagulation factor-related disease,
disorder or condition, wherein the administration results in the
improvement of one or more biochemical or physiological parameters
or clinical endpoints associated with the condition. In one
embodiment, the method of treatment comprises administering a
therapeutically-effective amount of an CFXTEN composition to a
subject suffering from hemophilia A wherein the administration
results in the improvement of one or more biochemical or
physiological parameters or clinical endpoints associated with the
condition. In another embodiment, the method of treatment comprises
administering a therapeutically-effective amount of an CFXTEN
composition to a subject suffering from hemophilia B wherein the
administration results in the improvement of one or more
biochemical or physiological parameters or clinical endpoints
associated with the condition. In another embodiment, the method of
treatment comprises administering a therapeutically-effective
amount of an CFXTEN composition to a subject suffering from factor
VII deficiency wherein said administration results in the
improvement of one or more biochemical or physiological parameters
or clinical endpoints associated with the condition. In another
embodiment, the method of treatment comprises administering a
therapeutically-effective amount of an CFXTEN composition to a
subject suffering from or at risk of developing uncontrolled
bleeding wherein the administration results in the improvement of
one or more biochemical or physiological parameters or clinical
endpoints associated with the condition. In most instances, the
embodiments of the disclosed method of treatments utilizing a
CFXTEN comprising a FVII are compositions in which the FVII has
been activated; i.e., FVIIa. However, the invention also
contemplates CFXTEN compositions in which the FVII has not been
activated. Because of the comparatively long-half life of CFXTEN
comprising FVII, it is believed that compositions comprising the
inactive form of FVII that can be activated by mammalian endogenous
proteases (because they include one or more cleavage sequences;
e.g., the sequences of Table 7) or the fusion protein undergoes
autoactivation such that 1) a bolus quantity of activated form of
FVII is available by activation via clotting proteins of the
intrinsic coagulation cascade that has been initiated; or 2) a
persistent quantity of activated form of FVII is available by
activation via proteases that are persistently or transiently
present in the circulation; e.g., MMP-12, MMP-17, etc.
Thus, the invention provides a method of treatment for a subject
with a coagulation factor-related disease, disorder or conditions
comprising administration of a CFXTEN comprising a FVII variant (as
described above) wherein the FVII is not activated but has one or
more cleavage sequences that, when cleaved by an endogenous
protease, converts the FVII component to the activated form. In one
embodiment of the foregoing, the method utilizes a CFXTEN
composition that has a terminal half-life of at least about 12 h,
or at least about 24 h, or at least about 48 h, or at least about
48 h, or at least about 96 h, or at least about 144 h, or at least
about 160 h. Accordingly, the method represents a means to treat
subjects with certain forms of chronic coagulopathies with what is
essentially a "prodrug" form of FVII.
[0244] In some embodiments, administration of the CFXTEN to a
subject results in an improvement in one or more of the
biochemical, physiologic, or clinical parameters that is of greater
magnitude than that of the corresponding CF component not linked to
XTEN, determined using the same assay or based on a measured
clinical parameter. In other embodiments, administration of the
CFXTEN to a subject using a therapeutically effective dose regimen
results in activity in one or more of the biochemical, physiologic,
or clinical parameters that is of longer duration than the activity
of the corresponding CF component not linked to XTEN, determined
using that same assay or based on a measured clinical parameter. In
one embodiment, the administration of a therapeutically effective
amount of a CFXTEN comprising a FVII to a subject results in a
reduction in prothrombin time at about 2-7 days after
administration of at least about 5%, or about 10%, or about 20%, or
about 30%, or about 40%, or about 50%, or about 60%, or about 70%,
or more in the subject compared to the prothrombin time in a
subject at a comparable time after administration of a comparable
amount of FVII not linked to XTEN. In another embodiment, the
administration of a CFXTEN comprising a FVII to a subject using a
therapeutically effective amount results in maintenance of
prothrombin times within 30% of normal in the subject for a period
of time that is at least two-fold, or about three-fold, or at least
about four-fold longer compared to a comparable dose regimen of
FVII not linked to XTEN administered to a subject. In another
embodiment, the administration of a therapeutically effective
amount of a CFXTEN comprising a FIX to a subject results in a
reduction in the activated partial prothrombin time at about 2-7
days after administration of at least about 5%, or about 10%, or
about 20%, or about 30%, or about 40%, or about 50%, or about 60%,
or about 70%, or more in the subject compared to the activated
partial prothrombin time in a subject at a comparable time after
administration of a comparable amount of FIX not linked to XTEN. In
another embodiment, the administration of a CFXTEN comprising a FIX
to a subject using a therapeutically effective amount results in
maintenance of activated partial prothrombin times within 30% of
normal in the subject for a period of time that is at least
two-fold, or at least about three-fold, or at least about four-fold
longer compared to a comparable dose regimen of FIX not linked to
XTEN administered to a subject. In another embodiment, the
administration of a CFXTEN comprising a FVII to a subject using a
therapeutically effective amount results in maintenance of a
bleeding time (in a bleeding time assay) within 30% of normal in
the subject for a period of time that is at least two-fold, or
about three-fold, or at least about four-fold longer compared to a
comparable amount of FVII not linked to XTEN administered to a
subject. In another embodiment, the administration of a CFXTEN
comprising a FIX to a subject using a therapeutically effective
amount results in maintenance of a bleeding time (in a bleeding
time assay) within 30% of normal in the subject for a period of
time that is at least two-fold, or about three-fold, or at least
about four-fold longer compared to a comparable amount of FIX not
linked to XTEN administered to a subject.
[0245] As a result of the enhanced PK parameters of CFXTEN, as
described herein, the CF is administered using longer intervals
between doses compared to the corresponding CF not linked to XTEN
to prevent, treat, alleviate, reverse or ameliorate symptoms or
clinical abnormalities of the coagulation factor-related disease,
disorder or condition or prolong the survival of the subject being
treated. In a particular application, CFXTEN comprising FVII have
utility in the treatment of hemophilia A and hemophilia B.
[0246] It has been observed that FVIIa administered in high
concentrations can function as a bypassing agent resulting in the
activation of FX even in the absence of FIX or FVIII. In order to
act as a bypassing agent FVIIa has to be dosed at concentrations
that exceed the level of FVIIa in healthy people by approximately
100-fold. These levels are generally safe because FVIIa has low
activity in the absence of tissue factor (TF), to which FVII binds.
Tissue factor is released or presented on injured tissues which
triggers clotting via the extrinsic system. The circulation
half-life of FVIIa is in part limited by its inactivation by
antithrombin (AT). Antithrombin can not bind to FVII but only to
FVIIa. Thus, in one embodiment, the invention provides a method of
treating hemophilia A or B by administering an amount of CFXTEN
comprising an activated form of FVII, wherein the ability to
activate FX in the circulation of a subject is maintained for a
period that is at least about two-fold longer, or at least about
three-fold, or at least about four-fold, or at least about
five-fold, or at least about 10-fold, or at least about 20-fold
longer compared to FVII not linked to XTEN and administered to a
comparable subject at a comparable dose. The current invention
further provides CFXTEN fusion proteins comprising FVII linked to
XTEN that can not be inactivated by AT by more than about 5% prior
to its activation to FVIIa-XTEN. In one embodiment, the invention
provides a method of treatment comprising administering a CFXTEN
with a FVII component that is not activated, wherein the CFXTEN
serves as a circulating depot wherein the area under the curve for
the FVII that is activated to FVIIa and not complexed with AT is at
least about two-fold greater, or at least about three-fold, or at
least about four-fold, or at least about five-fold, or at least
about 10-fold, or at least about 20-fold greater than a FVII not
linked to XTEN and administered at a comparable dose.
[0247] In some embodiments of the method of treatment, (i) a
smaller molar amount of (e.g. of about two-fold less, or about
three-fold less, or about four-fold less, or about five-fold less,
or about six-fold less, or about eight-fold less, or about
10-fold-less or greater) the fusion protein is administered in
comparison to the corresponding CF not linked to the XTEN under an
otherwise same dose regimen, and the fusion protein achieves a
comparable therapeutic effect as the corresponding CF not linked to
the XTEN; (ii) the fusion protein is administered less frequently
(e.g., every two days, about every seven days, about every 14 days,
about every 21 days, or about, monthly) in comparison to the
corresponding CF not linked to the XTEN under an otherwise same
dose amount, and the fusion protein achieves a comparable
therapeutic effect as the corresponding CF not linked to the XTEN;
or (iii) an accumulative smaller molar amount (e.g. about 5%, or
about 10%, or about 20%, or about 40%, or about 50%, or about 60%,
or about 70%, or about 80%, or about 90% less) of the fusion
protein is administered in comparison to the corresponding CF not
linked to the XTEN under the otherwise same dose regimen the fusion
protein achieves a comparable therapeutic effect as the
corresponding CF not linked to the XTEN. The accumulative smaller
molar amount is measure for a period of at least about one week, or
about 14 days, or about 21 days, or about one month. The
therapeutic effect can be determined by any of the measured
parameters or clinical endpoints described herein.
[0248] The methods of the invention includes administration of
consecutive doses of a therapeutically effective amount of the
CFXTEN for a period of time sufficient to achieve and/or maintain
the desired parameter or clinical effect, and such consecutive
doses of a therapeutically effective amount establishes the
therapeutically effective dose regimen for the CFXTEN, i.e., the
schedule for consecutively administered doses of the fusion protein
composition, wherein the doses are given in therapeutically
effective amounts to result in a sustained beneficial effect on any
clinical sign or symptom, aspect, measured parameter or
characteristic of a coagulation factor-related disease state or
condition, including, but not limited to, those described herein.
In one embodiment, the method comprises administering a
therapeutically-effective amount of a pharmaceutical composition
comprising a CFXTEN fusion protein composition comprising a CF
linked to an XTEN sequence(s) and at least one pharmaceutically
acceptable carrier to a subject in need thereof that results in
greater improvement in at least one parameter, physiologic
condition, or clinical outcome mediated by the CF component(s)
(non-limiting examples of which are described above) compared to
the effect mediated by administration of a pharmaceutical
composition comprising a CF not linked to XTEN and administered at
a comparable dose. In one embodiment, the pharmaceutical
composition is administered at a therapeutically effective dose. In
another embodiment, the pharmaceutical composition is administered
using multiple consecutive doses using a therapeutically effective
dose regimen (as defined herein) for the length of the dosing
period.
[0249] A therapeutically effective amount of the CFXTEN varies
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the administered
fusion protein to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the CFXTEN are outweighed by the
therapeutically beneficial effects. A prophylactically effective
amount refers to an amount of CFXTEN required for the period of
time necessary to achieve the desired prophylactic result; e.g.,
delayed onset of a bleeding episode. In the methods of treatment,
the dose of the CFXTEN that is administered to a subject ranges
from about 0.5 mg to 1000 mg/dose, or from about 1 mg to 400
mg/dose, or from about 10 mg to about 300 mg/dose for a 70 kg
subject as loading and maintenance doses, depending on the weight
of the subject and the severity of the condition.
[0250] The method of treatment comprises administration of a CFXTEN
using a therapeutically effective dose regimen to effect
improvements in one or more parameters associated with coagulation
factor diseases, disorders or conditions. In some embodiments,
administration of the CFXTEN to a subject results in an improvement
in one or more of the biochemical, physiologic, or clinical
parameters that is of greater magnitude than that of the
corresponding CF component not linked to XTEN, determined using the
same assay or based on a measured clinical parameter. In other
embodiments, administration of the CFXTEN to a subject using a
therapeutically effective dose regimen results in activity in one
or more of the biochemical, physiologic, or clinical parameters
that is of longer duration than the activity of one of the single
CF components not linked to XTEN, determined using that same assay
or based on a measured clinical parameter. In one embodiment of the
foregoing, the administration of the CFXTEN to a subject using a
therapeutically effective dose regimen results in an improvement in
prothrombin time or activated partial thromboplastin time of at
least about 10%, or about 20%, or about 30%, or about 40%, or about
50%, or about 60%, or about 70%, or about 80%, or about 90%, or
about 100% or more in the subject compared to a comparable dose of
CF not linked to XTEN administered to a subject. In another
embodiment of the foregoing, the administration of the CFXTEN to a
subject using a therapeutically effective dose regimen results in
decreased instances of bleeding in the subject of at least about
10%, or about 20%, or about 30%, or about 40%, or about 50% or more
compared to a comparable dose regimen of CF not linked to XTEN
administered to a subject.
[0251] The invention further contemplates that CFXTEN used in
accordance with the methods provided herein is administered in
conjunction with other treatment methods and compositions (e.g.,
other coagulation proteins) useful for treating coagulation
factor-related diseases, disorders, and conditions, or conditions
for which coagulation factor is adjunctive therapy; e.g., bleeding
episodes due to injury or surgery.
[0252] In another aspect, the invention provides a method of
designing the CFXTEN compositions with desired pharmacologic or
pharmaceutical properties. The CFXTEN fusion proteins are designed
and prepared with various objectives in mind (compared to the CF
components not linked to the fusion protein), including improving
the therapeutic efficacy for the treatment of coagulation
factor-related diseases, disorders, and conditions, enhancing the
pharmacokinetic characteristics of the fusion proteins compared to
the CF, lowering the dose or frequency of dosing required to
achieve a pharmacologic effect, enhancing the pharmaceutical
properties, and to enhance the ability of the CF components to
remain within the therapeutic window for an extended period of
time.
[0253] In general, the steps in the design and production of the
fusion proteins and the inventive compositions, as illustrated in
FIGS. 31-33, include: (1) the selection of CFs (e.g., native
proteins, sequences of Tables 1 and 2, analogs or derivatives with
activity) to treat the particular disease, disorder or condition;
(2) selecting the XTEN that will confer the desired PK and
physicochemical characteristics on the resulting CFXTEN (e.g., the
administration of the CFXTEN composition to a subject results in
the fusion protein being maintained within the therapeutic window
for a greater period compared to CF not linked to XTEN); (3)
establishing a desired N- to C-terminus configuration of the CFXTEN
to achieve the desired efficacy or PK parameters; (4) establishing
the design of the expression vector encoding the configured CFXTEN;
(5) transforming a suitable host with the expression vector; and
(6) expression and recovery of the resultant fusion protein. For
those CFXTEN for which an increase in half-life (greater than 24 h)
or an increased period of time spent within a therapeutic window is
desired, the XTEN chosen for incorporation generally has at least
about 100, or about 144, or about 288, or about 432, or about 576,
or about 864, or about 875, or about 912, or about 923 amino acid
residues where a single XTEN is to be incorporated into the CFXTEN.
In another embodiment, the CFXTEN comprises a first XTEN of the
foregoing lengths, and at least a second XTEN of about 36, or about
72, or about 144, or about 288, or about 576, or about 864, or
about 875, or about 912, or about 923 amino acid residues.
[0254] In other embodiments, where an increase in half-life is not
required, but an increase in a pharmaceutical property (e.g.,
solubility) is desired, a CFXTEN is designed to include XTEN of
shorter lengths. In some embodiments of the foregoing, the CFXTEN
comprises a CF linked to an XTEN having at least about 24, or about
36, or about 48, or about 60, or about 72, or about 84, or about 96
amino acid residues, in which the solubility of the fusion protein
under physiologic conditions is at least three-fold greater than
the corresponding CF not linked to XTEN, or alternatively, at least
four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold,
or nine-fold, or at least 10-fold, or at least 20-fold, or at least
30-fold, or at least 50-fold, or at least 60-fold or greater than
CF not linked to XTEN. In one embodiment of the foregoing, the CF
is factor IX. In another embodiment, the CF is factor VII. In
another embodiment, the XTEN is a sequence with at least about 80%,
or about 90%, or about 95% sequence identity compared to a sequence
from Tables 4, and 9-13.
[0255] In another aspect, the invention provides methods of making
CFXTEN compositions to improve ease of manufacture, result in
increased stability, increased water solubility, and/or ease of
formulation, as compared to the native CF. In one embodiment, the
invention includes a method of increasing the water solubility of a
CF comprising the step of linking the CF to one or more XTEN such
that a higher concentration in soluble form of the resulting CFXTEN
can be achieved, under physiologic conditions, compared to the CF
in an un-fused state. Factors that contribute to the property of
XTEN to confer increased water solubility of CFs when incorporated
into a fusion protein include the high solubility of the XTEN
fusion partner and the low degree of self-aggregation between
molecules of XTEN in solution. In some embodiments, the method
results in a CFXTEN fusion protein wherein the water solubility is
at least about 20%, or at least about 30% greater, or at least
about 50% greater, or at least about 75% greater, or at least about
90% greater, or at least about 100% greater, or at least about 150%
greater, or at least about 200% greater, or at least about 400%
greater, or at least about 600% greater, or at least about 800%
greater, or at least about 1000% greater, or at least about 2000%
greater, or at least about 4000% greater, or at least about 6000%
greater under physiologic conditions, compared to the un-fused CF.
In one embodiment, the XTEN of the CFXTEN fusion protein is a
sequence with at least about 80%, or about 90%, or about 95%
sequence identity compared to a sequence from Tables 4, and
9-13.
[0256] In another embodiment, the invention includes a method of
increasing the shelf-life of a CF comprising the step of linking
the CF with one or more XTEN selected such that the shelf-life of
the resulting CFXTEN is extended compared to the CF in an un-fused
state. As used herein, shelf-life refers to the period of time over
which the functional activity of a CF or CFXTEN that is in solution
or in some other storage formulation remains stable without undue
loss of activity. As used herein, "functional activity" refers to a
pharmacologic effect or biological activity, such as the ability to
bind a receptor or ligand, or substrate, or an enzymatic activity,
or to display one or more known functional activities associated
with a CF, as known in the art. A CF that degrades or aggregates
generally has reduced functional activity or reduced
bioavailability compared to one that remains in solution. Factors
that contribute to the ability of the method to extend the shelf
life of CFs when incorporated into a fusion protein include
increased water solubility, reduced self-aggregation in solution,
and increased heat stability of the XTEN fusion partner. In
particular, the low tendency of XTEN to aggregate facilitates
methods of formulating pharmaceutical preparations containing
higher drug concentrations of CFs, and the heat-stability of XTEN
contributes to the property of CFXTEN fusion proteins to remain
soluble and functionally active for extended periods. In one
embodiment, the method results in CFXTEN fusion proteins with
"prolonged" or "extended" shelf-life that exhibit greater activity
relative to a standard that has been subjected to the same storage
and handling conditions. The standard may be the un-fused
full-length CF. In one embodiment, the method includes the step of
formulating the isolated CFXTEN with one or more pharmaceutically
acceptable excipients that enhance the ability of the XTEN to
retain its unstructured conformation and for the CFXTEN to remain
soluble in the formulation for a time that is greater than that of
the corresponding un-fused CF. In one embodiment, the method
comprises linking a CF to one or more XTEN selected from Tables 4
and 9-13 to create a CFXTEN fusion protein results in a solution
that retains greater than about 100% of the functional activity, or
greater than about 105%, 110%, 120%, 130%, 150% or 200% of the
functional activity of a standard when compared at a given time
point and when subjected to the same storage and handling
conditions as the standard, thereby increasing its shelf-life.
[0257] Shelf-life may also be assessed in terms of functional
activity remaining after storage, normalized to functional activity
when storage began. CFXTEN fusion proteins of the invention with
prolonged or extended shelf-life as exhibited by prolonged or
extended functional activity retain about 50% more functional
activity, or about 60%, 70%, 80%, or 90% more of the functional
activity of the equivalent CF not linked to XTEN when subjected to
the same conditions for the same period of time. For example, a
CFXTEN fusion protein of the invention comprising coagulation
factor fused to one or more XTEN sequences selected from Tables 4
and 9-13 retains about 80% or more of its original activity in
solution for periods of up to 2 weeks, or 4 weeks, or 6 weeks or
longer under various temperature conditions. In some embodiments,
the CFXTEN retains at least about 50%, or about 60%, or at least
about 70%, or at least about 80%, and most preferably at least
about 90% or more of its original activity in solution when heated
at 80.degree. C. for 10 min. In other embodiments, the CFXTEN
retains at least about 50%, preferably at least about 60%, or at
least about 70%, or at least about 80%, or alternatively at least
about 90% or more of its original activity in solution when heated
or maintained at 37.degree. C. for about 7 days. In another
embodiment, CFXTEN fusion protein retains at least about 80% or
more of its functional activity after exposure to a temperature of
about 30.degree. C. to about 70.degree. C. over a period of time of
about one hour to about 18 hours. In the foregoing embodiments
hereinabove described in this paragraph, the retained activity of
the CFXTEN is at least about two-fold, or at least about
three-fold, or at least about four-fold, or at least about
five-fold, or at least about six-fold greater at a given time point
than that of the corresponding CF not linked to the fusion
protein.
VII). The Nucleic Acids Sequences of the Invention
[0258] The present invention provides isolated polynucleic acids
encoding CFXTEN chimeric fusion proteins and sequences
complementary to polynucleic acid molecules encoding CFXTEN
chimeric fusion proteins, including homologous variants thereof. In
another aspect, the invention encompasses methods to produce
polynucleic acids encoding CFXTEN chimeric fusion proteins and
sequences complementary to polynucleic acid molecules encoding
CFXTEN chimeric fusion protein, including homologous variants
thereof. In general, and as illustrated in FIGS. 4-6, the methods
of producing a polynucleotide sequence coding for a CFXTEN fusion
protein and expressing the resulting gene product include
assembling nucleotides encoding CF and XTEN, ligating the
components in frame, incorporating the encoding gene into an
expression vector appropriate for a host cell, transforming the
appropriate host cell with the expression vector, and culturing the
host cell under conditions causing or permitting the fusion protein
to be expressed in the transformed host cell, thereby producing the
biologically-active CFXTEN polypeptide, which is recovered as an
isolated fusion protein by standard protein purification methods
known in the art. Standard recombinant techniques in molecular
biology is used to make the polynucleotides and expression vectors
of the present invention.
[0259] In accordance with the invention, nucleic acid sequences
that encode CFXTEN (or its complement) is used to generate
recombinant DNA molecules that direct the expression of CFXTEN
fusion proteins in appropriate host cells. Several cloning
strategies are suitable for performing the present invention, many
of which is used to generate a construct that comprises a gene
coding for a fusion protein of the CFXTEN composition of the
present invention, or its complement. In some embodiments, the
cloning strategy is used to create a gene that encodes a monomeric
CFXTEN that comprises at least a first CF and at least a first XTEN
polypeptide, or their complement. In one embodiment of the
foregoing, the gene comprises a sequence encoding a CF or sequence
variant. In other embodiments, the cloning strategy is used to
create a gene that encodes a monomeric CFXTEN that comprises
nucleotides encoding at least a first molecule of CF or its
complement and a first and at least a second XTEN or their
complement that is used to transform a host cell for expression of
the fusion protein of the CFXTEN composition. In the foregoing
embodiments hereinabove described in this paragraph, the genes can
further comprise nucleotides encoding spacer sequences that also
encode cleavage sequence(s).
[0260] In designing a desired XTEN sequences, it was discovered
that the non-repetitive nature of the XTEN of the inventive
compositions is achieved despite use of a "building block"
molecular approach in the creation of the XTEN-encoding sequences.
This was achieved by the use of a library of polynucleotides
encoding peptide sequence motifs, described above, that are then
ligated and/or multimerized to create the genes encoding the XTEN
sequences (see FIGS. 4 and 5 and Examples). Thus, while the XTEN(s)
of the expressed fusion protein may consist of multiple units of as
few as four different sequence motifs, because the motifs
themselves consist of non-repetitive amino acid sequences, the
overall XTEN sequence is rendered non-repetitive. Accordingly, in
one embodiment, the XTEN-encoding polynucleotides comprise multiple
polynucleotides that encode non-repetitive sequences, or motifs,
operably linked in frame and in which the resulting expressed XTEN
amino acid sequences are non-repetitive.
[0261] In one approach, a construct is first prepared containing
the DNA sequence corresponding to CFXTEN fusion protein. DNA
encoding the CF of the compositions is obtained from a cDNA library
prepared using standard methods from tissue or isolated cells
believed to possess CF mRNA and to express it at a detectable
level. Libraries is screened with probes containing, for example,
about 20 to 100 bases designed to identify the CF gene of interest
by hybridization using conventional molecular biology techniques.
The best candidates for probes are those that represent sequences
that are highly homologous for coagulation factor, and should be of
sufficient length and sufficiently unambiguous that false positives
are minimized, but may be degenerate at one or more positions. If
necessary, the coding sequence can be obtained using conventional
primer extension procedures as described in Sambrook, et al.,
supra, to detect precursors and processing intermediates of mRNA
that may not have been reverse-transcribed into cDNA. One can then
use polymerase chain reaction (PCR) methodology to amplify the
target DNA or RNA coding sequence to obtain sufficient material for
the preparation of the CFXTEN constructs containing the CF gene(s).
Assays can then be conducted to confirm that hybridizing
full-length genes are the desired CF gene(s). By these conventional
methods, DNA can be conveniently obtained from a cDNA library
prepared from such sources. The CF encoding gene(s) is also be
obtained from a genomic library or created by standard synthetic
procedures known in the art (e.g., automated nucleic acid synthesis
using, for example one of the methods described in Engels et al.
(Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA
sequences obtained from publicly available databases, patents, or
literature references. Such procedures are well known in the art
and well described in the scientific and patent literature. For
example, sequences can be obtained from Chemical Abstracts Services
(CAS) Registry Numbers (published by the American Chemical Society)
and/or GenBank Accession Numbers (e.g., Locus ID, NP_XXXXX, and
XP_XXXXX) Model Protein identifiers available through the National
Center for Biotechnology Information (NCBI) webpage, available on
the world wide web at ncbi.nlm.nih.gov that correspond to entries
in the CAS Registry or GenBank database that contain an amino acid
sequence of the protein of interest or of a fragment or variant of
the protein. For such sequence identifiers provided herein, the
summary pages associated with each of these CAS and GenBank and
GenSeq Accession Numbers as well as the cited journal publications
(e.g., PubMed ID number (PMID)) are each incorporated by reference
in their entireties, particularly with respect to the amino acid
sequences described therein. In one embodiment, the CF encoding
gene encodes a protein from any one of Table 1 or Table 2, or a
fragment or variant thereof.
[0262] A gene or polynucleotide encoding the CF portion of the
subject CFXTEN protein, in the case of an expressed fusion protein
that comprises a single CF is then be cloned into a construct,
which is a plasmid or other vector under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system. In a later step, a second gene
or polynucleotide coding for the XTEN is genetically fused to the
nucleotides encoding the N- and/or C-terminus of the CF gene by
cloning it into the construct adjacent and in frame with the
gene(s) coding for the CF. This second step occurs through a
ligation or multimerization step. In the foregoing embodiments
hereinabove described in this paragraph, it is to be understood
that the gene constructs that are created can alternatively be the
complement of the respective genes that encode the respective
fusion proteins.
[0263] The gene encoding for the XTEN can be made in one or more
steps, either fully synthetically or by synthesis combined with
enzymatic processes, such as restriction enzyme-mediated cloning,
PCR and overlap extension, including methods more fully described
in the Examples. The methods disclosed herein can be used, for
example, to ligate short sequences of polynucleotides encoding XTEN
into longer XTEN genes of a desired length and sequence. In one
embodiment, the method ligates two or more codon-optimized
oligonucleotides encoding XTEN motif or segment sequences of about
9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to
36 amino acids, or about 48 to about 144 amino acids, or about 144
to about 288 or longer, or any combination of the foregoing ranges
of motif or segment lengths.
[0264] Alternatively, the disclosed method is used to multimerize
XTEN-encoding sequences into longer sequences of a desired length;
e.g., a gene encoding 36 amino acids of XTEN can be dimerized into
a gene encoding 72 amino acids, then 144, then 288, etc. Even with
multimerization, XTEN polypeptides can be constructed such that the
XTEN-encoding gene has low or virtually no repetitiveness through
design of the codons selected for the motifs of the shortest unit
being used, which can reduce recombination and increase stability
of the encoding gene in the transformed host. Genes encoding XTEN
with non-repetitive sequences is assembled from oligonucleotides
using standard techniques of gene synthesis. The gene design can be
performed using algorithms that optimize codon usage and amino acid
composition. In one method of the invention, a library of
relatively short XTEN-encoding polynucleotide constructs is created
and then assembled, as illustrated in FIGS. 4 and 5. This can be a
pure codon library such that each library member has the same amino
acid sequence but many different coding sequences are possible.
Such libraries can be assembled from partially randomized
oligonucleotides and used to generate large libraries of XTEN
segments comprising the sequence motifs. The randomization scheme
can be optimized to control amino acid choices for each position as
well as codon usage. Exemplary methods to achieve the foregoing are
disclosed in the Examples.
Polynucleotide Libraries
[0265] In another aspect, the invention provides libraries of
polynucleotides that encode XTEN sequences that are used to
assemble genes that encode XTEN of a desired length and
sequence.
[0266] In certain embodiments, the XTEN-encoding library constructs
comprise polynucleotides that encode polypeptide segments of a
fixed length. As an initial step, a library of oligonucleotides
that encode motifs of 9-14 amino acid residues can be assembled. In
a preferred embodiment, libraries of oligonucleotides that encode
motifs of 12 amino acids are assembled.
[0267] The XTEN-encoding sequence segments can be dimerized or
multimerized into longer encoding sequences. Dimerization or
multimerization can be performed by ligation, overlap extension,
PCR assembly or similar cloning techniques known in the art. This
process of can be repeated multiple times until the resulting
XTEN-encoding sequences have reached the organization of sequence
and desired length, providing the XTEN-encoding genes. As will be
appreciated, a library of polynucleotides that encodes, e.g., 12
amino acid motifs can be dimerized and/or ligated into a library of
polynucleotides that encode 36 amino acids. Libraries encoding
motifs of different lengths; e.g., 9-14 amino acid motifs leading
to libraries encoding 27 to 42 amino acids are contemplated by the
invention. In turn, the library of polynucleotides that encode 27
to 42 amino acids, and preferably 36 amino acids (as described in
the Examples) can be serially dimerized into a library containing
successively longer lengths of polynucleotides that encode XTEN
sequences of a desired length for incorporation into the gene
encoding the CFXTEN fusion protein, as disclosed herein. In some
embodiments, libraries are assembled of polynucleotides that encode
amino acids that are limited to specific sequence XTEN families;
e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. In other
embodiments, libraries comprise sequences that encode two or more
of the motif family sequences from Table 3. The names and sequences
of representative, non-limiting polynucleotide sequences of
libraries that encode 36 mers are presented in Tables 9-12, and the
methods used to create them are described more fully in the
respective Examples. In other embodiments, libraries that encode
XTEN are constructed from segments of polynucleotide codons linked
in a randomized sequence that encode amino acids wherein at least
about 80%, or at least about 90%, or at least about 91%, or at
least about 92%, or at least about 93%, or at least about 94%, or
at least about 95%, or at least about 97%, or at least about 98%,
or at least about 99% of the codons are selected from the group
consisting of condons for glycine (G), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) amino acids. The
libraries can be used, in turn, for serial dimerization or ligation
to achieve polynucleotide sequence libraries that encode XTEN
sequences, for example, of 48, 72, 144, 288, 576, 864, 875, 912,
923, 1318 amino acids, or up to a total length of about 3000 amino
acids, as well as intermediate lengths, in which the encoded XTEN
can have one or more of the properties disclosed herein, when
expressed as a component of a CFXTEN fusion protein. In some cases,
the polynucleotide library sequences may also include additional
bases used as "sequencing islands," described more fully below.
[0268] FIG. 5 is a schematic flowchart of representative,
non-limiting steps in the assembly of a XTEN polynucleotide
construct and a CFXTEN polynucleotide construct in the embodiments
of the invention. Individual oligonucleotides 501 are annealed into
sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which
is subsequently ligated with an oligo containing BbsI, and KpnI
restriction sites 503. Additional sequence motifs from a library
are annealed to the 12-mer until the desired length of the XTEN
gene 504 is achieved. The XTEN gene is cloned into a stuffer
vector. The vector optionally encodes a Flag sequence 506 followed
by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites
507 and, in this case, a single CF gene (encoding FIX in this
example) 508, resulting in the gene encoding a CFXTEN comprising a
single CF 500. A non-exhaustive list of the XTEN names for
polynucleotides encoding XTEN and precursor sequences is provided
in Table 8.
TABLE-US-00009 TABLE 8 DNA sequences of XTEN and precursor
sequences XTEN Name DNA Nucleotide Sequence AE48
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTA
CTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCT
TCTCCGGGCACCAGCTCTACCGGTTCT AM48
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCA
GCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGC
TCTACCCCGTCTGGTGCTACTGGCTCT AE144
GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTC
CTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCAGGTAGCCC
GGCAGGCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCTTCTGAGGGTAGC
GCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTAGCGAACCTGCTA
CCTCCGGCTCTGAAACTCCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGG
TACCTCTACCGAACCTTCCGAAGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCT
GAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTA
CCGAACCGTCCGAAGGTAGCGCACCA AF144
GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATC
TTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTTCTACCA
GCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCGTCTGGCACCGC
ACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGTACTTCCCCTAGCGGT
GAATCTTCTACTGCTCCAGGTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGGTTC
TACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCTAGCGGCGAATCTTCTA
CTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGC
GGTGAATCTTCTACCGCACCA AE288
GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCG
GCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGA
ACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCT
GGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCT
CTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGG
TAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCT
GAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGG
CTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGC
ACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCT
ACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTA
GCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTC
TGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACT
GAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTC
CAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC
TCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AE576
GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC
CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCC
AGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGC
GCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCG
CTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGG
TAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACC
TCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTA
CCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACC
GTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT
ACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACC
GGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCA
CCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAA
CCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTA
GCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGA
ATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAA
AGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTC
CAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTC
CGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACC
TCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTA
GCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGA
ACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA
GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCC
CGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
GCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCT
CTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGG
TAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG
GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA AF576
GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCCACTAGCTCTACCGCAGA
ATCTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCAGGTTCTACTA
GCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTCCTGGC
CCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCC
TTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTA
CTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACC
GCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATC
TCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTA
CCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGC
ACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCG
AATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA
GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGG
CTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACTA
GCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATC
TCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAATCCC
CGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTAC
TTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTA
CCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGA
ATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG
GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGG
TTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCA
GCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCT
CCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCC
GTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTACTT
CTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCG
GGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCCACTAGCTCTAC
TGCTGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTT
CTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCAGAATCT
CCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCC
TGAAAGCGGTTCTGCATCTCCA AE624
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTA
CTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCT
TCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTAC
TGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAG
GTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA
GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGA
ACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA
ACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCG
CAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAG
GTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTAC
CTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCT
ACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCG
GTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC
AACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGT
ACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAG
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA
AAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAA
GAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACC
TCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA
CCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG
TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACT
GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCAC
CAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCC
TACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC
TCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTG
AGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC
AACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTC
CTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACT
GAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAA
CCGTCTGAGGGCAGCGCACCA AM875
GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCG
GTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTAC
CAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCA
TCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATC
CCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTA
CCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTC
TGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCA
GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTC
CAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTC
CGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAG
CCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGT
AGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA
GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCC
AGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCC
GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTT
CTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA
AACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCG
TCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGG
GTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC
GGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAG
GAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTT
CCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTT
CTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCAC
CGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCT
ACCGCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG
GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGC
TTCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCC
CGTCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAAC
TCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACT
TCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTT
CTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCT
ACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTG
CAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACC
AGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGC
GGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTC
TACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGC
GCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGT
CTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGT
GCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTC
TGAAACCCCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCA
GGTTCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCC
AGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCT
CTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCT
ACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG CACCA
AE864 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC
CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCC
AGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGC
GCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCG
CTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGG
TAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACC
TCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTA
CCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACC
GTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT
ACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACC
GGCTACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCA
CCAGGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAA
CCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTA
GCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGA
ATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAA
AGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTC
CAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTC
CGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACC
TCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTA
GCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGA
ACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA
GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCC
CGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
GCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCT
CTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGG
TAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG
GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTG
AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGAC
TCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACC
TCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTA
CTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTC
CACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACC
GGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGC
CCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTC
CAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTAC
TTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAA
TCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGG
CTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC
AGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCC
GAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGC
GAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA AF864
GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATC
TTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTA
GCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCT
CCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGAATCTC
CTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACT
TCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCTCCGTCTGGCAC
TGCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGCG
GCGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGT
ACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTC
TACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTC
CGGAAAGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCC
AGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGT
CTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACC
AGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCAT
CTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTCCGAGCGGT
GAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTA
CTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCTGAAAGCGGTTCT
GCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTC
TACTGCTGAATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG
GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCT
TCTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAG
CGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTCCXXXXXXX
XXXXXTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAXXXXXXXXTAGCGAATCTCCT
TCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTAC
CAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCG
CTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCT
CCTTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTAC
TTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTA
CTGCTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGC
GGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG
TTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG
GTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACTAG
CGAATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTTCT
CCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCGAATCTC
CTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCC
ACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTGGCAC
CGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCG
GCGAATCTTCTACCGCACCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGT
ACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTT
CCGCTTCTCCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCT
AGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCAC
CAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCT
GAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTA
GCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGC
TCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA XXXX was inserted in two
areas where no sequence information is available. AG864
GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTTCTAC
TGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACCCCGG
GTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCT
CCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGGCACCA
GCTCTACTGGTTCTCCAGGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTAGC
TCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTC
TTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCA
CTAGCTCTACTGGTTCTCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGT
AGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTAC
CGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTC
CGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCA
GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCTACCCCTTCTGGTGC
TACCGGCTCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCAGGTGCATCTC
CGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCT
CCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGGCAGCGGTAC
CGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTC
TCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTT
CTCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGCAGCGGT
ACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGC
ATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTG
GTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCT
TCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAGG
TACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTA
CCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACT
CCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCC
AGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGC
TCTACCGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTC
TCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTT
CTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACC
AGCTCTACTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAG
CTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTT
CTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGC
GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGG
TAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCCTTCTGCATCCACCG
GTACCGGTCCAGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGCATCCCCG
GGCACTAGCTCTACCGGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCC
AGGTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCA
CCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT
CCGGGTACTAGCTCTACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTC
TCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCAT
CTACCGGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTTCT
AGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTTC
CTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTT
CTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA AM923
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGCACCA
GCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGC
TCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAG
CGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGT
TCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG
GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCT
GGCACTGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTAC
TCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTC
CAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTAC
TCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACC
TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGT
CCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGA
ACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTTCCG
AGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTC
TGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGC
GCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCG
CAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGG
TAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACC
TCCACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGG
GCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCT
CCAGGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGT
CTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAG
CCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA
CTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAA
GCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAG
GTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAAC
TTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTA
GCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGC
ACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCGTCTG
GTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAG
CGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGAA
TCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCT
CTACTGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCC
AGGTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCT
GGCTCTGAAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTT
CTACTGAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCC
TGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAA
TCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG
GTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGA
AGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGC
CCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTC
TCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCA
ACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTA
GCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGT
ACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAA
GCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCC
AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA AE912
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGCGGTA
CTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCT
TCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTAC
TGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAG
GTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA
GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGA
ACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA
ACTCCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCG
CAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAG
GTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTAC
CTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCT
ACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCG
GTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC
AACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGT
ACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAG
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA
AAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAA
GAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACC
TCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA
CCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG
TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACT
GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCAC
CAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCC
TACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC
TCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTG
AGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGC
AACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTC
CTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCTACT
GAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACCGAA
CCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAG
GTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCC
GGAATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT
GAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG
CACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGC
AACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGT
ACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTT
CCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTAC
CGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGC
CCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTA
CCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAG
CGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCC
ACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACTG
AACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCC
AGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC
GAGGGCAGCGCACCA AM1318
GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCG
GTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTAC
CAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCA
TCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATC
CCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGTA
CCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTC
TGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCA
GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTC
CAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTC
CGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAG
CCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGT
AGCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA
GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCC
AGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCC
GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTT
CTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA
AACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCG
TCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAGG
GTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC
GGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAG
GAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTT
CCGAAGGTAGCGCTCCAGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCG
AACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATC
CGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGC
GCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAG
GTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCC
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCG
GCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGG
CCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGT
GAATCTTCTACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTC
TACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTA
CCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAG
CGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTC
CGGAATCTGGTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACCTC
TACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCC
GGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACCTCCCCTAGCG
GCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGT
ACCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTCTACCGAACCGTCCGAGG
GTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTAC
CGAACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGC
CCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGG
TGCAACCGGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGTAGC
TCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGTACTAGCTCTACCG
GTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTCCGAGCG
GTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGT
ACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACTCCGG
AGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTAC
TGAACCGTCCGAAGGTAGCGCACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGC
CCAGGTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAG
CTCTACCGGTTCTCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTT
CTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACT
GCTCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTA
CTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGG
TTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTG
GCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCCCGGC
AGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGC
CCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTC
CAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTA
GCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAGCTCTACCCCGTCTGGTGCTAC
CGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCC
CGTCTGGTGCTACTGGCTCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCA
GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACCGCAGA
ATCTCCGGGTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCATCCC
CGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCT
CCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTC
CGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA BC864
GGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCATCCG
AACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCAGGTAGCG
GCGCATCCGAGCCTACCTCTACTGAACCAGGTAGCGAACCGGCTACCTCCGGTACTGA
GCCATCAGGTAGCGAACCGGCAACTTCCGGTACTGAACCATCAGGTAGCGAACCGGC
AACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCA
GGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCAGCTACTTCTG
GCACTGAACCATCAGGTACTTCTACTGAACCATCCGAACCAGGTAGCGCAGGTAGCGA
ACCTGCTACCTCTGGTACTGAGCCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAA
CCATCAGGTACTTCTACCGAACCATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAAC
CATCCGAGCCAGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAG
GTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTC
CGAACCAGGTGCAGGTAGCGGCGCATCCGAACCTACTTCCACTGAACCAGGTACTAGC
GAGCCATCCACCTCTGAACCAGGTGCAGGTAGCGAACCGGCAACTTCCGGCACTGAA
CCATCAGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAAC
CATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCAG
GTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAACTTCTGG
CACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCATCAGGTAGCGAA
CCGGCTACTTCCGGCACTGAACCATCAGGTAGCGAACCAGCAACCTCCGGTACTGAAC
CATCAGGTACTTCCACTGAACCATCCGAACCGGGTAGCGCAGGTAGCGAACCGGCAA
CTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGG
TACTTCTACTGAACCATCTGAGCCGGGCAGCGCAGGTAGCGAACCTGCAACCTCCGGC
ACTGAGCCATCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTACTTCCA
CCGAACCATCTGAGCCAGGCAGCGCAGGTAGCGGCGCATCTGAACCAACCTCTACTG
AACCAGGTAGCGAACCAGCAACTTCTGGTACTGAACCATCAGGTAGCGGCGCATCTG
AGCCTACTTCCACTGAACCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGG
TAGCGGTGCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAG
CCGGGCAGCGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGT
GCATCTGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCA
GCGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACC
ATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCAGGT
ACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAAC
CTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTTCTAC
TGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGAACCATCTGAACCTGGTAGC
GCAGGTACTTCCACTGAACCATCCGAACCAGGTAGCGCAGGTACTAGCGAACCATCCA
CCTCCGAACCAGGCGCAGGTAGCGGTGCATCTGAACCGACTTCTACTGAACCAGGTAC
TTCCACTGAACCATCTGAGCCAGGTAGCGCAGGTACTTCCACCGAACCATCCGAACCA
GGTAGCGCAGGTACTTCCACCGAACCATCCGAACCTGGCAGCGCAGGTAGCGAACCG
GCAACCTCTGGTACTGAACCATCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAAC
CAGGTAGCGAACCAGCAACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTC
TGGTACTGAACCATCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGC
GAACCAGCAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAAC
CAGGTGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCAT
CTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGC
AGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCTGAACC
AACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGCGCA BD864
GGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCAACTA
GCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCAGGTACTAG
CGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTACCTCTGGCTCCGA
GACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCAGGTACTTCCACTGAA
GCAAGTGAAGGCTCCGCATCAGGTACTTCCACCGAAGCAAGCGAAGGCTCCGCATCA
GGTACTAGTGAGTCCGCAACTAGCGAATCCGGTGCAGGTAGCGAAACCGCTACCTCTG
GTTCCGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCAC
TGCTGGTTCCGAGACTTCTACTGAAGCAGGTACTAGCGAATCTGCTACTAGCGAATCC
GGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCA
ACCTCTGGTTCCGAGACTGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAG
GTACTTCCACTGAAGCTAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGG
TTCCGAAACTGCAGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCT
ACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTG
AAGCAGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGC
TAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGCAGGT
AGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTCCGAAACTT
CCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGCAGGTACTAGCGA
ATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGC
GCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTACTAGCGAATCTGCTA
CTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTA
GCGAAACTGCAACCTCTGGTTCCGAGACTGCAGGTAGCGAAACCGCTACCTCTGGTTC
CGAAACTGCAGGTACTTCTACCGAGGCTAGCGAAGGTTCTGCATCAGGTAGCACTGCT
GGTTCCGAGACTTCTACTGAAGCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTG
CAGGTACTAGTGAATCCGCAACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGA
GACTTCCACTGAAGCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGC
ACTGCAGGTTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCT
CTGCATCAGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGG
TTCTGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATCA
GGTAGCACTGCAGGTTCTGAGACTTCCACCGAAGCAGGTAGCGAAACTGCTACTTCTG
GTTCCGAAACTGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCCGCATCAGGTACTAG
TGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGA
AACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTACTAGTGAGTC
CGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAACCTCCGGTTCTGAAACTGC
AGGTACTAGCGAATCCGCAACCAGCGAATCTGGCGCAGGTAGCGAAACTGCTACTTCC
GGCTCTGAGACTGCAGGTACTTCCACCGAAGCAAGCGAAGGTTCCGCATCAGGTACTT
CCACCGAGGCTAGTGAAGGCTCTGCATCAGGTAGCACTGCTGGCTCCGAGACTTCTAC
CGAAGCAGGTAGCACTGCAGGTTCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGC
TACCTCTGGCTCTGAGACTGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCA
GGTACTAGCGAATCCGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCT
GGTTCCGAGACTGCAGGTAGCGAAACTGCTACTTCCGGCTCCGAGACTGCAGGTAGCG
AAACTGCTACTTCTGGCTCCGAAACTGCAGGTACTTCTACTGAGGCTAGTGAAGGTTC
CGCATCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGC
TACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGCA
GGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGCTACCA
GCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGCA
[0269] One may clone the library of XTEN-encoding genes into one or
more expression vectors known in the art. To facilitate the
identification of well-expressing library members, one can
construct the library as fusion to a reporter protein. Non-limiting
examples of suitable reporter genes are green fluorescent protein,
luciferase, alkaline phosphatase, and beta-galactosidase. By
screening, one can identify short XTEN sequences that can be
expressed in high concentration in the host organism of choice.
Subsequently, one can generate a library of random XTEN dimers and
repeat the screen for high level of expression. Subsequently, one
can screen the resulting constructs for a number of properties such
as level of expression, protease stability, or binding to
antiserum.
[0270] One aspect of the invention is to provide polynucleotide
sequences encoding the components of the fusion protein wherein the
creation of the sequence has undergone codon optimization. Of
particular interest is codon optimization with the goal of
improving expression of the polypeptide compositions and to improve
the genetic stability of the encoding gene in the production hosts.
For example, codon optimization is of particular importance for
XTEN sequences that are rich in glycine or that have very
repetitive amino acid sequences. Codon optimization is performed
using computer programs (Gustafsson, C., et al. (2004) Trends
Biotechnol, 22: 346-53), some of which minimize ribosomal pausing
(Coda Genomics Inc.). In one embodiment, one can perform codon
optimization by constructing codon libraries where all members of
the library encode the same amino acid sequence but where codon
usage is varied. Such libraries can be screened for highly
expressing and genetically stable members that are particularly
suitable for the large-scale production of XTEN-containing
products. When designing XTEN sequences one can consider a number
of properties. One can minimize the repetitiveness in the encoding
DNA sequences. In addition, one can avoid or minimize the use of
codons that are rarely used by the production host (e.g. the AGG
and AGA arginine codons and one leucine codon in E. coli). In the
case of E. coli, two glycine codons, GGA and GGG, are rarely used
in highly expressed proteins. Thus codon optimization of the gene
encoding XTEN sequences can be very desirable. DNA sequences that
have a high level of glycine tend to have a high GC content that
can lead to instability or low expression levels. Thus, when
possible, it is preferred to choose codons such that the GC-content
of XTEN-encoding sequence is suitable for the production organism
that will be used to manufacture the XTEN.
[0271] Optionally, the full-length XTEN-encoding gene comprises one
or more sequencing islands. In this context, sequencing islands are
short-stretch sequences that are distinct from the XTEN library
construct sequences and that include a restriction site not present
or expected to be present in the full-length XTEN-encoding gene. In
one embodiment, a sequencing island is the sequence
5'-AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGT-3'. In another embodiment,
a sequencing island is the sequence
5'-AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGT-3'.
[0272] In one embodiment, polynucleotide libraries are constructed
using the disclosed methods wherein all members of the library
encode the same amino acid sequence but where codon usage for the
respective amino acids in the sequence is varied. Such libraries
can be screened for highly expressing and genetically stable
members that are particularly suitable for the large-scale
production of XTEN-containing products.
[0273] Optionally, one can sequence clones in the library to
eliminate isolates that contain undesirable sequences. The initial
library of short XTEN sequences allows some variation in amino acid
sequence. For instance one can randomize some codons such that a
number of hydrophilic amino acids can occur in a particular
position. During the process of iterative multimerization one can
screen the resulting library members for other characteristics like
solubility or protease resistance in addition to a screen for
high-level expression.
[0274] Once the gene that encodes the XTEN of desired length and
properties is selected, it is genetically fused at the desired
location to the nucleotides encoding the CF gene(s) by cloning it
into the construct adjacent and in frame with the gene coding for
CF, or alternatively between nucleotides encoding adjacent domains
of the CF, or alternatively within a sequence encoding a given CF
domain, or alternatively in frame with nucleotides encoding a
spacer/cleavage sequence linked to a terminal XTEN. The invention
provides various permutations of the foregoing, depending on the
CFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion
protein comprising a CF and two XTEN, such as embodied by formula
VI, as depicted above, the gene would have polynucleotides encoding
CF, encoding two XTEN, which can be identical or different in
composition and sequence length. In one non-limiting embodiment of
the foregoing, the CF polynucleotides would encode coagulation
factor and the polynucleotides encoding the C-terminus XTEN would
encode AE864 and the polynucleotides encoding an internal XTEN
adjacent to the C-terminus of EGF2 would encode AE144. The step of
cloning the CF genes into the XTEN construct can occur through a
ligation or multimerization step, as shown in FIG. 32. The
constructs encoding CFXTEN fusion proteins can be designed in
different configurations of the components XTEN, CF, and spacer
sequences, such as the configurations of formulae I-VI. In one
embodiment, the construct comprises polynucleotide sequences
complementary to, or those that encode a monomeric polypeptide of
components in the following order (5' to 3') CF and XTEN. In
another embodiment, the construct comprises polynucleotide
sequences complementary to, or those that encode a monomeric
polypeptide of components in the following order (5' to 3') CF,
spacer sequence, and XTEN. The spacer polynucleotides can
optionally comprise sequences encoding cleavage sequences. As will
be apparent to those of skill in the art, other permutations or
multimers of the foregoing are possible.
[0275] The invention also encompasses polynucleotides comprising
XTEN-encoding polynucleotide variants that have a high percentage
of sequence identity compared to (a) a polynucleotide sequence from
Table 8, or (b) sequences that are complementary to the
polynucleotides of (a). A polynucleotide with a high percentage of
sequence identity is one that has at least about an 80% nucleic
acid sequence identity, alternatively at least about 81%,
alternatively at least about 82%, alternatively at least about 83%,
alternatively at least about 84%, alternatively at least about 85%,
alternatively at least about 86%, alternatively at least about 87%,
alternatively at least about 88%, alternatively at least about 89%,
alternatively at least about 90%, alternatively at least about 91%,
alternatively at least about 92%, alternatively at least about 93%,
alternatively at least about 94%, alternatively at least about 95%,
alternatively at least about 96%, alternatively at least about 97%,
alternatively at least about 98%, and alternatively at least about
99% nucleic acid sequence identity compared to (a) or (b) of the
foregoing, or that can hybridize with the target polynucleotide or
its complement under stringent conditions.
[0276] Homology, sequence similarity or sequence identity of
nucleotide or amino acid sequences may also be determined
conventionally by using known software or computer programs such as
the BestFit or Gap pairwise comparison programs (GCG Wisconsin
Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.
53711). BestFit uses the local homology algorithm of Smith and
Waterman (Advances in Applied Mathematics. 1981.2: 482-489), to
find the best segment of identity or similarity between two
sequences. Gap performs global alignments: all of one sequence with
all of another similar sequence using the method of Needleman and
Wunsch, (Journal of Molecular Biology. 1970. 48:443-453). When
using a sequence alignment program such as BestFit, to determine
the degree of sequence homology, similarity or identity, the
default setting may be used, or an appropriate scoring matrix may
be selected to optimize identity, similarity or homology
scores.
[0277] Nucleic acid sequences that are "complementary" are those
that are capable of base-pairing according to the standard
Watson-Crick complementarity rules. As used herein, the term
"complementary sequences" means nucleic acid sequences that are
substantially complementary, as may be assessed by the same
nucleotide comparison set forth above, or as defined as being
capable of hybridizing to the polynucleotides that encode the
CFXTEN sequences under stringent conditions, such as those
described herein.
[0278] The resulting polynucleotides encoding the CFXTEN chimeric
fusion proteins can then be individually cloned into an expression
vector. The nucleic acid sequence is inserted into the vector by a
variety of procedures. In general, DNA is inserted into an
appropriate restriction endonuclease site(s) using techniques known
in the art. Vector components generally include, but are not
limited to, one or more of a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan. Such techniques are well known in the art and well
described in the scientific and patent literature.
[0279] Various vectors are publicly available. The vector may, for
example, be in the form of a plasmid, cosmid, viral particle, or
phage that may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e., a vector, which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0280] The invention provides for the use of plasmid vectors
containing replication and control sequences that are compatible
with and recognized by the host cell, and are operably linked to
the CFXTEN gene for controlled expression of the CFXTEN fusion
proteins. The vector ordinarily carries a replication site, as well
as sequences that encode proteins that are capable of providing
phenotypic selection in transformed cells. Such vector sequences
are well known for a variety of bacteria, yeast, and viruses.
Useful expression vectors that can be used include, for example,
segments of chromosomal, non-chromosomal and synthetic DNA
sequences. "Expression vector" refers to a DNA construct containing
a DNA sequence that is operably linked to a suitable control
sequence capable of effecting the expression of the DNA encoding
the fusion protein in a suitable host. The requirements are that
the vectors are replicable and viable in the host cell of choice.
Low- or high-copy number vectors may be used as desired.
[0281] Other suitable vectors include, but are not limited to,
derivatives of SV40 and pcDNA and known bacterial plasmids such as
col EI, pCR1, pBR322, pMa1-C2, pET, pGEX as described by Smith, et
al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids
such as RP4, phage DNAs such as the numerous derivatives of phage I
such as NM98 9, as well as other phage DNA such as M13 and
filamentous single stranded phage DNA; yeast plasmids such as the 2
micron plasmid or derivatives of the 2m plasmid, as well as
centromeric and integrative yeast shuttle vectors; vectors useful
in eukaryotic cells such as vectors useful in insect or mammalian
cells; vectors derived from combinations of plasmids and phage
DNAs, such as plasmids that have been modified to employ phage DNA
or the expression control sequences; and the like. Yeast expression
systems that can also be used in the present invention include, but
are not limited to, the non-fusion pYES2 vector (Invitrogen), the
fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.
[0282] The control sequences of the vector include a promoter to
effect transcription, an optional operator sequence to control such
transcription, a sequence encoding suitable mRNA ribosome binding
sites, and sequences that control termination of transcription and
translation. The promoter may be any DNA sequence, which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
[0283] Examples of suitable promoters for directing the
transcription of the DNA encoding the CF polypeptide variant in
mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell.
Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., Science 222 (1983), 809-814), the CMV promoter
(Boshart et al., Cell 41:521-530, 1985) or the adenovirus 2 major
late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319,
1982). The vector may also carry sequences such as UCOE (ubiquitous
chromatin opening elements).
[0284] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter or the tpiA
promoter. Examples of other useful promoters are those derived from
the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, A. niger neutral .alpha.-amylase, A. niger
acid stable .alpha.-amylase, A. niger or A. awamoriglucoamylase
(gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A.
oryzae triose phosphate isomerase or A. nidulans acetamidase.
Preferred are the TAKA-amylase and gluA promoters.
[0285] Promoters suitable for use in expression vectors with
prokaryotic hosts include the .beta.-lactamaseand lactose promoter
systems [Chang et al., Nature, 275:615 (1978); Goeddel et al.,
Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp)
promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP
36,776], and hybrid promoters such as the tac promoter [deBoer et
al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all is operably
linked to the DNA encoding CFXTEN polypeptides. Promoters for use
in bacterial systems can also contain a Shine-Dalgarno (S.D.)
sequence, operably linked to the DNA encoding CFXTEN
polypeptides.
[0286] The invention contemplates use of other expression systems
including, for example, a baculovirus expression system with both
non-fusion transfer vectors, such as, but not limited to pVL941
Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen),
pVL1392 (Summers, et al., Virology 84:390-402 (1978) and
Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer
vectors such as, but not limited to, pAc7 00 (Summers, et al.,
Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700,
with different reading frames), pAc360 Invitrogen) and
pBlueBacHisA, B, C (Invitrogen) can be used.
[0287] Examples of suitable promoters for directing the
transcription of the DNA encoding the CF polypeptide variant in
mammalian cells are the CMV promoter (Boshart et al., Cell
41:521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell.
Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2
major late promoter (Kaufman and Sharp, Mol. Cell. Biol,
2:1304-1319, 1982). The vector may also carry sequences such as
UCOE (ubiquitous chromatin opening elements).
[0288] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter or the tpiA
promoter.
[0289] The DNA sequences encoding the CFXTEN may also, if
necessary, be operably connected to a suitable terminator, such as
the hGH terminator (Palmiter et al., Science 222, 1983, pp.
809-814) or the TPI1 terminators (Alber and Kawasaki, J. Mol. Appl.
Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4,
1985, pp. 2093-2099). Expression vectors may also contain a set of
RNA splice sites located downstream from the promoter and upstream
from the insertion site for the CFXTEN sequence itself, including
splice sites obtained from adenovirus. Also contained in the
expression vectors is a polyadenylation signal located downstream
of the insertion site. Particularly preferred polyadenylation
signals include the early or late polyadenylation signal from SV40
(Kaufman and Sharp, ibid.), the polyadenylation signal from the
adenovirus 5 E1b region, the hGH terminator (DeNoto et al. Nucl.
Acids Res. 9:3719-3730, 1981). The expression vectors may also
include a noncoding viral leader sequence, such as the adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites; and enhancer sequences, such as the SV40 enhancer.
[0290] To direct the CFXTEN of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(a.k.a., a leader sequence, a prepro sequence, or a pre sequence)
may be included in the recombinant vector. The secretory signal
sequence is operably linked to the DNA sequences encoding the
CFXTEN, usually positioned 5' to the DNA sequence encoding the
CFXTEN fusion protein. The secretory signal sequence may be that,
normally associated with the protein or may be from a gene encoding
another secreted protein. Non-limiting examples include OmpA, PhoA,
and DsbA for E. coli expression, ppL-alpha, DEX4, invertase signal
peptide, acid phosphatase signal peptide, CPY, or INU1 for yeast
expression, and IL2L, SV40, IgG kappa and IgG lambda for mammalian
expression. Signal sequences are typically proteolytically removed
from the protein during the translocation and secretion process,
generating a defined N-terminus. Methods are disclosed in Arnau, et
al., Protein Expression and Purification 48: 1-13 (2006).
[0291] The procedures used to ligate the DNA sequences coding for
the CFXTEN, the promoter and optionally the terminator and/or
secretory signal sequence, respectively, and to insert them into
suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor, N.Y., 1989).
[0292] In other cases, the invention provides constructs and
methods of making constructs comprising an polynucleotide sequence
optimized for expression that encodes at least about 20 to about 60
amino acids with XTEN characteristics that can be included at the
N-terminus of an XTEN carrier encoding sequence (in other words,
the polynucleotides encoding the 20-60 encoded optimized amino
acids are linked in frame to polynucleotides encoding an XTEN
component that is N-terminal to CF) to promote the initiation of
translation to allow for expression of XTEN fusions at the
N-terminus of proteins without the presence of a helper domain. In
an advantage of the foregoing, the sequence does not require
subsequent cleavage, thereby reducing the number of steps to
manufacture XTEN-containing compositions. As described in more
detail in the Examples, the optimized N-terminal sequence has
attributes of an unstructured protein, but may include nucleotide
bases encoding amino acids selected for their ability to promote
initiation of translation and enhanced expression. In one
embodiment of the foregoing, the optimized polynucleotide encodes
an XTEN sequence with at least about 90% sequence identity compared
to AE912. In another embodiment of the foregoing, the optimized
polynucleotide encodes an XTEN sequence with at least about 90%
sequence identity compared to AM923. In another embodiment of the
foregoing, the optimized polynucleotide encodes an XTEN sequence
with at least about 90% sequence identity compared to AE48. In
another embodiment of the foregoing, the optimized polynucleotide
encodes an XTEN sequence with at least about 90% sequence identity
compared to AM48. In one embodiment, the optimized polynucleotide
NTS comprises a sequence that exhibits at least about 80%, at least
about 85%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 94%, at least about 95%, at
least about 96%, at least about 97%, at least about 98%, or at
least about 99%, sequence identity compared to a sequence or its
complement selected from
TABLE-US-00010 AE 48:
5'-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTAC
CCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTG
GTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCT CCA-3' and AM
48: 5'-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGC
ATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTG
GTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCT CCA-3'
[0293] In this manner, a chimeric DNA molecule coding for a
monomeric CFXTEN fusion protein is generated within the construct.
Optionally, this chimeric DNA molecule may be transferred or cloned
into another construct that is a more appropriate expression
vector. At this point, a host cell capable of expressing the
chimeric DNA molecule can be transformed with the chimeric DNA
molecule.
[0294] Examples of mammalian cell lines for use in the present
invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651),
BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al., J.
Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-K1
(ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293-F (Invitrogen
R790-7). A tk.sup.-ts13 BHK cell line is also available from the
ATCC under accession number CRL 1632. In addition, a number of
other cell lines may be used within the present invention,
including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat
hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB
8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells
(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220,
1980).
[0295] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous polypeptides there from are described, e.g.
in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos.
4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are
hereby incorporated by reference. Transformed cells are selected by
a phenotype determined by a selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient, e.g. leucine. A preferred vector for use in yeast is the
POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences
encoding the CFXTEN may be preceded by a signal sequence and
optionally a leader sequence, e.g. as described above. Further
examples of suitable yeast cells are strains of Kluyveromyces, such
as K lactis, Hansenula, e.g. H polymorpha, or Pichia, e.g. P.
pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp.
3459-3465; U.S. Pat. No. 4,882,279). Examples of other fungal cells
are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora
spp., Fusarium spp. or Trichoderma spp., in particular strains of
A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for
the expression of proteins is described in, e.g., EP 272 277, EP
238 023, EP 184 438 The transformation of F. oxysporum may, for
instance, be carried out as described by Malardier et al., 1989,
Gene 78: 147-156. The transformation of Trichoderma spp. may be
performed for instance as described in EP 244 234.
[0296] Other suitable cells that can be used in the present
invention include, but are not limited to, prokaryotic host cells
strains such as Escherichia coli, (e.g., strain DHS-.alpha.),
Bacillus subtilis, Salmonella typhimurium, or strains of the genera
of Pseudomonas, Streptomyces and Staphylococcus. Non-limiting
examples of suitable prokaryotes include those from the genera:
Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus;
Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus;
Paracoccus; Pseudomonas; Staphylococcus; Streptococcus;
Streptomyces; Thermoplasma; and Vibrio.
[0297] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in e.g., Kaufman
and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978),
725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603,
Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al.,
EMBO J. 1 (1982), 841-845.
[0298] Cloned DNA sequences are introduced into cultured mammalian
cells by, for example, calcium phosphate-mediated transfection
(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic
Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology
52d:456-467, 1973), transfection with many commercially available
reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) or
lipofectamine (Invitrogen) or by electroporation (Neumann et al.,
EMBO J. 1:841-845, 1982). To identify and select cells that express
the exogenous DNA, a gene that confers a selectable phenotype (a
selectable marker) is generally introduced into cells along with
the gene or cDNA of interest. Preferred selectable markers include
genes that confer resistance to drugs such as neomycin, hygromycin,
puromycin, zeocin, and methotrexate. The selectable marker may be
an amplifiable selectable marker. A preferred amplifiable
selectable marker is a dihydrofolate reductase (DHFR) sequence.
Further examples of selectable markers are well known to one of
skill in the art and include reporters such as enhanced green
fluorescent protein (EGFP), beta-galactosidase (.beta.-gal) or
chloramphenicol acetyltransferase (CAT). Selectable markers are
reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, Mass., incorporated herein by reference). The
person skilled in the art will easily be able to choose suitable
selectable markers. Any known selectable marker may be employed so
long as it is capable of being expressed simultaneously with the
nucleic acid encoding a gene product.
[0299] Selectable markers may be introduced into the cell on a
separate plasmid at the same time as the gene of interest, or they
may be introduced on the same plasmid. If, on the same plasmid, the
selectable marker and the gene of interest may be under the control
of different promoters or the same promoter, the latter arrangement
producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Pat. No.
4,713,339). It may also be advantageous to add additional DNA,
known as "carrier DNA," to the mixture that is introduced into the
cells.
[0300] After the cells have taken up the DNA, they are grown in an
appropriate growth medium, typically 1-2 days, to begin expressing
the gene of interest. As used herein the term "appropriate growth
medium" means a medium containing nutrients and other components
required for the growth of cells and the expression of the CFXTEN
of interest. Media generally include a carbon source, a nitrogen
source, essential amino acids, essential sugars, vitamins, salts,
phospholipids, protein and growth factors. For production of
gamma-carboxylated proteins, the medium will contain vitamin K,
preferably at a concentration of about 0.1 .mu.g/ml to about 5
.mu.g/ml. Drug selection is then applied to select for the growth
of cells that are expressing the selectable marker in a stable
fashion. For cells that have been transfected with an amplifiable
selectable marker the drug concentration may be increased to select
for an increased copy number of the cloned sequences, thereby
increasing expression levels. Clones of stably transfected cells
are then screened for expression of the CF polypeptide variant of
interest.
[0301] The transformed or transfected host cell is then cultured in
a suitable nutrient medium under conditions permitting expression
of the CF polypeptide variant after which the resulting peptide may
be recovered from the culture. The medium used to culture the cells
may be any conventional medium suitable for growing the host cells,
such as minimal or complex media containing appropriate
supplements. Suitable media are available from commercial suppliers
or may be prepared according to published recipes (e.g. in
catalogues of the American Type Culture Collection). The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0302] Gene expression may be measured in a sample directly, for
example, by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad.
Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in
situ hybridization, using an appropriately labeled probe, based on
the sequences provided herein. Alternatively, antibodies may be
employed that can recognize specific duplexes, including DNA
duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein
duplexes. The antibodies in turn may be labeled and the assay may
be carried out where the duplex is bound to a surface, so that upon
the formation of duplex on the surface, the presence of antibody
bound to the duplex can be detected.
[0303] Gene expression, alternatively, may be measured by
immunological of fluorescent methods, such as immunohistochemical
staining of cells or tissue sections and assay of cell culture or
body fluids or the detection of selectable markers, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of sample fluids may be
either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be prepared against a native
sequence CF polypeptide or against a synthetic peptide based on the
DNA sequences provided herein or against exogenous sequence fused
to CF and encoding a specific antibody epitope. Examples of
selectable markers are well known to one of skill in the art and
include reporters such as enhanced green fluorescent protein
(EGFP), beta-galactosidase (.beta.-gal) or chloramphenicol
acetyltransferase (CAT).
[0304] Expressed CFXTEN polypeptide product(s) may be purified via
methods known in the art or by methods disclosed herein. Procedures
such as gel filtration, affinity purification (e.g., using an
anti-CF antibody column), salt fractionation, ion exchange
chromatography, size exclusion chromatography, hydroxyapatite
adsorption chromatography, hydrophobic interaction chromatography
and gel electrophoresis may be used; each tailored to recover and
purify the fusion protein produced by the respective host cells.
Additional purification may be achieved by conventional chemical
purification means, such as high performance liquid chromatography.
Some expressed CFXTEN may require refolding during isolation and
purification. Methods of purification are described in Robert K.
Scopes, Protein Purification Principles and Practice, Charles R.
Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra.
Multi-step purification separations are also described in Baron, et
al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J.
Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it is
preferred that the CFXTEN fusion proteins of the invention are
substantially pure. Thus, in a preferred embodiment of the
invention the CFXTEN of the invention is purified to at least about
90 to 95% homogeneity, preferably to at least about 98%
homogeneity. Purity may be assessed by, e.g., gel electrophoresis,
HPLC, and amino-terminal amino acid sequencing.
VIII). Pharmaceutical Compositions
[0305] The present invention provides pharmaceutical compositions
comprising CFXTEN. In one embodiment, the pharmaceutical
composition comprises the CFXTEN fusion protein and at least one
pharmaceutically acceptable carrier. CFXTEN polypeptides of the
present invention can be formulated according to known methods to
prepare pharmaceutically useful compositions, whereby the
polypeptide is combined in admixture with a pharmaceutically
acceptable carrier vehicle, such as aqueous solutions or buffers,
pharmaceutically acceptable suspensions and emulsions. Examples of
non-aqueous solvents include propyl ethylene glycol, polyethylene
glycol and vegetable oils. Therapeutic formulations are prepared
for storage by mixing the active ingredient having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers, as described in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the
form of lyophilized formulations or aqueous solutions.
[0306] The pharmaceutical compositions can be administered orally,
intranasally, parenterally or by inhalation therapy, and may take
the form of tablets, lozenges, granules, capsules, pills, ampoules,
suppositories or aerosol form. They may also take the form of
suspensions, solutions and emulsions of the active ingredient in
aqueous or nonaqueous diluents, syrups, granulates or powders. In
addition, the pharmaceutical compositions can also contain other
pharmaceutically active compounds or a plurality of compounds of
the invention.
[0307] More particularly, the present pharmaceutical compositions
may be administered for therapy by any suitable route including
oral, rectal, nasal, topical (including transdermal, aerosol,
buccal and sublingual), vaginal, parenteral (including
subcutaneous, subcutaneous by infusion pump, intramuscular,
intravenous and intradermal), intravitreal, and pulmonary. It will
also be appreciated that the preferred route will vary with the
condition and age of the recipient, and the disease being
treated.
[0308] In one embodiment, the pharmaceutical composition is
administered subcutaneously. In this embodiment, the composition
may be supplied as a lyophilized powder to be reconstituted prior
to administration. The composition may also be supplied in a liquid
form, which can be administered directly to a patient. In one
embodiment, the composition is supplied as a liquid in a pre-filled
syringe such that a patient can easily self-administer the
composition.
[0309] Extended release formulations useful in the present
invention may be oral formulations comprising a matrix and a
coating composition. Suitable matrix materials may include waxes
(e.g., carnauba, bees wax, paraffin wax, ceresine, shellac wax,
fatty acids, and fatty alcohols), oils, hardened oils or fats
(e.g., hardened rapeseed oil, castor oil, beef tallow, palm oil,
and soya bean oil), and polymers (e.g., hydroxypropyl cellulose,
polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and
polyethylene glycol). Other suitable matrix tabletting materials
are microcrystalline cellulose, powdered cellulose, hydroxypropyl
cellulose, ethyl cellulose, with other carriers, and fillers.
Tablets may also contain granulates, coated powders, or pellets.
Tablets may also be multi-layered. Multi-layered tablets are
especially preferred when the active ingredients have markedly
different pharmacokinetic profiles. Optionally, the finished tablet
may be coated or uncoated.
[0310] The coating composition may comprise an insoluble matrix
polymer and/or a water soluble material. Water soluble materials
can be polymers such as polyethylene glycol, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone,
polyvinyl alcohol, or monomeric materials such as sugars (e.g.,
lactose, sucrose, fructose, mannitol and the like), salts (e.g.,
sodium chloride, potassium chloride and the like), organic acids
(e.g., fumaric acid, succinic acid, lactic acid, and tartaric
acid), and mixtures thereof. Optionally, an enteric polymer may be
incorporated into the coating composition. Suitable enteric
polymers include hydroxypropyl methyl cellulose, acetate succinate,
hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate
phthalate, cellulose acetate phthalate, cellulose acetate
trimellitate, shellac, zein, and polymethacrylates containing
carboxyl groups. The coating composition may be plasticised by
adding suitable plasticizers such as, for example, diethyl
phthalate, citrate esters, polyethylene glycol, glycerol,
acetylated glycerides, acetylated citrate esters, dibutylsebacate,
and castor oil. The coating composition may also include a filler,
which can be an insoluble material such as silicon dioxide,
titanium dioxide, talc, kaolin, alumina, starch, powdered
cellulose, MCC, or polacrilin potassium. The coating composition
may be applied as a solution or latex in organic solvents or
aqueous solvents or mixtures thereof. Solvents such as water, lower
alcohol, lower chlorinated hydrocarbons, ketones, or mixtures
thereof may be used.
[0311] The compositions of the invention may be formulated using a
variety of excipients. Suitable excipients include microcrystalline
cellulose (e.g. Avicel PH102, Avicel PH101), polymethacrylate,
poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl
methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl
methylcellulose (Methocel K100M, Premium CR Methocel K100M,
Methocel E5, Opadry.RTM.), magnesium stearate, talc, triethyl
citrate, aqueous ethylcellulose dispersion (Surelease.RTM.), and
protamine sulfate. The slow release agent may also comprise a
carrier, which can comprise, for example, solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents. Pharmaceutically acceptable salts can
also be used in these slow release agents, for example, mineral
salts such as hydrochlorides, hydrobromides, phosphates, or
sulfates, as well as the salts of organic acids such as acetates,
propionates, malonates, or benzoates. The composition may also
contain liquids, such as water, saline, glycerol, and ethanol, as
well as substances such as wetting agents, emulsifying agents, or
pH buffering agents. Liposomes may also be used as a carrier.
[0312] In another embodiment, the compositions of the present
invention are encapsulated in liposomes, which have demonstrated
utility in delivering beneficial active agents in a controlled
manner over prolonged periods of time. Liposomes are closed bilayer
membranes containing an entrapped aqueous volume. Liposomes may
also be unilamellar vesicles possessing a single membrane bilayer
or multilamellar vesicles with multiple membrane bilayers, each
separated from the next by an aqueous layer. The structure of the
resulting membrane bilayer is such that the hydrophobic (non-polar)
tails of the lipid are oriented toward the center of the bilayer
while the hydrophilic (polar) heads orient towards the aqueous
phase. In one embodiment, the liposome may be coated with a
flexible water soluble polymer that avoids uptake by the organs of
the mononuclear phagocyte system, primarily the liver and spleen.
Suitable hydrophilic polymers for surrounding the liposomes
include, without limitation, PEG, polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxethylacrylate,
hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and hydrophilic peptide sequences as described in
U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the
contents of which are incorporated by reference in their
entirety.
[0313] Liposomes may be comprised of any lipid or lipid combination
known in the art. For example, the vesicle-forming lipids may be
naturally-occurring or synthetic lipids, including phospholipids,
such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic
acid, phosphatidylserine, phasphatidylglycerol,
phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat.
Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also
be glycolipids, cerebrosides, or cationic lipids, such as
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE);
N-[1(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 3 [N--(N',N'-dimethylaminoethane) carbamoly] cholesterol
(DC-Chol); or dimethyldioctadecylammonium (DDAB) also as disclosed
in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the
proper range to impart stability to the vesicle as disclosed in
U.S. Pat. Nos. 5,916,588 and 5,874,104.
[0314] Additional liposomal technologies are described in U.S. Pat.
Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191;
6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104;
5,215,680; and 4,684,479, the contents of which are incorporated
herein by reference. These describe liposomes and lipid-coated
microbubbles, and methods for their manufacture. Thus, one skilled
in the art, considering both the disclosure of this invention and
the disclosures of these other patents could produce a liposome for
the extended release of the polypeptides of the present
invention.
[0315] For liquid formulations, a desired property is that the
formulation be supplied in a form that can pass through a 25, 28,
30, 31, 32 gauge needle for intravenous, intramuscular,
intraarticular, or subcutaneous administration.
[0316] Administration via transdermal formulations can be performed
using methods also known in the art, including those described
generally in, e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770,
4,861,800, 6,743,211, 6,945,952, 4,284,444, and WO 89/09051,
incorporated herein by reference in their entireties. A transdermal
patch is a particularly useful embodiment with polypeptides having
absorption problems. Patches can be made to control the release of
skin-permeable active ingredients over a 12 hour, 24 hour, 3 day,
and 7 day period. In one example, a 2-fold daily excess of a
polypeptide of the present invention is placed in a non-volatile
fluid. The compositions of the invention are provided in the form
of a viscous, non-volatile liquid. The penetration through skin of
specific formulations may be measures by standard methods in the
art (for example, Franz et al., J. Invest. Derm. 64:194-195
(1975)). Examples of suitable patches are passive transfer skin
patches, iontophoretic skin patches, or patches with microneedles
such as Nicoderm.
[0317] In other embodiments, the composition may be delivered via
intranasal, buccal, or sublingual routes to the brain to enable
transfer of the active agents through the olfactory passages into
the CNS and reducing the systemic administration. Devices commonly
used for this route of administration are included in U.S. Pat. No.
6,715,485. Compositions delivered via this route may enable
increased CNS dosing or reduced total body burden reducing systemic
toxicity risks associated with certain drugs. Preparation of a
pharmaceutical composition for delivery in a subdermally
implantable device can be performed using methods known in the art,
such as those described in, e.g., U.S. Pat. Nos. 3,992,518;
5,660,848; and 5,756,115.
[0318] Osmotic pumps may be used as slow release agents in the form
of tablets, pills, capsules or implantable devices. Osmotic pumps
are well known in the art and readily available to one of ordinary
skill in the art from companies experienced in providing osmotic
pumps for extended release drug delivery. Examples are ALZA's
DUROS.TM.; ALZA's OROS.TM.; Osmotica Pharmaceutical's Osmodex.TM.
system; Shire Laboratories' EnSoTrol.TM. system; and Alzet.TM..
Patents that describe osmotic pump technology are U.S. Pat. Nos.
6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532;
6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776;
4,200,0984; and 4,088,864, the contents of which are incorporated
herein by reference. One skilled in the art, considering both the
disclosure of this invention and the disclosures of these other
patents could produce an osmotic pump for the extended release of
the polypeptides of the present invention.
[0319] Syringe pumps may also be used as slow release agents. Such
devices are described in U.S. Pat. Nos. 4,976,696; 4,933,185;
5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585;
5,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the
contents of which are incorporated herein by reference. One skilled
in the art, considering both the disclosure of this invention and
the disclosures of these other patents could produce a syringe pump
for the extended release of the compositions of the present
invention.
IX). Pharmaceutical Kits
[0320] In another aspect, the invention provides a kit to
facilitate the use of the CFXTEN polypeptides. The kit comprises
the pharmaceutical composition provided herein, a label identifying
the pharmaceutical composition, and an instruction for storage,
reconstitution and/or administration of the pharmaceutical
compositions to a subject. In some embodiment, the kit comprises,
preferably: (a) an amount of a CFXTEN fusion protein composition
sufficient to treat a disease, condition or disorder upon
administration to a subject in need thereof; and (b) an amount of a
pharmaceutically acceptable carrier; together in a formulation
ready for injection or for reconstitution with sterile water,
buffer, or dextrose; together with a label identifying the CFXTEN
drug and storage and handling conditions, and a sheet of the
approved indications for the drug, instructions for the
reconstitution and/or administration of the CFXTEN drug for the use
for the prevention and/or treatment of a approved indication,
appropriate dosage and safety information, and information
identifying the lot and expiration of the drug. In another
embodiment of the foregoing, the kit can comprise a second
container that can carry a suitable diluent for the CFXTEN
composition, the use of which will provide the user with the
appropriate concentration of CFXTEN to be delivered to the
subject.
EXAMPLES
Example 1
Construction of XTEN_AD36 Motif Segments
[0321] The following example describes the construction of a
collection of codon-optimized genes encoding motif sequences of 36
amino acids. As a first step, a stuffer vector pCW0359 was
constructed based on a pET vector and that includes a T7 promoter.
pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease
recognition site followed by a stuffer sequence that is flanked by
BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted
such that they generate compatible overhangs after digestion. The
stuffer sequence is followed by a truncated version of the GFP gene
and a His tag. The stuffer sequence contains stop codons and thus
E. coli cells carrying the stuffer plasmid pCW0359 form
non-fluorescent colonies. The stuffer vector pCW0359 was digested
with BsaI and KpnI to remove the stuffer segment and the resulting
vector fragment was isolated by agarose gel purification. The
sequences were designated XTEN_AD36, reflecting the AD family of
motifs. Its segments have the amino acid sequence [X].sub.3 where X
is a 12 mer peptide with the sequences: GESPGGSSGSES, GSEGSSGPGESS,
GSSESGSSEGGP, or GSGGEPSESGSS. The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00011 AD1for: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC AD1rev:
ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC AD2for:
AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC AD2rev:
ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT AD3for:
AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC AD3rev:
ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA AD4for:
AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC
[0322] We also annealed the phosphorylated oligonucleotide 3
KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated
oligonucleotide pr.sub.--3 KpnIstopperRev: CCTCGAGTGAAGACGA. The
annealed oligonucleotide pairs were ligated, which resulted in a
mixture of products with varying length that represents the varying
number of 12 mer repeats ligated to one BbsI/KpnI segment. The
products corresponding to the length of 36 amino acids were
isolated from the mixture by preparative agarose gel
electrophoresis and ligated into the BsaI/KpnI digested stuffer
vector pCW0359. Most of the clones in the resulting library
designated LCW0401 showed green fluorescence after induction, which
shows that the sequence of XTEN_AD36 had been ligated in frame with
the GFP gene and that most sequences of XTEN_AD36 had good
expression levels.
[0323] We screened 96 isolates from library LCW0401 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 39 clones
were identified that contained correct XTEN_AD36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 9.
TABLE-US-00012 TABLE 9 DNA and Amino Acid Sequences for 36-mer
motifs File name Amino acid sequence Nucleotide sequence
LCW0401_001_GFP- GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAGC N_A01.ab1 SSGSESGESPGGSSGSES
TCAGGTGAATCTCCGGGTGGCTCTAGCGGTTCC GAGTCAGGTGAATCTCCTGGTGGTTCCAGCGGT
TCCGAGTCA LCW0401_002_GFP- GSEGSSGPGESSGESPGG
GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCT N_B01.ab1 SSGSESGSSESGSSEGGP
TCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCT GAATCAGGTTCCTCCGAAAGCGGTTCTTCCGAG
GGCGGTCCA LCW0401_003_GFP- GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGT N_C01.ab1 SSEGGPGESPGGSSGSES
CCAGGTTCCTCTGAAAGCGGTTCTTCTGAGGGT GGTCCAGGTGAATCTCCGGGTGGCTCCAGCGGT
TCCGAGTCA LCW0401_004_GFP- GSGGEPSESGSSGSSESG
GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGC N_D01.ab1 SSEGGPGSGGEPSESGSS
TCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGT GGTCCAGGTTCTGGTGGTGAACCTTCCGAGTCT
GGTAGCTCA LCW0401_007_GFP- GSSESGSSEGGPGSEGSS
GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGT N_F01.ab1 GPGESSGSEGSSGPGESS
CCAGGTAGCGAAGGTTCTTCCGGTCCAGGTGAG TCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGT
GAATCTTCA LCW0401_008_GFP- GSSESGSSEGGPGESPGG
GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGT N_G01.ab1 SSGSESGSEGSSGPGESS
CCAGGTGAATCTCCAGGTGGTTCCAGCGGTTCT GAGTCAGGTAGCGAAGGTTCTTCTGGTCCAGGT
GAATCCTCA LCW0401_012_GFP- GSGGEPSESGSSGSGGEP
GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGC N_H01.ab1 SESGSSGSEGSSGPGESS
TCAGGTTCCGGTGGCGAACCATCCGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCCGGTCCAGGT
GAGTCTTCA LCW0401_015_GFP- GSSESGSSEGGPGSEGSS
GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGT N_A02.ab1 GPGESSGESPGGSSGSES
CCAGGTAGCGAAGGTTCTTCTGGTCCAGGCGAA TCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGT
TCTGAGTCA LCW0401_016_GFP- GSSESGSSEGGPGSSESG
GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGT N_B02.ab1 SSEGGPGSSESGSSEGGP
CCAGGTTCCTCCGAAAGCGGTTCTTCCGAGGGC GGTCCAGGTTCTTCTGAAAGCGGTTCTTCCGAG
GGCGGTCCA LCW0401_020_GFP- GSGGEPSESGSSGSEGSS
GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGC N_E02.ab1 GPGESSGSSESGSSEGGP
TCAGGTAGCGAAGGTTCTTCTGGTCCAGGCGAA TCTTCAGGTTCCTCTGAAAGCGGTTCTTCTGAG
GGCGGTCCA LCW0401_022_GFP- GSGGEPSESGSSGSSESG
GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGC N_F02.ab1 SSEGGPGSGGEPSESGSS
TCAGGTTCTTCCGAAAGCGGTTCTTCTGAAGGT GGTCCAGGTTCCGGTGGCGAACCTTCTGAATCT
GGTAGCTCA LCW0401_024_GFP- GSGGEPSESGSSGSSESG
GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGC N_G02.ab1 SSEGGPGESPGGSSGSES
TCAGGTTCCTCCGAAAGCGGTTCTTCTGAAGGT GGTCCAGGTGAATCTCCAGGTGGTTCTAGCGGT
TCTGAATCA LCW0401_026_GFP- GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGC N_H02.ab1 SSGSESGSEGSSGPGESS
TCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCT GAATCAGGTAGCGAAGGTTCTTCTGGTCCTGGT
GAATCTTCA LCW0401_027_GFP- GSGGEPSESGSSGESPGG
GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGC N_A03.ab1 SSGSESGSGGEPSESGSS
TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCT GAGTCAGGTTCTGGTGGTGAACCTTCCGAGTCT
GGTAGCTCA LCW0401_028_GFP- GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGT N_B03.ab1 SSEGGPGSSESGSSEGGP
CCAGGTTCTTCCGAAAGCGGTTCTTCCGAGGGC GGTCCAGGTTCTTCCGAAAGCGGTTCTTCTGAA
GGCGGTCCA LCW0401_030_GFP- GESPGGSSGSESGSEGSS
GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAG N_C03.ab1 GPGESSGSEGSSGPGESS
TCAGGTAGCGAAGGTTCTTCCGGTCCGGGTGAG TCCTCAGGTAGCGAAGGTTCTTCCGGTCCTGGT
GAGTCTTCA LCW0401_031_GFP- GSGGEPSESGSSGSGGEP
GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGC N_D03.ab1 SESGSSGSSESGSSEGGP
TCAGGTTCCGGTGGTGAACCTTCTGAATCTGGT AGCTCAGGTTCTTCTGAAAGCGGTTCTTCCGAG
GGCGGTCCA LCW0401_033_GFP- GSGGEPSESGSSGSGGEP
GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGC N_E03.ab1 SESGSSGSGGEPSESGSS
TCAGGTTCCGGTGGCGAACCATCCGAGTCTGGT AGCTCAGGTTCCGGTGGTGAACCATCCGAGTCT
GGTAGCTCA LCW0401_037_GFP- GSGGEPSESGSSGSSESG
GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGC N_F03.ab1 SSEGGPGSEGSSGPGESS
TCAGGTTCCTCCGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTAGCGAAGGTTCTTCTGGTCCGGGC
GAGTCTTCA LCW0401_038_GFP- GSGGEPSESGSSGSEGSS
GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAGC N_G03.ab1 GPGESSGSGGEPSESGSS
TCAGGTAGCGAAGGTTCTTCTGGTCCGGGTGAG TCTTCAGGTTCTGGTGGCGAACCGTCCGAATCT
GGTAGCTCA LCW0401_039_GFP- GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGC N_H03.ab1 SSGSESGSGGEPSESGSS
TCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCC GAGTCAGGTTCTGGTGGCGAACCTTCCGAATCT
GGTAGCTCA LCW0401_040_GFP- GSSESGSSEGGPGSGGEP
GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGT N_A04.ab1 SESGSSGSSESGSSEGGP
CCAGGTTCCGGTGGTGAACCATCTGAATCTGGT AGCTCAGGTTCTTCTGAAAGCGGTTCTTCTGAA
GGTGGTCCA LCW0401_042_GFP- GSEGSSGPGESSGESPGG
GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCT N_C04.ab1 SSGSESGSEGSSGPGESS
TCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCC GAGTCAGGTAGCGAAGGTTCTTCTGGTCCTGGC
GAGTCCTCA LCW0401_046_GFP- GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGT N_D04.ab1 SSEGGPGSSESGSSEGGP
CCAGGTTCTTCCGAAAGCGGTTCTTCTGAGGGC GGTCCAGGTTCCTCCGAAAGCGGTTCTTCTGAG
GGTGGTCCA LCW0401_047_GFP- GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAGC N_E04.ab1 SSGSESGESPGGSSGSES
TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCC GAGTCAGGTGAATCTCCGGGTGGTTCCAGCGGT
TCTGAGTCA LCW0401_051_GFP- GSGGEPSESGSSGSEGSS
GGTTCTGGTGGCGAACCATCTGAGTCTGGTAGC N_F04.ab1 GPGESSGESPGGSSGSES
TCAGGTAGCGAAGGTTCTTCCGGTCCAGGCGAG TCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGT
TCTGAGTCA LCW0401_053_GFP- GESPGGSSGSESGESPGG
GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAG N_H04.ab1 SSGSESGESPGGSSGSES
TCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCC GAGTCAGGTGAATCTCCTGGTGGTTCTAGCGGT
TCTGAATCA LCW0401_054_GFP- GSEGSSGPGESSGSEGSS
GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCT N_A05.ab1 GPGESSGSGGEPSESGSS
TCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAA TCCTCAGGTTCCGGTGGCGAACCATCTGAATCT
GGTAGCTCA LCW0401_059_GFP- GSGGEPSESGSSGSEGSS
GGTTCTGGTGGCGAACCATCCGAATCTGGTAGC N_D05.ab1 GPGESSGESPGGSSGSES
TCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAA TCTTCAGGTGAATCTCCAGGTGGCTCTAGCGGT
TCCGAATCA LCW0401_060_GFP- GSGGEPSESGSSGSSESG
GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGC N_E05.ab1 SSEGGPGSGGEPSESGSS
TCAGGTTCCTCTGAAAGCGGTTCTTCCGAGGGT GGTCCAGGTTCCGGTGGTGAACCTTCTGAGTCT
GGTAGCTCA LCW0401_061_GFP- GSSESGSSEGGPGSGGEP
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGT N_F05.ab1 SESGSSGSEGSSGPGESS
CCAGGTTCTGGTGGCGAACCATCTGAATCTGGT AGCTCAGGTAGCGAAGGTTCTTCCGGTCCGGGT
GAATCTTCA LCW0401_063_GFP- GSGGEPSESGSSGSEGSS
GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGC N_H05.ab1 GPGESSGSEGSSGPGESS
TCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAG TCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGT
GAATCTTCA LCW0401_066_GFP- GSGGEPSESGSSGSSESG
GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGC N_B06.ab1 SSEGGPGSGGEPSESGSS
TCAGGTTCTTCCGAAAGCGGTTCTTCCGAAGGC GGTCCAGGTTCTGGTGGTGAACCGTCCGAATCT
GGTAGCTCA LCW0401_067_GFP- GSGGEPSESGSSGESPGG
GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGC N_C06.ab1 SSGSESGESPGGSSGSES
TCAGGTGAATCTCCGGGTGGTTCTAGCGGTTCC GAATCAGGTGAATCTCCAGGTGGTTCTAGCGGT
TCCGAATCA LCW0401_069_GFP- GSGGEPSESGSSGSGGEP
GGTTCCGGTGGTGAACCATCTGAGTCTGGTAGC N_D06.ab1 SESGSSGESPGGSSGSES
TCAGGTTCCGGTGGCGAACCGTCCGAGTCTGGT AGCTCAGGTGAATCTCCGGGTGGTTCCAGCGGT
TCCGAATCA LCW0401_070_GFP- GSEGSSGPGESSGSSESG
GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCC N_E06.ab1 SSEGGPGSEGSSGPGESS
TCAGGTTCCTCCGAAAGCGGTTCTTCCGAAGGT GGTCCAGGTAGCGAAGGTTCTTCCGGTCCTGGT
GAATCTTCA LCW0401_078_GFP- GSSESGSSEGGPGESPGG
GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGT N_F06.ab1 SSGSESGESPGGSSGSES
CCAGGTGAATCTCCGGGTGGCTCCAGCGGTTCT GAATCAGGTGAATCTCCTGGTGGCTCCAGCGGT
TCCGAGTCA LCW0401_079_GFP- GSEGSSGPGESSGSEGSS
GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCT N_G06.ab1 GPGESSGSGGEPSESGSS
TCAGGTAGCGAAGGTTCTTCCGGTCCTGGCGAG TCTTCAGGTTCCGGTGGCGAACCGTCCGAATCT
GGTAGCTCA
Example 2
Construction of XTEN_AE36 Segments
[0324] A codon library encoding XTEN sequences of 36 amino acid
length was constructed. The XTEN sequence was designated XTEN_AE36.
Its segments have the amino acid sequence [X].sub.3 where X is a 12
mer peptide with the sequence: GSPAGSPTSTEE, GSEPATSGSE TP, GTSESA
TPESGP, or GTSTEPSEGSAP. The insert was obtained by annealing the
following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00013 AE1for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA AE1rev:
ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT AE2for:
AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC AE2rev:
ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT AE3for:
AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC AE3rev:
ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT AE4for:
AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC AE4rev:
ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT
[0325] We also annealed the phosphorylated oligonucleotide 3
KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated
oligonucleotide pr.sub.--3 KpnIstopperRev: CCTCGAGTGAAGACGA. The
annealed oligonucleotide pairs were ligated, which resulted in a
mixture of products with varying length that represents the varying
number of 12 mer repeats ligated to one BbsUKpnI segment. The
products corresponding to the length of 36 amino acids were
isolated from the mixture by preparative agarose gel
electrophoresis and ligated into the BsaI/KpnI digested stuffer
vector pCW0359. Most of the clones in the resulting library
designated LCW0402 showed green fluorescence after induction which
shows that the sequence of XTEN_AE36 had been ligated in frame with
the GFP gene and most sequences of XTEN_AE36 show good
expression.
[0326] We screened 96 isolates from library LCW0402 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 37 clones
were identified that contained correct XTEN_AE36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 10.
TABLE-US-00014 TABLE 10 DNA and Amino Acid Sequences for 36-mer
motifs File name Amino acid sequence Nucleotide sequence
LCW0402_002_GFP- GSPAGSPTSTEEGTSE
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA N_A07.ab1 SATPESGPGTSTEPSE
GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA GSAP
GGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA LCW0402_003_GFP-
GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCA N_B07.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCA GSAP
GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA LCW0402_004_GFP-
GTSTEPSEGSAPGTSE GGTACCTCTACCGAACCGTCTGAAGGTAGCGCACCA N_C07.ab1
SATPESGPGTSESATP GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCA ESGP
GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA LCW0402_005_GFP-
GTSTEPSEGSAPGTSE GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCA N_D07.ab1
SATPESGPGTSESATP GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA ESGP
GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCW0402_006_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCA N_E07.ab1
SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCA STEE
GGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA LCW0402_008_GFP-
GTSESATPESGPGSEP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA N_F07.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA GSAP
GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA LCW0402_009_GFP-
GSPAGSPTSTEEGSPA GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAA N_G07.ab1
GSPTSTEEGSEPATSG GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAA SETP
GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA LCW0402_011_GFP-
GSPAGSPTSTEEGTSE GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAA N_A08.ab1
SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCA GSAP
GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA LCW0402_012_GFP-
GSPAGSPTSTEEGSPA GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAA N_B08.ab1
GSPTSTEEGTSTEPSE GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAA GSAP
GGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA LCW0402_013_GFP-
GTSESATPESGPGTST GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCA N_C08.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCA GSAP
GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCW0402_014_GFP-
GTSTEPSEGSAPGSPA GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCA N_D08.ab1
GSPTSTEEGTSTEPSE GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAA GSAP
GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA LCW0402_015_GFP-
GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA N_E08.ab1
GSPTSTEEGTSESATP GGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAA ESGP
GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA LCW0402_016_GFP-
GTSTEPSEGSAPGTSE GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA N_F08.ab1
SATPESGPGTSESATP GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA ESGP
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA LCW0402_020_GFP-
GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCA N_G08.ab1
ATSGSETPGSPAGSPT GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA STEE
GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA LCW0402_023_GFP-
GSPAGSPTSTEEGTSE GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA N_A09.ab1
SATPESGPGSEPATSG GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA LCW0402_024_GFP-
GTSESATPESGPGSPA GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA N_B09.ab1
GSPTSTEEGSPAGSPT GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA STEE
GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA LCW0402_025_GFP-
GTSTEPSEGSAPGTSE GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA N_C09.ab1
SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_026_GFP-
GSPAGSPTSTEEGTST GGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAA N_D09.ab1
EPSEGSAPGSEPATSG GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA SETP
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA LCW0402_027_GFP-
GSPAGSPTSTEEGTST GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA N_E09.ab1
EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA LCW0402_032_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCA N_H09.ab1
SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCA STEE
GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA LCW0402_034_GFP-
GTSESATPESGPGTST GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCA N_A10.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_036_GFP-
GSPAGSPTSTEEGTST GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA N_C10.ab1
EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA GSAP
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA LCW0402_039_GFP-
GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCA N_E10.ab1
EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCA GSAP
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW0402_040_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA N_F10.ab1
SATPESGPGTSTEPSE GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GSAP
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA LCW0402_041_GFP-
GTSTEPSEGSAPGSPA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA N_G10.ab1
GSPTSTEEGTSTEPSE GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA GSAP
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA LCW0402_050_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCA N_A11.ab1
SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCA SETP
GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA LCW0402_051_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCA N_B11.ab1
SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCA SETP
GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA LCW0402_059_GFP-
GSEPATSGSETPGSEP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCA N_E11.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCA GSAP
GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA LCW0402_060_GFP-
GTSESATPESGPGSEP GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA N_F11.ab1
ATSGSETPGSEPATSG GGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA SETP
GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA LCW0402_061_GFP-
GTSTEPSEGSAPGTST GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA N_G11.ab1
EPSEGSAPGTSESATP GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA ESGP
GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA LCW0402_065_GFP-
GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA N_A12.ab1
SATPESGPGTSESATP GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCA ESGP
GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA LCW0402_066_GFP-
GSEPATSGSETPGSEP GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCA N_B12.ab1
ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCA GSAP
GGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA LCW0402_067_GFP-
GSEPATSGSETPGTST GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA N_C12.ab1
EPSEGSAPGSEPATSG GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA SETP
GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA LCW0402_069_GFP-
GTSTEPSEGSAPGTST GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCA N_D12.ab1
EPSEGSAPGSEPATSG GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA LCW0402_073_GFP-
GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCA N_F12.ab1
ATSGSETPGSPAGSPT GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCA STEE
GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW0402_074_GFP-
GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA N_G12.ab1
GSPTSTEEGTSESATP GGTAGCCCAGCTGGTTCTCCAACCTCTACTGAGGAA ESGP
GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA LCW0402_075_GFP-
GTSESATPESGPGSEP GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA N_H12.ab1
ATSGSETPGTSESATP GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA ESGP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA
Example 3
Construction of XTEN_AF36 Segments
[0327] A codon library encoding sequences of 36 amino acid length
was constructed. The sequences were designated XTEN_AF36. Its
segments have the amino acid sequence [.alpha.]3 where X is a 12
mer peptide with the sequence: GSTSESPSGTAP, GTSTPESGSASP,
GTSPSGESSTAP, or GSTSSTAESPGP. The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00015 AF1for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC AF1rev:
ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA AF2for:
AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC AF2rev:
ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT AF3for:
AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC AF3rev:
ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT AF4for:
AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC AF4rev:
ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA
[0328] We also annealed the phosphorylated oligonucleotide 3
KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated
oligonucleotide pr.sub.--3 KpnIstopperRev: CCTCGAGTGAAGACGA. The
annealed oligonucleotide pairs were ligated, which resulted in a
mixture of products with varying length that represents the varying
number of 12 mer repeats ligated to one BbsI/KpnI segment The
products corresponding to the length of 36 amino acids were
isolated from the mixture by preparative agarose gel
electrophoresis and ligated into the BsaI/KpnI digested stuffer
vector pCW0359. Most of the clones in the resulting library
designated LCW0403 showed green fluorescence after induction which
shows that the sequence of XTEN_AF36 had been ligated in frame with
the GFP gene and most sequences of XTEN_AF36 show good
expression.
[0329] We screened 96 isolates from library LCW0403 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 44 clones
were identified that contained correct XTEN_AF36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 11.
TABLE-US-00016 TABLE 11 DNA and Amino Acid Sequences for 36-mer
motifs File name Amino acid sequence Nucleotide sequence
LCW0403_004_GFP- GTSTPESGSASPGTSP
GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA N_A01.ab1 SGESSTAPGTSPSGES
GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAG STAP
GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_GFP-
GTSPSGESSTAPGSTS GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCA N_B01.ab1
STAESPGPGTSPSGES GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG STAP
GTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_GFP-
GSTSSTAESPGPGTSP GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAG N_C01.ab1
SGESSTAPGTSTPESG GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGG SASP
TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAG N_D01.ab1
STAESPGPGTSPSGES GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAG STAP
GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_GFP-
GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_E01.ab1
SGESSTAPGTSTPESG GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA LCW0403_010_GFP-
GSTSSTAESPGPGTST GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG N_F01.ab1
PESGSASPGSTSESPS GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG GTAP
GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_011_GFP-
GSTSSTAESPGPGTST GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG N_G01.ab1
PESGSASPGTSTPESG GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG SASP
GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_GFP-
GSTSESPSGTAPGTSP GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG N_H01.ab1
SGESSTAPGSTSESPS GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG GTAP
TTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCA N_A02.ab1
STAESPGPGTSPSGES GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_GFP-
GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAG N_B02.ab1
PESGSASPGSTSESPS GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAG GTAP
GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG N_C02.ab1
STAESPGPGTSPSGES GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG STAP
TACCTCCCCGAGCGGTGAATCTTCTACTGCACCA LCW0403_017_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG N_D02.ab1
ESPSGTAPGSTSSTAE GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAG SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCA N_E02.ab1
STAESPGPGSTSSTAE GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAG SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCA LCW0403_019_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG N_F02.ab1
STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGG SPGP
TTCCACTAGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG N_H02.ab1
ESPSGTAPGSTSESPS GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG GTAP
TTCTACCAGCGAATCTCCTTCTGGTACTGCACCA LCW0403_024_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAG N_A03.ab1
STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP
TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_GFP-
GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAG N_B03.ab1
STAESPGPGTSPSGES GTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGG STAP
TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_GFP-
GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAG N_D03.ab1
PSGATGSPGSSTPSGA GTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGG TGSP
TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA LCW0403_029_GFP-
GTSPSGESSTAPGTST GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAG N_E03.ab1
PESGSASPGSTSSTAE GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAG SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAG N_F03.ab1
STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGG SASP
TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_031_GFP-
GTSPSGESSTAPGSTS GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAG N_G03.ab1
STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGG SASP
TACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA LCW0403_033_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAG N_H03.ab1
STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP
TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACCGCTGAATCTCCGGGCCCA N_A04.ab1
ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA SPGP
GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_GFP-
GSTSSTAESPGPGTSP GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG N_B04.ab1
SGESSTAPGTSTPESG GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAG SASP
GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG N_C04.ab1
ESPSGTAPGTSPSGES GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAG N_D04.ab1
ESPSGTAPGTSTPESG GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAG SASP
GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_GFP-
GTSTPESGSASPGSTS GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG N_E04.ab1
STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_GFP-
GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA N_F04.ab1
ESPSGTAPGTSPSGES GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAG STAP
GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAG N_G04.ab1
STAESPGPGSTSESPS GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG GTAP
GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_GFP-
GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCTGGCCCA N_H04.ab1
STAESPGPGTSTPESG GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAG SASP
GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_GFP-
GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG N_A05.ab1
STAESPGPGSTSESPS GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGG GTAP
TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_GFP-
GTSPSGESSTAPGSTS GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA N_B05.ab1
ESPSGTAPGSTSSTAE GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG SPGP
GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_GFP-
GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG N_C05.ab1
SGESSTAPGSTSSTAE GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGG SPGP
TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA LCW0403_057_GFP-
GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG N_D05.ab1
ESPSGTAPGTSPSGES GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG STAP
GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_GFP-
GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG N_E05.ab1
ESPSGTAPGTSTPESG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG SASP
GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_GFP-
GTSTPESGSASPGSTS GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCA N_F05.ab1
ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA SPGP
GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_GFP-
GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCA N_G05.ab1
SGESSTAPGTSPSGES GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAG STAP
GTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_GFP-
GTSPSGESSTAPGTSP GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAG N_H05.ab1
SGESSTAPGTSPSGES GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG STAP
TACCTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_GFP-
GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_A06.ab1
PESGSASPGSTSESPS GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG GTAP
TTCTACTAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_GFP-
GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAG N_B06.ab1
SGESSTAPGTSPSGES GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG STAP
TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG N_C06.ab1
PESGSASPGSTSSTAE GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGG SPGP
TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_068_GFP-
GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG N_D06.ab1
STAESPGPGSTSESPS GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGG GTAP
TTCTACCAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA N_E06.ab1
PESGSASPGTSTPESG GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAG SASP
GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_GFP-
GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG N_F06.ab1
PESGSASPGTSTPESG GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA
Example 4
Construction of XTEN_AG36 Segments
[0330] A codon library encoding sequences of 36 amino acid length
was constructed. The sequences were designated XTEN_AG36. Its
segments have the amino acid sequence [X].sub.3 where X is a 12 mer
peptide with the sequence: GTPGSGTASSSP, GSSTPSGATGSP,
GSSPSASTGTGP, or GASPGTSSTGSP. The insert was obtained by annealing
the following pairs of phosphorylated synthetic oligonucleotide
pairs:
TABLE-US-00017 AG1for: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC AG1rev:
ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT AG2for:
AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC AG2rev:
ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT AG3for:
AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC AG3rev:
ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA AG4for:
AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC AG4rev:
ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC
[0331] We also annealed the phosphorylated oligonucleotide 3
KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC and the non-phosphorylated
oligonucleotide pr.sub.--3 KpnIstopperRev: CCTCGAGTGAAGACGA. The
annealed oligonucleotide pairs were ligated, which resulted in a
mixture of products with varying length that represents the varying
number of 12 mer repeats ligated to one BbsI/KpnI segment. The
products corresponding to the length of 36 amino acids were
isolated from the mixture by preparative agarose gel
electrophoresis and ligated into the BsaI/KpnI digested stuffer
vector pCW0359. Most of the clones in the resulting library
designated LCW0404 showed green fluorescence after induction which
shows that the sequence of XTEN_AG36 had been ligated in frame with
the GFP gene and most sequences of XTEN_AG36 show good
expression.
[0332] We screened 96 isolates from library LCW0404 for high level
of fluorescence by stamping them onto agar plate containing IPTG.
The same isolates were evaluated by PCR and 48 isolates were
identified that contained segments with 36 amino acids as well as
strong fluorescence. These isolates were sequenced and 44 clones
were identified that contained correct XTEN_AG36 segments. The file
names of the nucleotide and amino acid constructs for these
segments are listed in Table 12.
TABLE-US-00018 TABLE 12 DNA and Amino Acid Sequences for 36-mer
motifs File name Amino acid sequence Nucleotide sequence
LCW0404_001_GFP- GASPGTSSTGSPGTPG
GGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCA N_A07.ab1 SGTASSSPGSSTPSGA
GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAG TGSP
GTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_GFP-
GSSTPSGATGSPGSSP GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG N_B07.ab1
SASTGTGPGSSTPSGA GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG TGSP
TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_GFP-
GASPGTSSTGSPGSSP GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG N_C07.ab1
SASTGTGPGSSTPSGA GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGG TGSP
TAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCA LCW0404_007_GFP-
GTPGSGTASSSPGSST GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAG N_D07.ab1
PSGATGSPGASPGTSS GTAGCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGG TGSP
TGCATCCCCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_GFP-
GTPGSGTASSSPGASP GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAG N_E07.ab1
GTSSTGSPGSRPSAST GTGCTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGG GTGP
TTCTAGACCTTCTGCATCCACCGGTACTGGTCCA LCW0404_011_GFP-
GASPGTSSTGSPGSST GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAG N_F07.ab1
PSGATGSPGASPGTSS GTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGG TGSP
TGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_012_GFP-
GTPGSGTASSSPGSST GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCA N_G07.ab1
PSGATGSPGSSTPSGA GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG TGSP
GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA LCW0404_014_GFP-
GASPGTSSTGSPGASP GGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAG N_H07.ab1
GTSSTGSPGASPGTSS GTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG TGSP
TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_GFP-
GSSTPSGATGSPGSSP GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA N_A08.ab1
SASTGTGPGASPGTSS GGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAG TGSP
GTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAG N_B08.ab1
PSGATGSPGTPGSGT GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG ASSSP
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG N_C08.ab1
PSGATGSPGASPGTSS GTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGG TGSP
TGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_018_GFP-
GTPGSGTASSSPGSSP GGTACTCCTGGTAGCGGTACCGCATCTTCCTCTCCAG N_D08.ab1
SASTGTGPGSSTPSGA GTTCTAGCCCTTCTGCATCTACCGGTACCGGTCCAGG TGSP
TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCA LCW0404_023_GFP-
GASPGTSSTGSPGSSP GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAG N_F08.ab1
SASTGTGPGTPGSGT GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGG ASSSP
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG N_G08.ab1
PSGATGSPGASPGTSS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG TGSP
TGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_GFP-
GTPGSGTASSSPGSST GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAG N_A09.ab1
PSGATGSPGSSPSAST GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG GTGP
TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCA LCW0404_030_GFP-
GSSTPSGATGSPGTPG GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAG N_B09.ab1
SGTASSSPGTPGSGTA GTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG SSSP
GTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCA LCW0404_031_GFP-
GTPGSGTASSSPGSST GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG N_C09.ab1
PSGATGSPGASPGTSS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG TGSP
TGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA LCW0404_034_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG N_D09.ab1
PSGATGSPGASPGTSS GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAG TGSP
GTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_GFP-
GASPGTSSTGSPGTPG GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG N_E09.ab1
SGTASSSPGSSTPSGA GTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAG TGSP
GTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCA LCW0404_036_GFP-
GSSPSASTGTGPGSST GGTTCTAGCCCGTCTGCTTCCACCGGTACTGGCCCAG N_F09.ab1
PSGATGSPGTPGSGT GTAGCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGG ASSSP
TACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_GFP-
GASPGTSSTGSPGSSP GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG N_G09.ab1
SASTGTGPGSSTPSGA GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG TGSP
TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_GFP-
GASPGTSSTGSPGSST GGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCA N_H09.ab1
PSGATGSPGSSTPSGA GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG TGSP
GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_GFP-
GTPGSGTASSSPGSST GGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAG N_A10.ab1
PSGATGSPGTPGSGT GTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGG ASSSP
TACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_GFP-
GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAG N_C10.ab1
PSGATGSPGSSTPSGA GTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGG TGSP
TAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_GFP-
GASPGTSSTGSPGSSP GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAG N_D10.ab1
SASTGTGPGSSPSAST GTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGG GTGP
TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA LCW0404_047_GFP-
GTPGSGTASSSPGASP GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG N_F10.ab1
GTSSTGSPGASPGTSS GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGG TGSP
TGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA LCW0404_048_GFP-
GSSTPSGATGSPGASP GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG N_G10.ab1
GTSSTGSPGSSTPSGA GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG TGSP
TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_GFP-
GSSTPSGATGSPGTPG GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG N_H10.ab1
SGTASSSPGSSTPSGA GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG TGSP
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_GFP-
GASPGTSSTGSPGSSP GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAG N_A11.ab1
SASTGTGPGSSTPSGA GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG TGSP
TAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAG N_B11.ab1
PSGATGSPGSSTPSGA GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG TGSP
TAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCA LCW0404_052_GFP-
GASPGTSSTGSPGTPG GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCA N_C11.ab1
SGTASSSPGASPGTSS GGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAG TGSP
GTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_GFP-
GSSTPSGATGSPGSSP GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAG N_D11.ab1
SASTGTGPGASPGTSS GTTCTAGCCCGTCTGCATCCACTGGTACCGGTCCAGG TGSP
TGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_057_GFP-
GASPGTSSTGSPGSST GGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAG N_E11.ab1
PSGATGSPGSSPSAST GTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGG GTGP
TTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_GFP-
GTPGSGTASSSPGSST GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG N_F11.ab1
PSGATGSPGASPGTSS GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGG TGSP
TGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_GFP-
GSSTPSGATGSPGTPG GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA N_G11.ab1
SGTASSSPGSSTPSGA GGTACTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAG TGSP
GTAGCTCTACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_GFP-
GSSPSASTGTGPGSSP GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAG N_H11.ab1
SASTGTGPGASPGTSS GTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGG TGSP
TGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_GFP-
GTPGSGTASSSPGSST GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAG N_A12.ab1
PSGATGSPGSNPSAST GTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGG GTGP
TTCTAACCCTTCTGCATCCACCGGTACCGGCCCA LCW0404_068_GFP-
GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAG N_B12.ab1
PSGATGSPGASPGTSS GTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGG TGSP
TGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_069_GFP-
GSSTPSGATGSPGASP GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAG N_C12.ab1
GTSSTGSPGTPGSGTA GTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAG SSSP
GTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCA LCW0404_070_GFP-
GSSTPSGATGSPGSST GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG N_D12.ab1
PSGATGSPGSSTPSGA GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG TGSP
TAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCA LCW0404_073_GFP-
GASPGTSSTGSPGTPG GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAG N_E12.ab1
SGTASSSPGSSTPSGA GTACCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGG TGSP
TAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCA LCW0404_075_GFP-
GSSTPSGATGSPGSSP GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAG N_F12.ab1
SASTGTGPGSSPSAST GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG GTGP
TTCTAGCCCGTCTGCATCTACTGGTACTGGTCCA LCW0404_080_GFP-
GASPGTSSTGSPGSSP GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAG N_G12.ab1
SASTGTGPGSSPSAST GTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGG GTGP
TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA LCW0404_081_GFP-
GASPGTSSTGSPGSSP GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAG N_H12.ab1
SASTGTGPGTPGSGT GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG ASSSP
TACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA
Example 5
Construction of XTEN_AE864
[0333] XTEN_AE864 was constructed from serial dimerization of
XTEN_AE36 to AE72, 144, 288, 576 and 864. A collection of XTEN_AE72
segments was constructed from 37 different segments of XTEN_AE36.
Cultures of E. coli harboring all 37 different 36-amino acid
segments were mixed and plasmid was isolated. This plasmid pool was
digested with BsaI/NcoI to generate the small fragment as the
insert. The same plasmid pool was digested with BbsI/NcoI to
generate the large fragment as the vector. The insert and vector
fragments were ligated resulting in a doubling of the length and
the ligation mixture was transformed into BL21Gold(DE3) cells to
obtain colonies of XTEN_AE72.
[0334] This library of XTEN_AE72 segments was designated LCW0406.
All clones from LCW0406 were combined and dimerized again using the
same process as described above yielding library LCW0410 of
XTEN_AE144. All clones from LCW0410 were combined and dimerized
again using the same process as described above yielding library
LCW0414 of XTEN_AE288. Two isolates LCW0414.001 and LCW0414.002
were randomly picked from the library and sequenced to verify the
identities. All clones from LCW0414 were combined and dimerized
again using the same process as described above yielding library
LCW0418 of XTEN_AE576. We screened 96 isolates from library LCW0418
for high level of GFP fluorescence. 8 isolates with right sizes of
inserts by PCR and strong fluorescence were sequenced and 2
isolates (LCW0418.018 and LCW0418.052) were chosen for future use
based on sequencing and expression data.
[0335] The specific clone pCW0432 of XTEN_AE864 was constructed by
combining LCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288
using the same dimerization process as described above.
Example 6
Construction of XTEN_AM144
[0336] A collection of XTEN_AM144 segments was constructed starting
from 37 different segments of XTEN_AE36, 44 segments of XTEN_AF36,
and 44 segments of XTEN_AG36.
[0337] Cultures of E. coli harboring all 125 different 36-amino
acid segments were mixed and plasmid was isolated. This plasmid
pool was digested with BsaI/NcoI to generate the small fragment as
the insert. The same plasmid pool was digested with BbsI/NcoI to
generate the large fragment as the vector. The insert and vector
fragments were ligated resulting in a doubling of the length and
the ligation mixture was transformed into BL21Gold(DE3) cells to
obtain colonies of XTEN_AM72.
[0338] This library of XTEN_AM72 segments was designated LCW0461.
All clones from LCW0461 were combined and dimerized again using the
same process as described above yielding library LCW0462. 1512
Isolates from library LCW0462 were screened for protein expression.
Individual colonies were transferred into 96 well plates and
cultured overnight as starter cultures. These starter cultures were
diluted into fresh autoinduction medium and cultured for 20-30 h.
Expression was measured using a fluorescence plate reader with
excitation at 395 nm and emission at 510 nm. 192 isolates showed
high level expression and were submitted to DNA sequencing. Most
clones in library LCW0462 showed good expression and similar
physicochemical properties suggesting that most combinations of
XTEN_AM36 segments yield useful XTEN sequences. 30 isolates from
LCW0462 were chosen as a preferred collection of XTEN_AM144
segments for the construction of multifunctional proteins that
contain multiple XTEN segments. The file names of the nucleotide
and amino acid constructs for these segments are listed in Table
13.
TABLE-US-00019 TABLE 13 DNA and amino acid sequences for AM144
segments Clone Sequence Trimmed Protein Sequence LCW462_r1
GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG GTPGSGTASSSPGSST
GTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGG PSGATGSPGSSTPSGA
TAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGT TGSPGSPAGSPTSTEE
AGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTA GTSESATPESGPGTST
CTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTAC EPSEGSAPGSSPSAST
CTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTTCT GTGPGSSPSASTGTGP
AGCCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTA GASPGTSSTGSPGTST
GCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT EPSEGSAPGTSTEPSE
CCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTA GSAPGSEPATSGSETP
CCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTAC
TGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC GGCAACCTCCGGTTCTGAAACTCCA
LCW462_r5 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAG GSTSESPSGTAPGSTS
GTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAGG ESPSGTAPGTSPSGES
TACTTCTCCGAGCGGCGAATCTTCTACTGCTCCAGGT STAPGTSTEPSEGSAP
ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTSE
CCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC SATPESGPGASPGTSS
TTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTGCA TGSPGSSTPSGATGSP
TCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTC GASPGTSSTGSPGSTS
TACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC ESPSGTAPGSTSESPS
CGGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAG GTAPGTSTPESGSASP
CGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC
GAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCC CTGAAAGCGGTTCCGCTTCTCCA
LCW462_r9 GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAG GTSTEPSEGSAPGTSE
GTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGG SATPESGPGTSESATP
TACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT ESGPGTSTEPSEGSAP
ACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA GTSESATPESGPGTST
CTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTAC EPSEGSAPGTSTEPSE
TTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACT GSAPGSEPATSGSETP
TCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCG GSPAGSPTSTEEGASP
AACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCC GTSSTGSPGSSPSAST
GGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCT GTGPGSSPSASTGTGP
CCTGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCC
CTTCTGCTTCTACCGGTACTGGTCCAGGTTCTAGCCCT TCTGCATCCACTGGTACTGGTCCA
LCW462_r10 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAG GSEPATSGSETPGTSE
GTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG SATPESGPGTSESATP
TACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGT ESGPGSTSESPSGTAP
TCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTT GSTSESPSGTAPGTSP
CTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTAC SGESSTAPGASPGTSS
TTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTGCA TGSPGSSPSASTGTGP
TCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTTCTAG GSSTPSGATGSPGSST
CCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCT PSGATGSPGSSTPSGA
ACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTA TGSPGASPGTSSTGSP
CTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTAC
TCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTG GCACCAGCTCTACCGGTTCTCCA
LCW462_r15 GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG GASPGTSSTGSPGSSP
GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG SASTGTGPGSSTPSGA
TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGT TGSPGTSESATPESGP
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTA GSEPATSGSETPGSEP
GCGAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAG ATSGSETPGTSESATP
CGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTACT ESGPGTSTEPSEGSAP
TCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCT GTSTEPSEGSAPGTST
CTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTC EPSEGSAPGTSTEPSE
TACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCT GSAPGSEPATSGSETP
ACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTA
CTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACC GGCAACCTCCGGTTCTGAAACTCCA
LCW462_r16 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAG GTSTEPSEGSAPGSPA
GTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGG GSPTSTEEGTSTEPSE
TACTTCTACCGAACCTTCTGAGGGTAGCGCACCAGGT GSAPGTSESATPESGP
ACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTA GSEPATSGSETPGTSE
GCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTAC SATPESGPGSPAGSPT
CTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC STEEGTSESATPESGP
CCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTT GTSTEPSEGSAPGSEP
CTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC ATSGSETPGTSTEPSE
TACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGA GSAPGSEPATSGSETP
ACCTGCTACTTCTGGTTCTGAAACTCCAGGTACTTCTA
CCGAACCGTCCGAGGGTAGCGCTCCAGGTAGCGAAC CTGCTACTTCTGGTTCTGAAACTCCA
LCW462_r20 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGT GSAPGTSTEPSEGSAP
ACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTST
CCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTAC EPSEGSAPGTSTEPSE
CTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACT GSAPGTSESATPESGP
TCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTT GTSESATPESGPGTST
CTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTC EPSEGSAPGSEPATSG
TGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCT SETPGSPAGSPTSTEE
ACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAAC
CTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGC TGGCTCTCCGACCTCCACCGAGGAA
LCW462_r23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT GSAPGSTSESPSGTAP
TCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTC GSTSESPSGTAPGTST
TACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACT PESGSASPGSEPATSG
TCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCG SETPGTSESATPESGP
AACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC GTSTEPSEGSAPGTST
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCT EPSEGSAPGTSESATP
ACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTA ESGPGTSESATPESGP
CTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGA
AAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA AAGCGCAACCCCGGAGTCCGGCCCA
LCW462_r24 GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG GSSTPSGATGSPGSSP
GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG SASTGTGPGSSTPSGA
TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCAGGTA TGSPGSPAGSPTSTEE
GCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAG GSPAGSPTSTEEGTST
CCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACT EPSEGSAPGASPGTSS
TCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCTT TGSPGSSPSASTGTGP
CCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCTAG GTPGSGTASSSPGSTS
CCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCG STAESPGPGTSPSGES
GGCAGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAG STAPGTSTPESGSASP
CTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTCCTA
GCGGTGAATCTTCTACCGCTCCAGGTACCTCTACTCC GGAAAGCGGTTCTGCATCTCCA
LCW462_r27 GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG GTSTEPSEGSAPGTSE
GTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGG SATPESGPGTSTEPSE
TACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT GSAPGTSTEPSEGSAP
ACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTA GTSESATPESGPGTSE
CTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTAC SATPESGPGTPGSGTA
CTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACT SSSPGASPGTSSTGSP
CCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTC GASPGTSSTGSPGSPA
TCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTC GSPTSTEEGSPAGSPT
CGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGC STEEGTSTEPSEGSAP
TGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCT
GGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG AACCTTCCGAAGGTAGCGCTCCA
LCW462_r28 GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAG GSPAGSPTSTEEGTST
GTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGG EPSEGSAPGTSTEPSE
TACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGT GSAPGTSTEPSEGSAP
ACCTCTACCGAACCGTCTGAAGGTAGCGCACCAGGTA GTSESATPESGPGTSE
CCTCTGAAAGCGCAACTCCTGAGTCCGGTCCAGGTAC SATPESGPGTPGSGTA
TTCTGAAAGCGCAACCCCGGAGTCTGGCCCAGGTACC SSSPGSSTPSGATGSP
CCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCT GASPGTSSTGSPGTST
CTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCT EPSEGSAPGTSESATP
CCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTA ESGPGTSTEPSEGSAP
CTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA
AAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT GAACCGTCCGAAGGTAGCGCACCA
LCW462_r38 GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAG GSEPATSGSETPGTSE
GTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGG SATPESGPGSEPATSG
TAGCGAACCGGCTACTTCCGGCTCTGAAACCCCAGGT SETPGSSTPSGATGSP
AGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTA GTPGSGTASSSPGSST
CTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGC PSGATGSPGASPGTSS
TCTACTCCGTCTGGTGCTACCGGCTCCCCAGGTGCAT TGSPGSSTPSGATGSP
CTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCT GASPGTSSTGSPGSEP
ACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC ATSGSETPGTSTEPSE
GGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCT GSAPGSEPATSGSETP
GCTACTTCTGGTTCTGAAACTCCAGGTACTTCTACCG
AACCGTCCGAGGGTAGCGCTCCAGGTAGCGAACCTG CTACTTCTGGTTCTGAAACTCCA
LCW462_r39 GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAG EPSEGSAPGTSESATP
GTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG ESGPGSPAGSPTSTEE
TAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGT GSPAGSPTSTEEGTST
AGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTA EPSEGSAPGSPAGSPT
CTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAG STEEGTSTEPSEGSAP
CCCGGCTGGTTCTCCGACTTCCACCGAGGAAGGTACC GTSTEPSEGSAPGASP
TCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCT GTSSTGSPGSSPSAST
CTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTC GTGPGSSPSASTGTGP
CCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGC
CCGTCTGCTTCTACTGGTACTGGTCCAGGTTCTAGCCC TTCTGCTTCCACTGGTACTGGTCCA
LCW462_r41 GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG GSSTPSGATGSPGASP
GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGT GTSSTGSPGSSTPSGA
AGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTA TGSPGSPAGSPTSTEE
GCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTAC GTSESATPESGPGSEP
CTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGC ATSGSETPGASPGTSS
GAACCGGCAACCTCCGGTTCTGAAACCCCAGGTGCAT TGSPGSSTPSGATGSP
CTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCT GSSPSASTGTGPGSTS
ACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCC ESPSGTAPGSTSESPS
CTTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAG GTAPGTSTPESGSASP
CGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGC
GAATCCCCTTCTGGCACCGCACCAGGTACTTCTACCC CTGAAAGCGGCTCCGCTTCTCCA
LCW462_r42 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG GSTSESPSGTAPGSTS
GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGG ESPSGTAPGTSPSGES
TACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGT STAPGTSESATPESGP
ACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA GTSTEPSEGSAPGTST
CCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAC EPSEGSAPGTSTEPSE
TTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACC GSAPGTSESATPESGP
TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTT GTSTEPSEGSAPGSST
CTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTC PSGATGSPGASPGTSS
TACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCT TGSPGSSTPSGATGSP
ACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCC
TGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACC CCGTCTGGTGCTACTGGCTCTCCA
LCW462_r43 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAG GSTSSTAESPGPGTSP
GTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG SGESSTAPGTSPSGES
TACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTT STAPGSTSSTAESPGP
CTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTC GSTSSTAESPGPGTST
TACCAGCTCTACTGCAGAATCTCCTGGCCCAGGTACT PESGSASPGTSPSGES
TCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACTT STAPGSTSSTAESPGP
CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCTACC GTSTPESGSASPGSTS
AGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTA STAESPGPGSTSESPS
CCCCGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAG GTAPGTSPSGESSTAP
CTCTACCGCTGAATCTCCTGGCCCAGGTTCTACTAGC
GAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTA GCGGTGAATCTTCTACTGCACCA
LCW462_r45 GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG GTSTPESGSASPGSTS
GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGG ESPSGTAPGSTSSTAE
TTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAGGT SPGPGTSTEPSEGSAP
ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTSE
CCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC SATPESGPGTSESATP
TTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACC ESGPGTSTEPSEGSAP
TCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCT GTSTEPSEGSAPGTSE
CTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTC SATPESGPGTSTEPSE
TACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCT GSAPGTSTEPSEGSAP
GAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTA
CCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTAC TGAACCTTCTGAGGGTAGCGCTCCC
LCW462_r47 GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAG GTSTEPSEGSAPGTST
GTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGG EPSEGSAPGSEPATSG
TAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGT SETPGTSTEPSEGSAP
ACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTA GTSESATPESGPGTSE
CTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTAC SATPESGPGASPGTSS
CTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTGC TGSPGSSPSASTGTGP
ATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGTTCTA GSSTPSGATGSPGSST
GCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC PSGATGSPGSSTPSGA
TACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCT TGSPGASPGTSSTGSP
ACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTA
CTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCT GGCACCAGCTCTACCGGTTCTCCA
LCW462_r54 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAG GSEPATSGSETPGSEP
GTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGG ATSGSETPGTSTEPSE
TACTTCTACTGAACCTTCTGAGGGCAGCGCACCAGGT GSAPGSEPATSGSETP
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA GTSESATPESGPGTST
CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTAC EPSEGSAPGSSTPSGA
TTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGC TGSPGSSTPSGATGSP
TCTACTCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTC GASPGTSSTGSPGSST
TACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTCTC PSGATGSPGASPGTSS
CGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTAC TGSPGSSTPSGATGSP
CCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTG
GTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC
GTCTGGTGCTACTGGCTCTCCA LCW462_r55
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT GSAPGTSESATPESGP
ACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGTA GTSTEPSEGSAPGTST
CCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTAC EPSEGSAPGSTSESPS
TTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTTCT GTAPGTSPSGESSTAP
ACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTT GTSPSGESSTAPGSPA
CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC GSPTSTEEGTSESATP
CCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGG ESGPGTSTEPSEGSAP
CTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA
AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACT GAACCGTCCGAAGGTAGCGCTCCA
LCW462_r57 GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG GTSTEPSEGSAPGSEP
GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGG ATSGSETPGSPAGSPT
TAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGT STEEGSPAGSPTSTEE
AGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTA GTSESATPESGPGTST
CTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC EPSEGSAPGTSTEPSE
CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACC GSAPGTSTEPSEGSAP
TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCT GTSESATPESGPGTSST
CTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTC PSGATGSPGSSPSAST
TGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCT GTGPGASPGTSSTGSP
ACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCC
CGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCC GGGCACCAGCTCTACTGGTTCTCCA
LCW462_r61 GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAG GSEPATSGSETPGSPA
GTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAAGG GSPTSTEEGTSESATP
TACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCAGGT ESGPGTSTEPSEGSAP
ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTSE
CCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC SATPESGPGTSTPESG
TTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACC SASPGSTSESPSGTAP
TCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA GSTSSTAESPGPGTSE
CCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTAC SATPESGPGTSTEPSE
TAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCT GSAPGTSTEPSEGSAP
GAAAGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTA
CCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCTAC TGAACCTTCTGAGGGTAGCGCTCCA
LCW462_r64 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACTTCTACTGAACCTTCCGAAGGTAGCGCACCAGGT GSAPGTSTEPSEGSAP
ACCTCTACCGAACCGTCTGAAGGTAGCGCACCAGGTA GTSESATPESGPGTSE
CCTCTGAAAGCGCAACTCCTGAGTCCGGTCCAGGTAC SATPESGPGTPGSGTA
TTCTGAAAGCGCAACCCCGGAGTCTGGCCCAGGTACT SSSPGSSTPSGATGSP
CCTGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCT GASPGTSSTGSPGSTS
CTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTCT STAESPGPGTSPSGES
CCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCA STAPGTSTPESGSASP
GCTCTACTGCTGAATCTCCTGGTCCAGGTACCTCTCCT
AGCGGTGAATCTTCTACTGCTCCAGGTACTTCTACTCC TGAAAGCGGCTCTGCTTCTCCA
LCW462_r67 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAG GSPAGSPTSTEEGTSE
GTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGG SATPESGPGTSTEPSE
TACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGT GSAPGTSESATPESGP
ACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTA GSEPATSGSETPGTST
GCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTAC EPSEGSAPGSPAGSPT
TTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTAGC STEEGTSTEPSEGSAP
CCGGCTGGTTCTCCGACTTCCACCGAGGAAGGTACCT GTSTEPSEGSAPGTST
CTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTC EPSEGSAPGTSTEPSE
TACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCT GSAPGTSTEPSEGSAP
ACCGAACCGTCCGAGGGCAGCGCTCCAGGTACTTCTA
CTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTAC TGAACCTTCCGAAGGTAGCGCACCA
LCW462_r69 GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAG GTSPSGESSTAPGSTS
GTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGG STAESPGPGTSPSGES
TACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGT STAPGTSESATPESGP
ACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTA GTSTEPSEGSAPGTST
CCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAC EPSEGSAPGSSPSAST
TTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTTCT GTGPGSSTPSGATGSP
AGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCT GASPGTSSTGSPGTST
CTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCT PESGSASPGTSPSGES
CCGGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTA STAPGTSPSGESSTAP
CTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCC
TAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTA GCGGCGAATCTTCTACTGCTCCA
LCW462_r70 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAG GTSESATPESGPGTST
GTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT GSAPGSPAGSPTSTEE
AGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTA GSPAGSPTSTEEGTST
GCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTAC EPSEGSAPGSSPSAST
TTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTTCT GTGPGSSTPSGATGSP
AGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCT GSSTPSGATGSPGSEP
CTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT ATSGSETPGTSESATP
ACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAAC ESGPGSEPATSGSETP
CGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGA
AAGCGCTACTCCTGAGTCTGGCCCAGGTAGCGAACCT GCTACCTCTGGCTCTGAAACCCCA
LCW462_r72 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAG GTSTEPSEGSAPGTST
GTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGG EPSEGSAPGTSTEPSE
TACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGT GSAPGSSTPSGATGSP
AGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTG GASPGTSSTGSPGSST
CTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGC PSGATGSPGTSESATP
TCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTC ESGPGSEPATSGSETP
TGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGA GTSTEPSEGSAPGSTS
ACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT ESPSGTAPGSTSESPS
ACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTA GTAPGTSTPESGSASP
GCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAG
CGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACC CCTGAAAGCGGTTCCGCTTCTCCA
LCW462_r73 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG GTSTPESGSASPGSTS
GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGG STAESPGPGSTSSTAE
TTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTT SPGPGSSPSASTGTGP
CTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAG GSSTPSGATGSPGASP
CTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTT GTSSTGSPGSEPATSG
CTCCGGGTACTAGCTCTACCGGTTCTCCAGGTAGCGA SETPGTSESATPESGP
ACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCT GSPAGSPTSTEEGSTS
GAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGG ESPSGTAPGSTSESPS
CAGGTTCTCCGACTTCCACTGAGGAAGGTTCTACTAG GTAPGTSTPESGSASP
CGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGC
GAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCC CTGAAAGCGGTTCCGCTTCTCCC
LCW462_r78 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG GSPAGSPTSTEEGTSE
GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGG SATPESGPGTSTEPSE
TACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGT GSAPGSTSESPSGTAP
TCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTT GSTSESPSGTAPGTSP
CTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTAC SGESSTAPGTSTEPSE
TTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTACC GSAPGSPAGSPTSTEE
TCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGCC GTSTEPSEGSAPGSEP
CGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTC ATSGSETPGTSESATP
TACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGA ESGPGTSTEPSEGSAP
ACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT
GAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA CTGAACCGTCCGAGGGCAGCGCACCA
LCW462_r79 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAG GTSTEPSEGSAPGSPA
GTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGG GSPTSTEEGTSTEPSE
TACTTCTACCGAACCTTCTGAGGGTAGCGCACCAGGT GSAPGTSPSGESSTAP
ACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTA GTSPSGESSTAPGTSP
CCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAC SGESSTAPGSTSESPS
CTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTTCT GTAPGSTSESPSGTAP
ACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTA GTSTPESGSASPGSEP
CCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTC ATSGSETPGTSESATP
TACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAA ESGPGTSTEPSEGSAP
CCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG
AAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTAC TGAACCGTCCGAGGGCAGCGCACCA
LCW462_r87 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAG GSEPATSGSETPGTSE
GTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG SATPESGPGTSESATP
TACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGT ESGPGTSPSGESSTAP
ACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTT GSTSSTAESPGPGTSP
CTACTAGCTCTACCGCTGAATCTCCGGGCCCAGGTAC SGESSTAPGSTSESPS
TTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCTA GTAPGTSPSGESSTAP
CTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTC GSTSSTAESPGPGSST
CCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACC PSGATGSPGSSTPSGA
AGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTA TGSPGSSTPSGANWLS
CTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTAC
CCCTTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACC CCTTCTGGTGCAAACTGGCTCTCC
LCW462_r88 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAG GSPAGSPTSTEEGSPA
GTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGG GSPTSTEEGTSTEPSE
TACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GSAPGTSTEPSEGSAP
ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTA GTSTEPSEGSAPGTSE
CCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTAC SATPESGPGASPGTSS
TTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTGCA TGSPGSSTPSGATGSP
TCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTAGCTC GASPGTSSTGSPGSST
TACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC PSGATGSPGTPGSGT
CGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTAC ASSSPGSSTPSGATGSP
CCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGC
AGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC TTCTGGTGCTACTGGCTCTCCA
LCW462_r89 GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG GSSTPSGATGSPGTPG
GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG SGTASSSPGSSTPSGA
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTA TGSPGSPAGSPTSTEE
GCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTAC GTSESATPESGPGTST
TTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACC EPSEGSAPGTSESATP
TCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTACCT ESGPGSEPATSGSETP
CTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGA GTSESATPESGPGTST
ACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT EPSEGSAPGTSESATP
GAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTA ESGPGTSESATPESGP
CTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGA
AAGCGCAACCCCGGAATCCGGCCCAGGTACCTCTGA AAGCGCAACCCCGGAGTCCGGCCCA
Example 7
Construction of XTEN_AM288
[0339] The entire library LCW0462 was dimerized as described in
Example 6 resulting in a library of XTEN_AM288 clones designated
LCW0463. 1512 isolates from library LCW0463 were screened using the
protocol described in Example 6. 176 highly expressing clones were
sequenced and 40 preferred XTEN_AM288 segments were chosen for the
construction of multifunctional proteins that contain multiple XTEN
segments with 288 amino acid residues.
Example 8
Construction of XTEN_AM432
[0340] We generated a library of XTEN_AM432 segments by recombining
segments from library LCW0462 of XTEN_AM144 segments and segments
from library LCW0463 of XTEN_AM288 segments. This new library of
XTEN_AM432 segment was designated LCW0464. Plasmid was isolated
from cultures of E. coli harboring LCW0462 and LCW0463,
respectively. 1512 isolates from library LCW0464 were screened
using the protocol described in Example 6. 176 highly expressing
clones were sequenced and 39 preferred XTEN_AM432 segment were
chosen for the construction of longer XTENs and for the
construction of multifunctional proteins that contain multiple XTEN
segments with 432 amino acid residues.
[0341] In parallel we constructed library LMS0100 of XTEN_AM432
segments using preferred segments of XTEN_AM144 and XTEN_AM288.
Screening of this library yielded 4 isolates that were selected for
further construction
Example 9
Construction of XTEN_AM875
[0342] The stuffer vector pCW0359 was digested with BsaI and KpnI
to remove the stuffer segment and the resulting vector fragment was
isolated by agarose gel purification.
[0343] We annealed the phosphorylated oligonucleotide
BsaI-AscI-KpnIforP:
AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC and the
non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev:
CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC for introducing
the sequencing island A (SI-A) which encodes amino acids
GASASGAPSTG and has the restriction enzyme AscI recognition
nucleotide sequence GGCGCGCC inside. The annealed oligonucleotide
pairs were ligated with BsaI and KpnI digested stuffer vector
pCW0359 prepared above to yield pCW0466 containing SI-A. We then
generated a library of XTEN_AM443 segments by recombining 43
preferred XTEN_AM432 segments from Example 8 and SI-A segments from
pCW0466 at C-terminus using the same dimerization process described
in Example 5. This new library of XTEN_AM443 segments was
designated LCW0479.
[0344] We generated a library of XTEN_AM875 segments by recombining
segments from library LCW0479 of XTEN_AM443 segments and 43
preferred XTEN_AM432 segments from Example 8 using the same
dimerization process described in Example 5. This new library of
XTEN_AM875 segment was designated LCW0481.
Example 10
Construction of XTEN_AM1318
[0345] We annealed the phosphorylated oligonucleotide
BsaI-FseI-KpnlforP:
AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC and the
non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev:
CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG for introducing
the sequencing island B (SI-B) which encodes amino acids
GPEPTGPAPSG and has the restriction enzyme FseI recognition
nucleotide sequence GGCCGGCC inside. The annealed oligonucleotide
pairs were ligated with BsaI and KpnI digested stuffer vector
pCW0359 as used in Example 9 to yield pCW0467 containing SI-B. We
then generated a library of XTEN_AM443 segments by recombining 43
preferred XTEN_AM432 segments from Example 8 and SI-B segments from
pCW0467 at C-terminus using the same dimerization process described
in Example 5. This new library of XTEN_AM443 segments was
designated LCW0480.
[0346] We generated a library of XTEN_AM1318 segments by
recombining segments from library LCW0480 of XTEN_AM443 segments
and segments from library LCW0481 of XTEN_AM875 segments using the
same dimerization process as in Example 5. This new library of
XTEN_AM1318 segment was designated LCW0487.
Example 11
Construction of XTEN_AD864
[0347] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AD864 sequences starting from
segments of XTEN_AD36 listed in Example 1. These sequences were
assembled as described in Example 5. Several isolates from
XTEN_AD864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. One
intermediate construct of XTEN_AD576 was sequenced. This clone was
evaluated in a PK experiment in cynomolgus monkeys and a half-life
of about 20 h was measured.
Example 12
Construction of XTEN_AF864
[0348] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AF864 sequences starting from
segments of XTEN_AF36 listed in Example 3. These sequences were
assembled as described in Example 5. Several isolates from
XTEN_AF864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. One
intermediate construct of XTEN_AF540 was sequenced. This clone was
evaluated in a PK experiment in cynomolgus monkeys and a half-life
of about 20 h was measured. A full length clone of XTEN_AF864 had
excellent solubility and showed half-life exceeding 60 h in
cynomolgus monkeys. A second set of XTEN_AF sequences was assembled
including a sequencing island as described in Example 9.
Example 13
Construction of XTEN_AG864
[0349] Using the several consecutive rounds of dimerization, we
assembled a collection of XTEN_AG864 sequences starting from
segments of XTEN_AD36 listed in Example 1. These sequences were
assembled as described in Example 5. Several isolates from
XTEN_AG864 were evaluated and found to show good expression and
excellent solubility under physiological conditions. A full-length
clone of XTEN_AG864 had excellent solubility and showed half-life
exceeding 60 h in cynomolgus monkeys.
Example 14
Construction of N-Terminal Extensions of XTEN--Construction and
Screening of 12 mer Addition Libraries
[0350] This example details a step in the optimization of the
N-terminus of the XTEN protein to promote the initiation of
translation to allow for expression of XTEN fusions at the
N-terminus of fusion proteins without the presence of a helper
domain. Historically expression of proteins with XTEN at the
N-terminus was poor, yielding values that would essentially
undetectable in the GFP fluorescence assay (<25% of the
expression with the N-terminal CBD helper domain). To create
diversity at the codon level, seven amino acid sequences were
selected and prepared with a diversity of codons. Seven pairs of
oligonucleotides encoding 12 amino acids with codon diversities
were designed, annealed and ligated into the NdeUBsaI restriction
enzyme digested stuffer vector pCW0551 (Stuffer-XTEN_AM875-GFP),
and transformed into E. coli BL21Gold(DE3) competent cells to
obtain colonies of seven libraries. The resulting clones have
N-terminal XTEN 12 mers fused in-frame to XTEN_AM875-GFP to allow
use of GFP fluorescence for screening the expression. Individual
colonies from the seven created libraries were picked and grown
overnight to saturation in 500 .mu.l of super broth media in a 96
deep well plate. The number of colonies picked ranged from
approximately half to a third of the theoretical diversity of the
library (see Table 14).
TABLE-US-00020 TABLE 14 Theoretical Diversity and Sampling Numbers
for 12mer Addition Libraries. The amino acid residues with
randomized codons are underlined. Amino Acid Theoretical Number
Library Motif Family Sequence Diversity screened LCW546 AE12
MASPAGSPTSTEE 572 2 plates (168) LCW547 AE12 MATSESATPESGP 1536 5
plates (420) LCW548 AF12 MATSPSGESSTAP 192 2 plates (168) LCW549
AF12 MESTSSTAESPGP 384 2 plates (168) LCW552 AG12 MASSTPSGATGSP 384
2 plates (168) LCW553 AG12 MEASPGTSSTGSP 384 2 plates (168) LCW554
(CBD-like) MASTPESGSSG 32 1 plate (84)
[0351] The saturated overnight cultures were used to inoculate
fresh 500 .mu.l cultures in auto-induction media in which they were
grown overnight at 26.degree. C. These expression cultures were
then assayed using a fluorescence plate reader (excitation 395 nm,
emission 510 nm) to determine the amount of GFP reporter present
(see FIG. 28 for results of expression assays). The results
indicated that while median expression levels were approximately
half of the expression levels compared to the "benchmark" CBD
N-terminal helper domain, the best clones from the libraries were
much closer to the benchmarks, indicating that further optimization
around those sequences was warranted. This is in contrast to
previous XTEN versions that were <25% of the expression levels
of the CBD N-terminal benchmark. The results also show that the
libraries starting with amino acids MA had better expression levels
than those beginning with ME. This was most apparent when looking
at the best clones, which were closer to the benchmarks as they
mostly start with MA. Of the 176 clones within 33% of the CBD-AM875
benchmark, 87% begin with MA, where as only 75% of the sequences in
the libraries beginning with MA, a clear over representation of the
clones beginning with MA at the highest level of expression. 96 of
the best clones were sequenced to confirm identity and twelve
sequences (see Table 15), 4 from LCW546, 4 from LCW547 and 4 from
LCW552 were selected for further optimization.
TABLE-US-00021 TABLE 15 Advanced 12mer DNA Nucleotide Sequences
Clone DNA Nucleotide Sequence LCW546_02
ATGGCTAGTCCGGCTGGCTCTCCGACCTCCACTGAGGAAGG TACTTCTACT LCW546_06
ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACTGAGGAAGG TACTTCTACT LCW546_07
ATGGCTAGTCCAGCAGGCTCTCCTACCTCCACCGAGGAAGG TACTTCTACT LCW546_09
ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACTGAGGAAGG TACTTCTACT LCW547_03
ATGGCTACATCCGAAAGCGCAACCCCTGAGTCCGGTCCAG GTACTTCTACT LCW547_06
ATGGCTACATCCGAAAGCGCAACCCCTGAATCTGGTCCAGG TACTTCTACT LCW547_10
ATGGCTACGTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG TACTTCTACT LCW547_17
ATGGCTACGTCCGAAAGCGCTACCCCTGAATCCGGTCCAGG TACTTCTACT LCW552_03
ATGGCTAGTTCTACCCCGTCTGGTGCAACCGGTTCCCCAGG TACTTCTACT LCW552_05
ATGGCTAGCTCCACTCCGTCTGGTGCTACCGGTTCCCCAGG TACTTCTACT LCW552_10
ATGGCTAGCTCTACTCCGTCTGGTGCTACTGGTTCCCCAGG TACTTCTACT LCW552_11
ATGGCTAGTTCTACCCCTTCTGGTGCTACTGGTTCTCCAGG TACTTCTACT
Example 15
Construction of N-Terminal Extensions of XTEN--Construction and
Screening of Libraries Optimizing Codons 3 and 4
[0352] This example details a step in the optimization of the
N-terminus of the XTEN protein to promote the initiation of
translation to allow for expression of XTEN fusions at the
N-terminus of proteins without the presence of a helper domain.
With preferences for the first two codons established (see Example
supra), the third and fourth codons were randomized to determine
preferences. Three libraries, based upon best clones from LCW546,
LCW547 and LCW552, were designed with the third and fourth residues
modified such that all combinations of allowable XTEN codons were
present at these positions (see FIG. 29). In order to include all
the allowable XTEN codons for each library, nine pairs of
oligonucleotides encoding 12 amino acids with codon diversities of
third and fourth residues were designed, annealed and ligated into
the NdeI/BsaI restriction enzyme digested stuffer vector pCW0551
(Stuffer-XTEN_AM875-GFP), and transformed into E. coli
BL21Gold(DE3) competent cells to obtain colonies of three libraries
LCW0569-571. With 24.times.TEN codons the theoretical diversity of
each library is 576 unique clones. A total of 504 individual
colonies from the three created libraries were picked and grown
overnight to saturation in 5000 of super broth media in a 96 deep
well plate. This provided sufficient coverage to understand
relative library performance and sequence preferences. The
saturated overnight cultures were used to inoculate new 500 .mu.l
cultures in auto-induction media in which were grown overnight at
26.degree. C. These expression cultures were then assayed using a
fluorescence plate reader (excitation 395 nm, emission 510 nm) to
determine the amount of GFP reporter present. The top 75 clones
from the screen were sequenced and retested for GFP reporter
expression versus the benchmark samples (see FIG. 28). 52 clones
yielded usable sequencing data and were used for subsequent
analysis. The results were broken down by library and indicate that
LCW546 was the superior library. The results are presented in Table
16. Surprisingly, it was discovered that base-lined fluorescence
readings for the best clones were .about.900 AU, whereas the CBD
N-terminal benchmark was only .about.600 AU. This indicates that
this library had instituted an approximately 33% improvement over
the best clones from the previous library which were approximately
equal in expression to the CBD N-terminal benchmark (Example
14).
TABLE-US-00022 TABLE 16 Third and Fourth Codon Optimization Library
Comparison LCW569 LCW570 LCW571 N 21 15 16 Mean Fluorescence (AU)
628 491 537 SD 173 71 232 CV 28% 15% 43%
[0353] Further trends were seen in the data showing preferences for
particular codons at the third and fourth position. Within the
LCW569 library the glutamate codon GAA at the third position and
the threonine codon ACT were associated with higher expression as
seen in Table 17.
TABLE-US-00023 TABLE 17 Preferred Third and Fourth Codons in LCW569
3 = GAA Rest 4 = ACT Rest N 8 13 4 17 Mean Fluorescence (AU) 749
554 744 601 SD 234 47 197 162 CV 31% 9% 26% 27%
[0354] Additionally, the retest of the top 75 clones indicated that
several were now superior to the benchmark clones.
Example 16
Construction of N-Terminal Extensions of XTEN--Construction and
Screening of Combinatorial 12 mer and 36 mer Libraries
[0355] This example details a step in the optimization of the
N-terminus of the XTEN protein to promote the initiation of
translation to allow for expression of XTEN fusions at the
N-terminus of proteins without the presence of a helper domain.
With preferences for the first two codons established (see Example
supra), the N-terminus was examined in a broader context by
combining the 12 selected 12 mer sequences (see Example supra) at
the very N-terminus followed by 125 previously constructed 36 mer
segments (see example supra) in a combinatorial manner. This
created novel 48 mers at the N-terminus of the XTEN protein and
enabled the assessment of the impact of longer-range interactions
at the N-terminus on expression of the longer sequences (FIG. 29).
Similar to the dimerization procedures used to assemble 36 mers
(see Example infra), the plasmids containing the 125 selected 36
mer segments were digested with restriction enzymes BbsI/NcoI and
the appropriate fragment was gel-purified. The plasmid from clone
AC94 (CBD-XTEN_AM875-GFP) was also digested with BsaI/NcoI and the
appropriate fragments were gel-purified. These fragments were
ligated together and transformed into E. coli BL21Gold(DE3)
competent cells to obtain colonies of the library LCW0579, which
also served as the vector for further cloning 12 selected 12 mers
at the very N-terminus. The plasmids of LCW0579 were digested with
NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. 12
pairs of oligonucleotides encoding 12 selected 12 mer sequences
were designed, annealed and ligated with the NdeI/EcoRI/BsaI
digested LCW0579 vector, and transformed into E. coli BL21Gold(DE3)
competent cells to obtain colonies of the library LCW0580. With a
theoretical diversity of 1500 unique clones, a total of 1512
individual colonies from the created library were picked and grown
overnight to saturation in 500 .mu.l of super broth media in a 96
deep well plate. This provided sufficient coverage to understand
relative library performance and sequence preferences. The
saturated overnight cultures were used to inoculate new 500 .mu.l
cultures in auto-induction media that were grown overnight at
26.degree. C. These expression cultures were then assayed using a
fluorescence plate reader (excitation 395 nm, emission 510 nm) to
determine the amount of GFP reporter present. The top 90 clones
were sequenced and retested for GFP reporter expression. 83 clones
yielded usable sequencing data and were used for subsequent
analysis. The sequencing data was used to determine the lead 12 mer
that was present in each clone and the impact of each 12 mer on
expression was assessed. Clones LCW54606 and LCW54609 stood out as
being the superior N-terminus (see Table 18).
TABLE-US-00024 TABLE 18 Relative Performance of Clones Starting
with LCW546_06 and LCW459_09 All All LCW546_06 Others LCW546_09
Others N 11 72 9 74 Mean Fluorescence (AU) 1100 752 988 775 SD 275
154 179 202 CV 25% 20% 18% 26%
[0356] The sequencing and retest also revealed several instances of
independent replicates of the same sequence in the data producing
similar results, thus increasing confidence in the assay.
Additionally, 10 clones with 6 unique sequences were superior to
the benchmark clone. They are presented in Table 19. It was noted
that these were the only occurrences of these sequences and in no
case did one of these sequences occur and fail to beat the
bench-mark clone. These six sequences were advanced for further
optimization.
TABLE-US-00025 TABLE 19 Combinatorial 12mer and 36mer Clones
Superior to Benchmark Clone Clone Name First 60 codons 12mer Name
36mer Name LCW580_51 ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACT LCW546_06
LCW0404_040 GAGGAAGGTGCATCCCCGGGCACCAGCTCTACC
GGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCT ACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT
GCTACTGGCTCTCCAGGTACTTCTACTGAACCG TCTGAAGGCAGCGCA LCW580_81
ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACT LCW546_06 LCW0404_040
GAGGAAGGTGCATCCCCGGGCACCAGCTCTACC GGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCT
ACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGT GCTACTGGCTCTCCAGGTACTTCTACTGAACCG
TCTGAAGGCAGCGCA LCW580_38 ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACT
LCW546_06 LCW0402_041 GAGGAAGGTACTTCTACCGAACCGTCCGAGGGT
AGCGCACCAGGTAGCCCAGCAGGTTCTCCTACC TCCACCGAGGAAGGTACTTCTACCGAACCGTCC
GAGGGTAGCGCACCAGGTACTTCTACTGAACCG TCTGAAGGCAGCGCA LCW580_63
ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT LCW546_09 LCW0402_020
GAGGAAGGTACTTCTACTGAACCGTCTGAAGGC AGCGCACCAGGTAGCGAACCGGCTACTTCCGGT
TCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCA ACTTCTACTGAAGAAGGTACTTCTACTGAACCG
TCTGAAGGCAGCGCA LCW580_06 ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACT
LCW546_06 LCW0404_031 GAGGAAGGTACCCCGGGTAGCGGTACTGCTTCT
TCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAA
CCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCT CTACCGGTTCTCCAGGTACTTCTACTGAACCGT
CTGAAGGCAGCGCA LCW580_35 ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT
LCW546_09 LCW0402_020 GAGGAAGGTACTTCTACTGAACCGTCTGAAGGC
AGCGCACCAGGTAGCGAACCGGCTACTTCCGGT TCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCA
ACTTCTACTGAAGAAGGTACTTCTACTGAACCG TCTGAAGGCAGCGCA LCW580_67
ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT LCW546_09 LCW0403_064
GAGGAAGGTACCTCCCCTAGCGGCGAATCTTCT ACTGCTCCAGGTACCTCTCCTAGCGGCGAATCT
TCTACCGCTCCAGGTACCTCCCCTAGCGGTGAA TCTTCTACCGCACCAGGTACTTCTACTGAACCG
TCTGAAGGCAGCGCA LCW580_13 ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT
LCW546_09 LCW0403_060 GAGGAAGGTACCTCTACTCCGGAAAGCGGTTCC
GCATCTCCAGGTTCTACCAGCGAATCCCCGTCT GGCACCGCACCAGGTTCTACTAGCTCTACTGCT
GAATCTCCGGGCCCAGGTACTTCTACTGAACCG TCTGAAGGCAGCGCA LCW580_88
ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT LCW546_09 LCW0403_064
GAGGAAGGTACCTCCCCTAGCGGCGAATCTTCT ACTGCTCCAGGTACCTCTCCTAGCGGCGAATCT
TCTACCGCTCCAGGTACCTCCCCTAGCGGTGAA TCTTCTACCGCACCAGGTACTTCTACTGAACCG
TCTGAAGGCAGCGCA LCW580_11 ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACT
LCW546_09 LCW0403_060 GAGGAAGGTACCTCTACTCCGGAAAGCGGTTCC
GCATCTCCAGGTTCTACCAGCGAATCCCCGTCT GGCACCGCACCAGGTTCTACTAGCTCTACTGCT
GAATCTCCGGGCCCAGGTACTTCTACTGAACCG TCTGAAGGCAGCGCA
Example 17
Construction of N-Terminal Extensions of XTEN--Construction and
Screening of Combinatorial 12 mer and 36 mer Libraries for
XTEN-AM875 and XTEN-AE864
[0357] This example details a step in the optimization of the
N-terminus of the XTEN protein to promote the initiation of
translation to allow for expression of XTEN fusions at the
N-terminus of proteins without the presence of a helper domain.
With preferences for the first four codons (see Examples supra, and
for the best pairing of N-terminal 12 mers and 36 mers (see Example
supra) established, a combinatorial approach was undertaken to
examine the union of these preferences. This created novel 48 mers
at the N-terminus of the XTEN protein and enabled the testing of
the confluence of previous conclusions. Additionally, the ability
of these leader sequences to be a universal solution for all XTEN
proteins was assessed by placing the new 48 mers in front of both
XTEN-AE864 and XTEN-AM875. Instead of using all 125 clones of 36
mer segment, the plasmids from 6 selected clones of 36 mer segment
with best GFP expression in the combinatorial library were digested
with NdeI/EcoRI/BsaI and the appropriate fragments were
gel-purified. The plasmids from clones AC94 (CBD-XTEN_AM875-GFP)
and AC104 (CBD-XTEN_AE864-GFP) were digested with digested with
NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified.
These fragments were ligated together and transformed into E. coli
BL21Gold(DE3) competent cells to obtain colonies of the libraries
LCW0585 (--XTEN_AM875-GFP) and LCW0586 (--XTEN_AE864-GFP), which
could also serve as the vectors for further cloning 8 selected 12
mers at the very N-terminus. The plasmids of LCW0585 and LCW0586
were digested with NdeI/EcoRI/BsaI and the appropriate fragments
were gel-purified. 8 pairs of oligonucleotides encoding 8 selected
12 mer sequences with best GFP expression in the previous
(Generation 2) screening were designed, annealed and ligated with
the NdeI/EcoRI/BsaI digested LCW0585 and LCW0586 vectors, and
transformed into E. coli BL21Gold(DE3) competent cells to obtain
colonies of the final libraries LCW0587 (XTEN_AM923-GFP) and
LCW0588 (XTEN_AE912-GFP). With a theoretical diversity of 48 unique
clones, a total of 252 individual colonies from the created
libraries were picked and grown overnight to saturation in 500
.mu.l of super broth media in a 96 deep well plate. This provided
sufficient coverage to understand relative library performance and
sequence preferences. The saturated overnight cultures were used to
inoculate new 500 .mu.l cultures in auto-induction media in which
were grown overnight at 26.degree. C. These expression cultures
were then assayed using a fluorescence plate reader (excitation 395
nm, emission 510 nm) to determine the amount of GFP reporter
present. The top 36 clones were sequenced and retested for GFP
reporter expression. 36 clones yielded usable sequencing data and
these 36 were used for the subsequent analysis. The sequencing data
determined the 12 mer, the third codon, the fourth codon and the 36
mer present in the clone and revealed that many of the clones were
independent replicates of the same sequence. Additionally, the
retest results for these clones are close in value, indicating the
screening process was robust. Preferences for certain combinations
at the N-terminus were seen and were consistently yielding higher
fluorescence values approximately 50% greater than the benchmark
controls (see Tables 20 and 21). These date support the conclusion
that the inclusion of the sequences encoding the optimized
N-terminal XTEN into the fusion protein genes conferred a marked
enhancement on the expression of the fusion proteins.
TABLE-US-00026 TABLE 20 Preferred N-terminal Combinations for
XTEN-AM875 Number of Clone Name Replicates 12mer 36mer Mean SD CV
CBD-AM875 NA NA NA 1715 418 16% LCW587_08 7 LCW546_06_3 = GAA
LCW404_40 2333 572 18% LCW587_17 5 LCW546_09_3 = GAA LCW403_64 2172
293 10%
TABLE-US-00027 TABLE 21 Preferred N-terminal Combinations for
XTEN-AE864 Number of Clone Name Replicates 12mer 36mer Mean SD CV
AC82 NA NA NA 1979 679 24% LCW588_14 8 LCW546_06_opt3 LCW404_31
2801 240 6% LCW588_27 2 LCW546_06_opt34 LCW404_40 2839 556 15%
[0358] Notably, the preferred combination of the N-terminal for the
XTEN-AM875 and the preferred combination for the XTEN-AE864 are not
the same, indicating more complex interactions further than 150
bases from the initiation site influence expression levels. The
sequences for the preferred nucleotide sequences are listed in
Table 22 and the preferred clones were analyzed by SDS-PAGE to
independently confirm expression (see FIG. 30). The complete
sequences of XTEN_AM923 and XTEN_AE912 were selected for further
analysis.
TABLE-US-00028 TABLE 22 Preferred DNA Nucleotide Sequences for
first 48 Amino Acid Residues of N-terminal XTEN-AM875 and
XTEN-AE864 XTEN Clone Name Modified DNA Nucleotide Sequence
LCW587_08 AM875 ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATC
CCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTG
GTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGC
TCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW587_17 AM875
ATGGCTGAACCTGCTGGCTCTCCGACCTCTACTGAGGAAGGTACCTC
CCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCG
AATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACC
GCACCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA LCW588_14 AE864
ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCC
GGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGG
TGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTT
CTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG LCW588_27 AE864
ATGGCTGAAACTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATC
CCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTG
GTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGC
TCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG
Example 18
Methods of Producing and Evaluating CFXTEN; XTEN-CF as Example
[0359] A general schema for producing and evaluating CFXTEN
compositions is presented in FIG. 33, and forms the basis for the
general description of this Example. Using the disclosed methods
and those known to one of ordinary skill in the art, together with
guidance provided in the illustrative examples, a skilled artesian
can create and evaluate a range of CFXTEN fusion proteins
comprising, XTENs, CF and variants of CF known in the art. The
Example is, therefore, to be construed as merely illustrative, and
not limitative of the methods in any way whatsoever; numerous
variations will be apparent to the ordinarily skilled artisan. In
this Example, a CFXTEN of coagulation factor linked to an XTEN of
the AE family of motifs is created.
[0360] The general scheme for producing polynucleotides encoding
XTEN is presented in FIGS. 31 and 32. FIG. 32 is a schematic
flowchart of representative steps in the assembly of a XTEN
polynucleotide construct in one of the embodiments of the
invention. Individual oligonucleotides 501 are annealed into
sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which
is subsequently ligated with an oligo containing BbsI, and KpnI
restriction sites 503. The motif libraries can be limited to
specific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ
sequences of Table 3. In this case, the motifs of the AE family are
used as the motif library, which are annealed to the 12-mer to
create a "building block" length; e.g., a segment that encodes 36
amino acids. The gene encoding the XTEN sequence can be assembled
by ligation and multimerization of the "building blocks" until the
desired length of the XTEN gene 504 is achieved. As illustrated in
FIG. 32, the XTEN length in this case is 48 amino acid residues,
but longer lengths can be achieved by this process. For example,
multimerization can be performed by ligation, overlap extension,
PCR assembly or similar cloning techniques known in the art. The
XTEN gene can be cloned into a stuffer vector. In the example
illustrated in FIG. 32, the vector can encode a Flag sequence 506
followed by a stuffer sequence that is flanked by BsaI, BbsI, and
KpnI sites 507 and a CF gene (e.g., FVII) 508, resulting in the
gene encoding the CFXTEN 500, which, in this case encodes the
fusion protein in the configuration, N- to C-terminus, XTEN-FVII.
As is apparent to one of ordinary skill in the art, the methods can
be applied to create constructs in alternative configurations and
with varying XTEN lengths.
[0361] DNA sequences encoding CF can be conveniently obtained by
standard procedures known in the art from a cDNA library prepared
from an appropriate cellular source, from a genomic library, or may
be created synthetically (e.g., automated nucleic acid synthesis)
using DNA sequences obtained from publicly available databases,
patents, or literature references. A gene or polynucleotide
encoding the CF portion of the protein or its complement can be
then be cloned into a construct, such as those described herein,
which can be a plasmid or other vector under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system. A second gene or polynucleotide
coding for the XTEN portion or its complement (in the case of FIG.
32 illustrated as an XTEN with 48 amino acid residues) can be
genetically fused to the nucleotides encoding the terminus of the
CF gene by cloning it into the construct adjacent and in frame with
the gene coding for the CF, through a ligation or multimerization
step. In this manner, a chimeric DNA molecule coding for (or
complementary to) the CFXTEN fusion protein Re generated within the
construct. Optionally, a gene encoding for a second XTEN are
inserted and ligated in-frame to the nucleotides encoding the
opposite terminus of the CFXTEN gene or can be inserted within the
CF-encoding region. The construct can be designed in different
configurations to encode the various permutations of the fusion
partners as a monomeric polypeptide. For example, the gene can be
created to encode the fusion protein in the order (N- to
C-terminus): CF-XTEN; XTEN-CF; CF-XTEN--CF; XTEN--CF-XTEN; as well
as multimers of the foregoing. Optionally, this chimeric DNA
molecule is transferred or cloned into another construct that is a
more appropriate expression vector. At this point, a host cell
capable of expressing the chimeric DNA molecule is transformed with
the chimeric DNA molecule. The vectors containing the DNA segments
of interest can be transferred into an appropriate host cell by
well-known methods, depending on the type of cellular host, as
described supra.
[0362] Host cells containing the XTEN-CF expression vector is
cultured in conventional nutrient media modified as appropriate for
activating the promoter. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan. After expression of the fusion protein,
culture broth is harvested and separated from the cell mass and the
resulting crude extract retained for purification of the fusion
protein.
[0363] Gene expression are measured in a sample directly, for
example, by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad.
Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in
situ hybridization, using an appropriately labeled probe, based on
the sequences provided herein. Alternatively, gene expression is
measured by immunological of fluorescent methods, such as
immunohistochemical staining of cells to quantitate directly the
expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of sample fluids may be
either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be prepared against the CF
sequence polypeptide using a synthetic peptide based on the
sequences provided herein or against exogenous sequence fused to CF
and encoding a specific antibody epitope. Examples of selectable
markers are well known to one of skill in the art and include
reporters such as enhanced green fluorescent protein (EGFP),
beta-galactosidase (.beta.-gal) or chloramphenicol
acetyltransferase (CAT).
[0364] The CFXTEN polypeptide product is purified via methods known
in the art. Procedures such as gel filtration, affinity
purification, salt fractionation, ion exchange chromatography, size
exclusion chromatography, hydroxyapatite adsorption chromatography,
hydrophobic interaction chromatography or gel electrophoresis are
all techniques that may be used in the purification. Specific
methods of purification are described in Robert K. Scopes, Protein
Purification: Principles and Practice, Charles R. Castor, ed.,
Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step
purification separations are also described in Baron, et al., Crit.
Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr.
A. 679:67-83 (1994).
[0365] As illustrated in FIG. 33, the isolated CFXTEN fusion
proteins would then be characterized for their chemical and
activity properties. Isolated fusion protein is characterized,
e.g., for sequence, purity, apparent molecular weight, solubility
and stability using standard methods known in the art. The fusion
protein meeting expected standards would then be evaluated for
activity, which can be measured in vitro or in vivo by measuring
one of the coagulation factor-associated parameters described
herein, using one or more assays disclosed herein, or using the
assays of the Examples or Table 40.
[0366] In addition, the XTEN-CF fusion protein is administered to
one or more animal species to determine standard pharmacokinetic
parameters and pharmacodynamic properties, as described in Examples
30-33.
[0367] By the iterative process of producing, expressing, and
recovering CFXTEN constructs, followed by their characterization
using methods disclosed herein or others known in the art, the
CFXTEN compositions comprising CF and an XTEN can be produced and
evaluated by one of ordinary skill in the art to confirm the
expected properties such as enhanced solubility, enhanced
stability, improved pharmacokinetics and reduced immunogenicity,
leading to an overall enhanced therapeutic activity compared to the
corresponding unfused CF. For those fusion proteins not possessing
the desired properties, a different sequence can be constructed,
expressed, isolated and evaluated by these methods in order to
obtain a composition with such properties.
Example 19
Construction of Expression Plasmids for FVII-XTEN
[0368] Construction of FVII-TEV-XTEN 864 Expression Vectors
[0369] The cloning vector containing the gene encoding FVII was
purchased from OriGene (SC109205). PCR reactions were performed to
abolish BbsI and BsaI restriction sites within the FVII coding
region. The resulting FVII coding region was then amplified using
primers that introduced NheI and TEV-BsaI sequences on the 5' and
3' end respectively. The digested FVII fragment was fused to
BsaI/HindIII digested XTEN_AE864 fragment and inserted into
NheI/HindIII digested pSecTag2C expression vector. The ligated DNA
mixture was electroporated into XL1-Blue bacterial cells.
Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The final construct is
pCW0647.001 which encodes the FVII-TEV-XTEN_AE864 protein (Table
23).
[0370] Construction of FVII-XTEN 864 Expression Vectors
[0371] FVII was amplified with pCW0647.001 as a template. The PCR
primers introduced NheI and BsaI restriction enzyme recognition
sequences on the 5' and 3' end respectively and deleted the TEV
site. The NheI/BsaI digested FVII fragment was fused to
BsaI/HindIII digested XTEN_AE864 fragment and inserted into
NheI/HindIII digested pSecTag2C expression vector. The ligated DNA
mixture was electroporated into XL1-Blue bacterial cells.
Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The final construct is
pCW0645.001 which encodes the FVII-XTEN_AE864 protein (Table
23).
[0372] Construction of Expression Vectors Encoding FVII-XTEN 864
Genes Using Millipore Plasmids
[0373] Expression vector pCW0645.001 was digested with NheI and
SalI. The resulting 4091 bp fragment included nucleotides that
encode the FVII-XTEN_AE864 protein. This fragment was ligated with
NheI/SalI digested CET1019-AS-puro, CET1019-HS-puro, SC AS-puro, or
DC HS-puro (licensed from Millipore). These vectors feature a CMV
promoter that lies upstream of the gene insertion site, and the
CET1019 vectors also contain a UCOE element upstream of the
promoter. The ligated DNA mixture was electroporated into XL1-Blue
bacterial cells. Transformants were screened by DNA miniprep and
the desired constructs were confirmed by DNA sequencing. The
resulting expression vectors were AC397 (pBC0013, SC AS
puro-FVII-XTEN_AE864), AC402 (pBC0014, SC HS puro-FVII-XTEN_AE864),
AC403 (pBC0015, CET1019 AS puro-FVII-XTEN_AE864), and AC404
(pBC0016, CET1019 HS puro-FVII-XTEN_AE864)
[0374] Construction of Expression Vectors Encoding FVII-XTEN 288
Genes
[0375] Expression vector pCW0645.001 was digested with BsaI and
HindIII. The resulting 6400 bp fragment was ligated with
BsaI/HindIII digested XTEN_AE288 fragment. The ligated DNA mixture
was electroporated into XL1-Blue bacterial cells. Transformants
were screened by DNA miniprep and the desired constructs were
confirmed by DNA sequencing. The resulting expression vector was
pBC0019 (pSecTag2C-FVII-XTEN_AE288).
[0376] Expression vector pBC0019 was digested with NheI and SalI.
The resulting 2363 bp fragment included nucleotides that encode the
FVII-XTEN_AE288 protein. This fragment was ligated with NheI/SalI
digested CET1019-AS-puro, or CET1019-HS-puro (licensed from
Millipore). These vectors feature a CMV promoter and a UCOE element
that lie upstream of the gene insertion site. The ligated DNA
mixture was electroporated into XL1-Blue bacterial cells.
Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The resulting
expression vectors were AC405 (pBC0017, CET1019 AS
puro-FVII-XTEN_AE288), and AC398 (pBC0018, CET1019 HS
puro-FVII-XTEN_AE288) (Table 23).
TABLE-US-00029 TABLE 23 FVII amino acid and nucleic acid sequences
Name Amino Acid Sequence Nucleic Acid Sequences FVII-TEV-
MVSQALRLLCLLLGLQ ATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCT XTEN_AE864,
GCLAAVFVTQEEAHGV TGGGCTTCAGGGCTGCCTGGCTGCAGTGTTCGTAACCC pCW0647.001
LHRRRRANAFLEELRP AGGAGGAAGCCCACGGCGTCCTGCACCGGCGCCGGCG
GSLERECKEEQCSFEEA CGCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCC
REIFKDAERTKLFWISY CTGGAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCG
SDGDQCASSPCQNGGS AGGAGGCCCGGGAGATCTTCAAGGACGCGGAGAGGA
CKDQLQSYICFCLPAFE CGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAG
GRNCETHKDDQLICVN TGTGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCA
ENGGCEQYCSDHTGTK AGGACCAGCTCCAGTCCTATATCTGCTTCTGCCTCCCT
RSCRCHEGYSLLADGV GCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG
SCTPTVEYPCGKIPILEK ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGA
RNASKPQGRIVGGKVC GCAGTACTGCAGTGACCACACGGGCACCAAGCGCTCC
PKGECPWQVLLLVNGA TGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAGACG
QLCGGTLINTIWVVSAA GGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGA
HCFDKIKNWRNLIAVL AAAATACCTATTCTAGAAAAAAGAAATGCCAGCAAAC
GEHDLSEHDGDEQSRR CCCAAGGCCGAATTGTGGGGGGCAAGGTGTGCCCCAA
VAQVIIPSTYVPGTTNH AGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAAT
DIALLRLHQPVVLTDH GGAGCTCAGTTGTGTGGGGGGACCCTGATCAACACCA
VVPLCLPERTFSERTLA TCTGGGTGGTCTCCGCGGCCCACTGTTTCGACAAAATC
FVRFSLVSGWGQLLDR AAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGC
GATALELMVLNVPRLM ACGACCTCAGCGAGCACGACGGGGATGAGCAGAGCCG
TQDCLQQSRKVGDSPNI GCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGTC
TEYMFCAGYSDGSKDS CCGGGCACCACCAACCACGACATCGCGCTGCTCCGCC
CKGDSGGPHATHYRGT TGCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCC
WYLTGIVSWGQGCATV CTCTGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCT
GHFGVYTRVSQYIEWL GGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCC
QKLMRSEPRPGVLLRA AGCTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCAT
PFPGPEGPSENLYFQGG GGTCCTCAACGTGCCCCGGCTGATGACCCAGGACTGC
SPGSPAGSPTSTEEGTSE CTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA
SATPESGPGTSTEPSEGS TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGC
APGSPAGSPTSTEEGTS AGCAAGGACTCCTGCAAGGGGGACAGTGGAGGCCCAC
TEPSEGSAPGTSTEPSEG ATGCCACCCACTACCGGGGCACGTGGTACCTGACGGG
SAPGTSESATPESGPGS CATCGTCAGCTGGGGCCAGGGCTGCGCAACCGTGGGC
EPATSGSETPGSEPATS CACTTTGGGGTGTACACCAGGGTCTCCCAGTACATCGA
GSETPGSPAGSPTSTEE GTGGCTGCAAAAGCTCATGCGCTCAGAGCCACGCCCA
GTSESATPESGPGTSTEP GGAGTCCTCCTGCGAGCCCCATTTCCCGGCCCAGAAG
SEGSAPGTSTEPSEGSA GCCCATCCGAAAATCTGTATTTTCAGGGTGGGTCTCCA
PGSPAGSPTSTEEGTSTE GGTTCTCCAGCCGGGTCCCCAACTTCGACCGAGGAAG
PSEGSAPGTSTEPSEGS GGACCTCCGAGTCAGCTACCCCGGAGTCCGGTCCTGG
APGTSESATPESGPGTS CACCTCCACCGAACCATCGGAGGGCAGCGCCCCTGGG
TEPSEGSAPGTSESATPE AGCCCTGCCGGGAGCCCTACAAGCACCGAAGAGGGCA
SGPGSEPATSGSETPGT CCAGTACAGAGCCAAGTGAGGGGAGCGCCCCTGGTAC
STEPSEGSAPGTSTEPSE TAGTACTGAACCATCCGAGGGGTCAGCTCCAGGCACG
GSAPGTSESATPESGPG AGTGAGTCCGCTACCCCCGAGAGCGGACCGGGCTCAG
TSESATPESGPGSPAGSP AGCCCGCCACGAGTGGCAGTGAAACTCCAGGCTCAGA
TSTEEGTSESATPESGP ACCCGCCACTAGTGGGTCAGAGACTCCAGGCAGCCCT
GSEPATSGSETPGTSES GCCGGATCCCCTACGTCCACCGAGGAGGGAACATCTG
ATPESGPGTSTEPSEGS AGTCCGCAACACCCGAATCCGGTCCAGGCACCTCCAC
APGTSTEPSEGSAPGTS GGAACCTAGTGAAGGCTCGGCACCAGGTACAAGCACC
TEPSEGSAPGTSTEPSEG GAACCTAGCGAGGGCAGCGCTCCCGGCAGCCCTGCCG
SAPGTSTEPSEGSAPGT GCAGCCCAACCTCAACTGAGGAGGGCACCAGTACTGA
STEPSEGSAPGSPAGSPT GCCCAGCGAGGGATCAGCACCTGGCACCAGCACCGAA
STEEGTSTEPSEGSAPG CCTAGCGAGGGGAGCGCCCCTGGGACTAGCGAGTCAG
TSESATPESGPGSEPATS CTACACCAGAGAGCGGGCCTGGAACTTCTACCGAACC
GSETPGTSESATPESGP CAGTGAGGGATCCGCTCCAGGCACCTCCGAATCCGCA
GSEPATSGSETPGTSES ACCCCCGAATCCGGACCTGGCTCAGAGCCCGCCACCA
ATPESGPGTSTEPSEGS GCGGGAGCGAAACCCCTGGCACATCCACCGAGCCTAG
APGTSESATPESGPGSP CGAAGGGTCCGCACCCGGCACCAGTACAGAGCCTAGC
AGSPTSTEEGSPAGSPT GAGGGATCAGCACCTGGCACCAGTGAATCTGCTACAC
STEEGSPAGSPTSTEEG CAGAGAGCGGCCCTGGAACCTCCGAGTCCGCTACCCC
TSESATPESGPGTSTEPS CGAGAGCGGGCCAGGTTCTCCTGCTGGCTCCCCCACCT
EGSAPGTSESATPESGP CAACAGAAGAGGGGACAAGCGAAAGCGCTACGCCTG
GSEPATSGSETPGTSES AGAGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCT
ATPESGPGSEPATSGSE GAAACCCCTGGCACTAGTGAGTCTGCCACGCCTGAGT
TPGTSESATPESGPGTST CCGGACCCGGGACCTCTACTGAGCCCTCGGAGGGGAG
EPSEGSAPGSPAGSPTST CGCTCCTGGCACGAGTACAGAACCTTCCGAAGGAAGT
EEGTSESATPESGPGSEP GCACCGGGCACAAGCACCGAGCCTTCCGAAGGCTCTG
ATSGSETPGTSESATPES CTCCCGGAACCTCTACCGAACCCTCTGAAGGGTCTGCA
GPGSPAGSPTSTEEGSP CCCGGCACGAGCACCGAACCCAGCGAAGGGTCAGCGC
AGSPTSTEEGTSTEPSE CTGGGACCTCAACAGAGCCCTCGGAAGGATCAGCGCC
GSAPGTSESATPESGPG TGGAAGCCCTGCAGGGAGTCCAACTTCCACGGAAGAA
TSESATPESGPGTSESAT GGAACGTCTACAGAGCCATCAGAGGGGTCCGCACCAG
PESGPGSEPATSGSETP GTACCAGCGAATCCGCTACTCCCGAATCTGGCCCTGGG
GSEPATSGSETPGSPAG TCCGAACCTGCCACCTCCGGCTCTGAAACTCCAGGGAC
SPTSTEEGTSTEPSEGSA CTCCGAATCTGCCACACCCGAGAGCGGCCCTGGCTCC
PGTSTEPSEGSAPGSEP GAGCCCGCAACATCTGGCAGCGAGACACCTGGCACCT
ATSGSETPGTSESATPES CCGAGAGCGCAACACCCGAGAGCGGCCCTGGCACCAG
GPGTSTEPSEGSAPGSSS CACCGAGCCATCCGAGGGATCCGCCCCAGGCACTTCT
GAGTCAGCCACACCCGAAAGCGGACCAGGATCACCCG
CTGGCTCCCCCACCAGTACCGAGGAGGGGTCCCCCGC
TGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCTGCC
GGCTCCCCCACAAGTACCGAAGAGGGCACAAGTGAGA
GCGCCACTCCCGAGTCCGGGCCTGGCACCAGCACAGA
GCCTTCCGAGGGGTCCGCACCAGGTACCTCAGAGTCT
GCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAGCCA
CCTCCGGGTCTGAGACACCCGGGACTTCCGAGAGTGC
CACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCCACTT
CCGGCTCCGAAACTCCCGGCACAAGCGAGAGCGCTAC
CCCAGAGTCAGGACCAGGAACATCTACAGAGCCCTCT
GAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTCCCA
CTAGCACCGAGGAGGGAACCTCTGAAAGCGCCACACC
CGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGCGGC
AGCGAGACACCAGGCACCTCTGAGTCCGCCACACCAG
AGTCCGGACCCGGATCTCCCGCTGGGAGCCCCACCTCC
ACTGAGGAGGGATCTCCTGCTGGCTCTCCAACATCTAC
TGAGGAAGGTACCTCAACCGAGCCATCCGAGGGATCA
GCTCCCGGCACCTCAGAGTCGGCAACCCCGGAGTCTG
GACCCGGAACTTCCGAAAGTGCCACACCAGAGTCCGG
TCCCGGGACTTCAGAATCAGCAACACCCGAGTCCGGC
CCTGGGTCTGAACCCGCCACAAGTGGTAGTGAGACAC
CAGGATCAGAACCTGCTACCTCAGGGTCAGAGACACC
CGGATCTCCGGCAGGCTCACCAACCTCCACTGAGGAG
GGCACCAGCACAGAACCAAGCGAGGGCTCCGCACCCG
GAACAAGCACTGAACCCAGTGAGGGTTCAGCACCCGG
CTCTGAGCCGGCCACAAGTGGCAGTGAGACACCCGGC
ACTTCAGAGAGTGCCACCCCCGAGAGTGGCCCAGGCA
CTAGTACCGAGCCCTCTGAAGGCAGTGCGCCAGGTTC GTCTTCATAA FVII-
MVSQALRLLCLLLGLQ ATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCT XTEN_AE864,
GCLAAVFVTQEEAHGV TGGGCTTCAGGGCTGCCTGGCTGCAGTGTTCGTAACCC pCW0645.001
LHRRRRANAFLEELRP AGGAGGAAGCCCACGGCGTCCTGCACCGGCGCCGGCG
GSLERECKEEQCSFEEA CGCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCC
REIFKDAERTKLFWISY CTGGAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCG
SDGDQCASSPCQNGGS AGGAGGCCCGGGAGATCTTCAAGGACGCGGAGAGGA
CKDQLQSYICFCLPAFE CGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAG
GRNCETHKDDQLICVN TGTGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCA
ENGGCEQYCSDHTGTK AGGACCAGCTCCAGTCCTATATCTGCTTCTGCCTCCCT
RSCRCHEGYSLLADGV GCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG
SCTPTVEYPCGKIPILEK ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGA
RNASKPQGRIVGGKVC GCAGTACTGCAGTGACCACACGGGCACCAAGCGCTCC
PKGECPWQVLLLVNGA TGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAGACG
QLCGGTLINTIWVVSAA GGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGA
HCFDKIKNWRNLIAVL AAAATACCTATTCTAGAAAAAAGAAATGCCAGCAAAC
GEHDLSEHDGDEQSRR CCCAAGGCCGAATTGTGGGGGGCAAGGTGTGCCCCAA
VAQVIIPSTYVPGTTNH AGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAAT
DIALLRLHQPVVLTDH GGAGCTCAGTTGTGTGGGGGGACCCGATCAACACCAT
VVPLCLPERTFSERTLA CTGGGTGGTCTCCGCGGCCCACTGTTTCGACAAAATCA
FVRFSLVSGWGQLLDR AGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCA
GATALELMVLNVPRLM CGACCTCAGCGAGCACGACGGGGATGAGCAGAGCCGG
TQDCLQQSRKVGDSPNI CGGGTGGCGCAGGTCATCATCCCCAGCACGTACGTCC
TEYMFCAGYSDGSKDS CGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT
CKGDSGGPHATHYRGT GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCC
WYLTGIVSWGQGCATV TCTGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTG
GHFGVYTRVSQYIEWL GCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCA
QKLMRSEPRPGVLLRA GCTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATG
PFPGSPGSPAGSPTSTEE GTCCTCAACGTGCCCCGGCTGATGACCCAGGACTGCCT
GTSESATPESGPGTSTEP GCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATATC
SEGSAPGSPAGSPTSTE ACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCA
EGTSTEPSEGSAPGTST GCAAGGACTCCTGCAAGGGGGACAGTGGAGGCCCACA
EPSEGSAPGTSESATPES TGCCACCCACTACCGGGGCACGTGGTACCTGACGGGC
GPGSEPATSGSETPGSE ATCGTCAGCTGGGGCCAGGGCTGCGCAACCGTGGGCC
PATSGSETPGSPAGSPTS ACTTTGGGGTGTACACCAGGGTCTCCCAGTACATCGAG
TEEGTSESATPESGPGT TGGCTGCAAAAGCTCATGCGCTCAGAGCCACGCCCAG
STEPSEGSAPGTSTEPSE GAGTCCTCCTGCGAGCCCCATTTCCCGGAGGTAGCCCG
GSAPGSPAGSPTSTEEG GCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA
TSTEPSEGSAPGTSTEPS AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG
EGSAPGTSESATPESGP AACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGG
GTSTEPSEGSAPGTSES CTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC
ATPESGPGSEPATSGSE CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCT
TPGTSTEPSEGSAPGTST TCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTA
EPSEGSAPGTSESATPES CCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCT
GPGTSESATPESGPGSP GGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCG
AGSPTSTEEGTSESATP GTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACC
ESGPGSEPATSGSETPG TCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG
TSESATPESGPGTSTEPS AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGG
EGSAPGTSTEPSEGSAP CAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGT
GTSTEPSEGSAPGTSTEP AGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCAC
SEGSAPGTSTEPSEGSA CGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGC
PGTSTEPSEGSAPGSPA GCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCG
GSPTSTEEGTSTEPSEGS CTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGT
APGTSESATPESGPGSE CCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCAC
PATSGSETPGTSESATPE CAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCC
SGPGSEPATSGSETPGT AGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA
SESATPESGPGTSTEPSE GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAG
GSAPGTSESATPESGPG GTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGT
SPAGSPTSTEEGSPAGSP ACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTA
TSTEEGSPAGSPTSTEE CCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAG
GTSESATPESGPGTSTEP CCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCT
SEGSAPGTSESATPESG CTGAAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGA
PGSEPATSGSETPGTSES ACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTG
ATPESGPGSEPATSGSE AAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACT
TPGTSESATPESGPGTST GAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGA
EPSEGSAPGSPAGSPTST ACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAA
EEGTSESATPESGPGSEP CCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC
ATSGSETPGTSESATPES CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCT
GPGSPAGSPTSTEEGSP TCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTC
AGSPTSTEEGTSTEPSE CGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCT
GSAPGTSESATPESGPG ACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCG
TSESATPESGPGTSESAT AGGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCC
PESGPGSEPATSGSETP TGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT
GSEPATSGSETPGSPAG CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGA
SPTSTEEGTSTEPSEGSA ATCTGGTCCAGGTAGCGAACCTGCAACCTCTGGCTCTG
PGTSTEPSEGSAPGSEP AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCT
ATSGSETPGTSESATPES GGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG
GPGTSTEPSEGSAPGSSS CACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGC
CCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGG
AAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA
AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA
GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAG
GTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGG
TACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTA
GCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACC
TCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCG
AACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT
GAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTAC
TGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCT
GGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGC
AACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCG
CTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCT
CCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTC
CAACTTCTACTGAAGAAGGTACTTCTACCGAACCTTCC
GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC
CTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCT
GAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCGG
AATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTG
AAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT
GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCG
CACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCT
CCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCC
CAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAG GTTAA FVII- MVSQALRLLCLLLGLQ
ATGGTGTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCT XTEN_AE288, GCLAAVFVTQEEAHGV
TGGGCTTCAGGGCTGCCTGGCTGCAGTGTTCGTAACCC pBC0019 LHRRRRANAFLEELRP
AGGAGGAAGCCCACGGCGTCCTGCACCGGCGCCGGCG GSLERECKEEQCSFEEA
CGCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCC REIFKDAERTKLFWISY
CTGGAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCG SDGDQCASSPCQNGGS
AGGAGGCCCGGGAGATCTTCAAGGACGCGGAGAGGA CKDQLQSYICFCLPAFE
CGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAG GRNCETHKDDQLICVN
TGTGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCA ENGGCEQYCSDHTGTK
AGGACCAGCTCCAGTCCTATATCTGCTTCTGCCTCCCT RSCRCHEGYSLLADGV
GCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG SCTPTVEYPCGKIPILEK
ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGA RNASKPQGRIVGGKVC
GCAGTACTGCAGTGACCACACGGGCACCAAGCGCTCC PKGECPWQVLLLVNGA
TGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAGACG QLCGGTLINTIWVVSAA
GGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGA HCFDKIKNWRNLIAVL
AAAATACCTATTCTAGAAAAAAGAAATGCCAGCAAAC GEHDLSEHDGDEQSRR
CCCAAGGCCGAATTGTGGGGGGCAAGGTGTGCCCCAA VAQVIIPSTYVPGTTNH
AGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAAT DIALLRLHQPVVLTDH
GGAGCTCAGTTGTGTGGGGGGACCCTGATCAACACCA VVPLCLPERTFSERTLA
TCTGGGTGGTGTCCGCGGCCCACTGTTTCGACAAAATC FVRFSLVSGWGQLLDR
AAGAACTGGAGAACCTGATCGCGGTGCTGGGCGAGCA GATALELMVLNVPRLM
CGACCTCAGCGAGCACGACGGGGATGAGCAGAGCCGG TQDCLQQSRKVGDSPNI
CGGGTGGCGCAGGTCATCATCCCCAGCACGTACGTCC TEYMFCAGYSDGSKDS
CGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT CKGDSGGPHATHYRGT
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCC WYLTGIVSWGQGCATV
TCTGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTG GHFGVYTRVSQYIEWL
GCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCA QKLMRSEPRPGVLLRA
GCTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATG PFPGSPGTSESATPESGP
GTCCTCAACGTGCCCCGGCTGATGACCCAGGACTGCCT GSEPATSGSETPGTSES
GCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATATC
ATPESGPGSEPATSGSE ACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCA
TPGTSESATPESGPGTST GCAAGGACTCCTGCAAGGGGGACAGTGGAGGCCCACA
EPSEGSAPGSPAGSPTST TGCCACCCACTACCGGGGCACGTGGTACCTGACGGGC
EEGTSESATPESGPGSEP ATCGTCAGCTGGGGCCAGGGCTGCGCAACCGTGGGCC
ATSGSETPGTSESATPES ACTTTGGGGTGTACACCAGGGTGTCCCAGTACATCGA
GPGSPAGSPTSTEEGSP GTGGCTGCAAAAGCTCATGCGCTCAGAGCCACGCCCA
AGSPTSTEEGTSTEPSE GGAGTCCTCCTGCGAGCCCCATTTCCCGGGTCTCCAGG
GSAPGTSESATPESGPG TACCTCAGAGTCTGCTACCCCCGAGTCAGGGCCAGGA
TSESATPESGPGTSESAT TCAGAGCCAGCCACCTCCGGGTCTGAGACACCCGGGA
PESGPGSEPATSGSETP CTTCCGAGAGTGCCACCCCTGAGTCCGGACCCGGGTCC
GSEPATSGSETPGSPAG GAGCCCGCCACTTCCGGCTCCGAAACTCCCGGCACAA
SPTSTEEGTSTEPSEGSA GCGAGAGCGCTACCCCAGAGTCAGGACCAGGAACATC
PGTSTEPSEGSAPGSEP TACAGAGCCCTCTGAAGGCTCCGCTCCAGGGTCCCCA
ATSGSETPGTSESATPES GCCGGCAGTCCCACTAGCACCGAGGAGGGAACCTCTG
GPGTSTEPSEGSAPGSSS AAAGCGCCACACCCGAATCAGGGCCAGGGTCTGAGCC
TGCTACCAGCGGCAGCGAGACACCAGGCACCTCTGAG
TCCGCCACACCAGAGTCCGGACCCGGATCTCCCGCTG
GGAGCCCCACCTCCACTGAGGAGGGATCTCCTGCTGG
CTCTCCAACATCTACTGAGGAAGGTACCTCAACCGAG
CCATCCGAGGGATCAGCTCCCGGCACCTCAGAGTCGG
CAACCCCGGAGTCTGGACCCGGAACTTCCGAAAGTGC
CACACCAGAGTCCGGTCCCGGGACTTCAGAATCAGCA
ACACCCGAGTCCGGCCCTGGGTCTGAACCCGCCACAA
GTGGTAGTGAGACACCAGGATCAGAACCTGCTACCTC
AGGGTCAGAGACACCCGGATCTCCGGCAGGCTCACCA
ACCTCCACTGAGGAGGGCACCAGCACAGAACCAAGCG
AGGGCTCCGCACCCGGAACAAGCACTGAACCCAGTGA
GGGTTCAGCACCCGGCTCTGAGCCGGCCACAAGTGGC
AGTGAGACACCCGGCACTTCAGAGAGTGCCACCCCCG
AGAGTGGCCCAGGCACTAGTACCGAGCCCTCTGAAGG CAGTGCGCCAGGTTCGTCTTCATAA
Example 20
Construction of Expression Plasmids for FIX-XTEN
[0377] Construction of FIX-XTEN 864 Genes and Vectors
[0378] The cloning vector containing the gene encoding FIX was
purchased from OriGene (SC126517). PCR reactions were performed to
abolish two BbsI restriction sites within the FIX coding region.
The resulting FIX coding region was then amplified using primers
that introduced NheI and BsaI restriction enzyme recognition
sequences on the 5' and 3' end respectively. The digested FIX
fragment was fused to BsaI/HindIII digested XTEN_AM864, AE864,
AF864, or AG864 fragments and inserted into NheI/HindIII digested
pSecTag2C expression vector. The final constructs are AC282
(pCW0562, FIX-XTEN_AM864), AC283 (pCW0563, FIX-XTEN_AE864), pCW0564
(FIX-XTEN_AF864), and pCW0565 (FIX-XTEN_AG864) (Table 24).
[0379] Construction of Expression Vectors for FIX Helper Genes
[0380] The cloning vector containing the gene encoding PC5 was
purchased from OriGene (SC310051). The PC5 coding region was
amplified using primers that introduced NotI and BstBI restriction
enzyme recognition sequences. The NotI/BstBI digested PC5 fragment
was ligated with NotI/BstBI digested CET1019-HD-puro or DC-HD-puro
vectors. Both CET1019-HD-puro and DC-HD-puro feature dual cassettes
where a CMV promoter lies upstream of the gene insertion site,
CET1019-HD-puro also contains a UCOE element upstream of the
promoter. The ligated DNA mixture was electroporated into XL1-Blue
bacterial cells. Transformants were screened by DNA miniprep and
the desired constructs were confirmed by DNA sequencing. The
resulting expression vectors are pBC0037 (DC-HD-puro-PC5) and
pBC0038 (CET1019 HD-puro-PC5).
[0381] Construction of FIX-XTEN and PC5 Dual Expression Vectors
[0382] The pBC0037 and pBC0038 constructs were digested with NheI
and SalI and ligated with the NheI/SalI digested FIX-XTEN_AE864.
The ligated DNA mixture was electroporated into XL1-Blue bacterial
cells. Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The resulting
expression vectors were pBC0035 (DC-HD-puro-FIX-XTEN_AE864-PC5) and
pBC0036 (CET1019-HD-puro-FIX-XTEN_AE864-PC5).
TABLE-US-00030 TABLE 24 FIX amino acid and nucleic acid sequences
Name Amino Acid Sequence Nucleic Acid Sequences FIX-
MQRVNMIMAESPGLITI ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAG XTEN_AM864,
CLLGYLLSAECTVFLDH GCCTCATCACCATCTGCCTTTTAGGATATCTACTCAGT pCW0562
ENANKILNRPKRYNSG GCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAA
KLEEFVQGNLERECME CAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGT
EKCSFEEAREVFENTER AAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAG
TTEFWKQYVDGDQCES AATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACG
NPCLNGGSCKDDINSYE AGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTT
CWCPFGFEGKNCELDV TGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCA
TCNIKNGRCEQFCKNS ATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATT
ADNKVVCSCTEGYRLA AATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG
ENQKSCEPAVPFPCGRV AAAGAACTGTGAATTAGATGTAACATGTAACATTAAG
SVSQTSKLTRAETVFPD AATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTG
VDYVNSTEAETILDNIT ATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA
QSTQSFNDFTRVVGGE CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGC
DAKPGQFPWQVVLNG CATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCT
KVDAFCGGSIVNEKWI AAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGA
VTAAHCVETGVKITVV CTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATA
AGEHNIEETEHTEQKRN ACATCACTCAAAGCACCCAATCATTTAATGACTTCACT
VIRIIPHHNYNAAINKY CGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAAT
NHDIALLELDEPLVLNS TCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCA
YVTPICIADKEYTNIFLK TTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGT
FGSGYVSGWGRVFHKG AACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTA
RSALVLQYLRVPLVDR CAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGA
ATCLRSTKFTIYNNMFC ACATACAGAGCAAAAGCGAAATGTGATTCGAATTATT
AGFHEGGRDSCQGDSG CCTCACCACAACTACAATGCAGCTATTAATAAGTACA
GPHVTEVEGTSFLTGIIS ACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTA
WGEECAMKGKYGIYT GTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGA
KVSRYVNWIKEKTKLT CAAGGAATACACGAACATCTTCCTCAAATTTGGATCTG
GGTSTEPSEGSAPGSEP GCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGG
ATSGSETPGSPAGSPTS GAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCAC
TEEGSTSSTAESPGPGTS TTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC
TPESGSASPGSTSESPSG ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGA
TAPGSTSESPSGTAPGT AGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGA
STPESGSASPGTSTPESG CCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAAC
SASPGSEPATSGSETPG TGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAA
TSESATPESGPGSPAGSP GGCAAATATGGAATATATACCAAGGTATCCCGGTATG
TSTEEGTSTEPSEGSAP TCAACTGGATTAAGGAAAAAACAAAGCTCACTGGAGG
GTSESATPESGPGTSTEP TACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGT
SEGSAPGTSTEPSEGSA AGCGAACCGGCTACTTCCGGTTCTGAAACCCCAGGTA
PGSPAGSPTSTEEGTSTE GCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCT
PSEGSAPGTSTEPSEGS ACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTC
APGTSESATPESGPGTS TACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTA
ESATPESGPGTSTEPSEG GCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGC
SAPGTSTEPSEGSAPGT GAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCC
SESATPESGPGTSTEPSE TGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCCGG
GSAPGSEPATSGSETPG AAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAAC
SPAGSPTSTEEGSSTPSG CTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA
ATGSPGTPGSGTASSSP CTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCG
GSSTPSGATGSPGTSTE ACTTCCACTGAGGAAGGTACCTCTACTGAACCTTCTGA
PSEGSAPGTSTEPSEGS GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCG
APGSEPATSGSETPGSP GAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG
AGSPTSTEEGSPAGSPT GTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGG
STEEGTSTEPSEGSAPG TAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCA
ASASGAPSTGGTSESAT CCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG
PESGPGSPAGSPTSTEE CGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGC
GSPAGSPTSTEEGSTSST GCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCG
AESPGPGSTSESPSGTAP GCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGT
GTSPSGESSTAPGTPGS CCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCC
GTASSSPGSSTPSGATG AGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA
SPGSSPSASTGTGPGSEP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAG
ATSGSETPGTSESATPES GTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGT
GPGSEPATSGSETPGST AGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAG
SSTAESPGPGSTSSTAES CCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGC
PGPGTSPSGESSTAPGSE TCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCC
PATSGSETPGSEPATSG GGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTA
SETPGTSTEPSEGSAPGS CCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACC
TSSTAESPGPGTSTPESG GAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTG
SASPGSTSESPSGTAPGT AACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCGGC
STEPSEGSAPGTSTEPSE AACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCT
GSAPGTSTEPSEGSAPG CTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCT
SSTPSGATGSPGSSPSAS CCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC
TGTGPGASPGTSSTGSP CGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCG
GSEPATSGSETPGTSES CCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTG
ATPESGPGSPAGSPTST AGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC
EEGSSTPSGATGSPGSSP ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA
SASTGTGPGASPGTSST CTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCT
GSPGTSESATPESGPGT GGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGC
STEPSEGSAPGTSTEPSE ACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCAC GSAPG
CAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCA
GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG
TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTA
GCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTAC
TTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGC
GAACCGGCTACTTCCGGCTCTGAAACCCCAGGTTCCAC
CAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTA
GCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCT
AGCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGG
CAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCTGC
AACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAAC
CTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACC
GCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAG
CGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTT
CTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAA
GGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGG
GCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGT
AGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCG
GCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACT
GGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTC
TCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCC
CAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCC
AGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAG
GTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGT
TCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGC
TTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCT
CTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCT
ACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTAC
TGAACCGTCCGAAGGTAGCGCACCAGGTTAA FIX- MQRVNMIMAESPGLITI
ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAG XTEN_AE864, CLLGYLLSAECTVFLDH
GCCTCATCACCATCTGCCTTTTAGGATATCTACTCAGT pCW0563 ENANKILNRPKRYNSG
GCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAA KLEEFVQGNLERECME
CAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGT EKCSFEEAREVFENTER
AAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAG TTEFWKQYVDGDQCES
AATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACG NPCLNGGSCKDDINSYE
AGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTT CWCPFGFEGKNCELDV
TGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCA TCNIKNGRCEQFCKNS
ATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATT ADNKVVCSCTEGYRLA
AATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG ENQKSCEPAVPFPCGRV
AAAGAACTGTGAATTAGATGTAACATGTAACATTAAG SVSQTSKLTRAETVFPD
AATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTG VDYVNSTEAETILDNIT
ATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA QSTQSFNDFTRVVGGE
CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGC DAKPGQFPWQVVLNG
CATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCT KVDAFCGGSIVNEKWI
AAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGA VTAAHCVETGVKITVV
CTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATA AGEHNIEETEHTEQKRN
ACATCACTCAAAGCACCCAATCATTTAATGACTTCACT VIRIIPHHNYNAAINKY
CGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAAT NHDIALLELDEPLVLNS
TCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCA YVTPICIADKEYTNIFLK
TTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGT FGSGYVSGWGRVFHKG
AACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTA RSALVLQYLRVPLVDR
CAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGA ATCLRSTKFTIYNNMFC
ACATACAGAGCAAAAGCGAAATGTGATTCGAATTATT AGFHEGGRDSCQGDSG
CCTCACCACAACTACAATGCAGCTATTAATAAGTACA GPHVTEVEGTSFLTGIIS
ACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTA WGEECAMKGKYGIYT
GTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGA KVSRYVNWIKEKTKLT
CAAGGAATACACGAACATCTTCCTCAAATTTGGATCTG GGSPAGSPTSTEEGTSE
GCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGG SATPESGPGTSTEPSEGS
GAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCAC APGSPAGSPTSTEEGTS
TTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC TEPSEGSAPGTSTEPSEG
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGA SAPGTSESATPESGPGS
AGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGA EPATSGSETPGSEPATS
CCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAAC GSETPGSPAGSPTSTEE
TGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAA GTSESATPESGPGTSTEP
GGCAAATATGGAATATATACCAAGGTATCCCGGTATG SEGSAPGTSTEPSEGSA
TCAACTGGATTAAGGAAAAAACAAAGCTCACTGGAGG PGSPAGSPTSTEEGTSTE
TAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTA PSEGSAPGTSTEPSEGS
CTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACC APGTSESATPESGPGTS
TCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCC TEPSEGSAPGTSESATPE
CAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCT SGPGSEPATSGSETPGT
ACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTA STEPSEGSAPGTSTEPSE
CTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAA GSAPGTSESATPESGPG
AGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGG TSESATPESGPGSPAGSP
CTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCT TSTEEGTSESATPESGP
ACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTC GSEPATSGSETPGTSES
TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA ATPESGPGTSTEPSEGS
ACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGT APGTSTEPSEGSAPGTS
CTGAGGGCAGCGCACCAGGTACTTCTACCGAACCGTC TEPSEGSAPGTSTEPSEG
CGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCT SAPGTSTEPSEGSAPGT
ACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCG STEPSEGSAPGSPAGSPT
AGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTGA STEEGTSTEPSEGSAPG
GGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCG TSESATPESGPGSEPATS
GAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAG GSETPGTSESATPESGP
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGA GSEPATSGSETPGTSES
ATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTG ATPESGPGTSTEPSEGS
AGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAG APGTSESATPESGPGSP
CGCACCAGGTACTTCTACTGAACCGTCTGAAGGTAGC AGSPTSTEEGSPAGSPT
GCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCG STEEGSPAGSPTSTEEG
GCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGG TSESATPESGPGTSTEPS
CCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAG EGSAPGTSESATPESGP
AAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC GSEPATSGSETPGTSES
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA ATPESGPGSEPATSGSE
GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAG TPGTSESATPESGPGTST
GTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGT EPSEGSAPGSPAGSPTST
ACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTA EEGTSESATPESGPGSEP
CTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTAC ATSGSETPGTSESATPES
CTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCT GPGSPAGSPTSTEEGSP
CTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCT AGSPTSTEEGTSTEPSE
ACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAG GSAPGTSESATPESGPG
CAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACC TSESATPESGPGTSESAT
GAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAA PESGPGSEPATSGSETP
GCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC GSEPATSGSETPGSPAG
TACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG SPTSTEEGTSTEPSEGSA
CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC PGTSTEPSEGSAPGSEP
CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTA ATSGSETPGTSESATPES
CTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCC GPGTSTEPSEGSAPG
GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACTC
CTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTC
TACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT
ACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGT
CCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCT
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGA
CTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGG
TCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCC
CAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAG
GTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGT
ACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAGGTA
GCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAC
TTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCC
CGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTA
CCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGA
AAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA
AGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAG
CGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCT
ACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTAC
CTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTC
CGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCC
GAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTG
AGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTG
AATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC AGCGCACCAGGTTAA FIX-
MQRVNMIMAESPGLITI ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAG XTEN_AF864,
CLLGYLLSAECTVFLDH GCCTCATCACCATCTGCCTTTTAGGATATCTACTCAGT pCW0564
ENANKILNRPKRYNSG GCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAA
KLEEFVQGNLERECME CAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGT
EKCSFEEAREVFENTER AAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAG
TTEFWKQYVDGDQCES AATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACG
NPCLNGGSCKDDINSYE AGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTT
CWCPFGFEGKNCELDV TGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCA
TCNIKNGRCEQFCKNS ATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATT
ADNKVVCSCTEGYRLA AATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG
ENQKSCEPAVPFPCGRV AAAGAACTGTGAATTAGATGTAACATGTAACATTAAG
SVSQTSKLTRAETVFPD AATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTG
VDYVNSTEAETILDNIT ATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA
QSTQSFNDFTRVVGGE CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGC
DAKPGQFPWQVVLNG CATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCT
KVDAFCGGSIVNEKWI AAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGA
VTAAHCVETGVKITVV CTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATA
AGEHNIEETEHTEQKRN ACATCACTCAAAGCACCCAATCATTTAATGACTTCACT
VIRIIPHHNYNAAINKY CGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAAT
NHDIALLELDEPLVLNS TCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCA
YVTPICIADKEYTNIFLK TTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGT
FGSGYVSGWGRVFHKG AACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTA
RSALVLQYLRVPLVDR CAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGA
ATCLRSTKFTIYNNMFC ACATACAGAGCAAAAGCGAAATGTGATTCGAATTATT
AGFHEGGRDSCQGDSG CCTCACCACAACTACAATGCAGCTATTAATAAGTACA
GPHVTEVEGTSFLTGIIS ACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTA
WGEECAMKGKYGIYT GTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGA
KVSRYVNWIKEKTKLT CAAGGAATACACGAACATCTTCCTCAAATTTGGATCTG
GGSTSESPSGTAPGTSPS GCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGG
GESSTAPGSTSESPSGT GAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCAC
APGSTSESPSGTAPGTS TTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC
TPESGSASPGTSTPESGS ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGA
ASPGSTSESPSGTAPGST AGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGA
SESPSGTAPGTSPSGESS CCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAAC
TAPGSTSESPSGTAPGT TGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAA
SPSGESSTAPGTSPSGES GGCAAATATGGAATATATACCAAGGTATCCCGGTATG
STAPGSTSSTAESPGPG TCAACTGGATTAAGGAAAAAACAAAGCTCACTGGAGG
TSPSGESSTAPGTSPSGE TTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTA
SSTAPGSTSSTAESPGPG CCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCT
TSTPESGSASPGTSTPES ACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTAC
GSASPGSTSESPSGTAP TAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTA
GSTSESPSGTAPGTSTPE CTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACT
SGSASPGSTSSTAESPGP CCGGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGA
GTSTPESGSASPGSTSES ATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAAT
PSGTAPGTSPSGESSTAP CCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGC
GSTSSTAESPGPGTSPSG GAATCTTCTACCGCACCAGGTTCTACTAGCGAATCTCC
ESSTAPGTSTPESGSASP GTCTGGCACTGCTCCAGGTACTTCTCCTAGCGGTGAAT
GSTSSTAESPGPGSTSST CTTCTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCT
AESPGPGSTSSTAESPGP TCTACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATC
GSTSSTAESPGPGTSPSG TCCGGGCCCAGGTACCTCTCCTAGCGGTGAATCTTCTA
ESSTAPGSTSESPSGTAP CCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACC
GSTSESPSGTAPGTSTPE GCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTGG
SGPGGGGASASGAPST CCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTC
GGGGSESPSGTAPGSTS CAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA
ESPSGTAPGSTSESPSGT GGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGG
APGSTSESPSGTAPGSTS TTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGGTA
ESPSGTAPGSTSESPSGT CCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCT
APGTSTPESGSASPGTSP ACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTC
SGESSTAPGTSPSGESST TACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTA
APGSTSSTAESPGPGTSP GCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTCCG
SGESSTAPGTSTPESGS AGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTC
ASPGSTSESPSGTAPGST TACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCG
SESPSGTAPGTSPSGESS GTGAATCTTCTACTGCTCCAGGTACCTCTACTCCTGAA
TAPGSTSESPSGTAPGT AGCGGTTCTGCATCTCCAGGTTCCACTAGCTCTACCGC
STPESGSASPGTSTPESG AGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCTG
SASPGSTSESPSGTAPGT AATCTCCTGGCCCAGGTTCTACTAGCTCTACTGCTGAA
STPESGSASPGSTSSTAE TCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATC
SPGPGSTSESPSGTAPGS TCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTA
TSESPSGTAPGTSPSGES CTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGCACT
STAPGSTSSTAESPGPG GCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGC
TSPSGESSTAPGTSTPES ACCAGGTACCTCTACCCCTGAAAGCGGTCCGGGGGGG
GSASPGTSPSGESSTAP GGGGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGA
GTSPSGESSTAPGTSPSG GGGGGGGGTAGCGAATCTCCTTCTGGTACCGCTCCAG
ESSTAPGSTSSTAESPGP GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGT
GSTSSTAESPGPGTSPSG TCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTC
ESSTAPGSSPSASTGTGP TACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTCTA
GSSTPSGATGSPGSSTPS CCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACC GATGSPG
AGCGAATCTCCTTCTGGTACTGCACCAGGTACTTCTAC
TCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTA
GCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGC
GGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTAC
TGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTG
AATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGC
GGTTCCGCTTCTCCAGGTTCTACCAGCGAATCTCCTTC
TGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG
GTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCT
ACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTAC
CGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTT
CTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCT
CCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCC
AGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAG
GTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGT
TCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTC
TACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTT
CTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACC
AGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCC
GAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTC
CGGAAAGCGGTTCCGCTTCTCCAGGTACCTCCCCTAGC
GGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGG
CGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTG
AATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCT
GAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGA
ATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTT
CTACTGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGT
ACCGGCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGG
CTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCT CCCCAGGTTAA FIX-
MQRVNMIMAESPGLITI ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAG XTEN_AG864,
CLLGYLLSAECTVFLDH GCCTCATCACCATCTGCCTTTTAGGATATCTACTCAGT pCW0565
ENANKILNRPKRYNSG GCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAA
KLEEFVQGNLERECME CAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGT
EKCSFEEAREVFENTER AAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAG
TTEFWKQYVDGDQCES AATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACG
NPCLNGGSCKDDINSYE AGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTT
CWCPFGFEGKNCELDV TGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCA
TCNIKNGRCEQFCKNS ATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATT
ADNKVVCSCTEGYRLA AATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG
ENQKSCEPAVPFPCGRV AAAGAACTGTGAATTAGATGTAACATGTAACATTAAG
SVSQTSKLTRAETVFPD AATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTG
VDYVNSTEAETILDNIT ATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA
QSTQSFNDFTRVVGGE CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGC
DAKPGQFPWQVVLNG CATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCT
KVDAFCGGSIVNEKWI AAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGA
VTAAHCVETGVKITVV CTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATA
AGEHNIEETEHTEQKRN ACATCACTCAAAGCACCCAATCATTTAATGACTTCACT
VIRIIPHHNYNAAINKY CGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAAT
NHDIALLELDEPLVLNS TCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCA
YVTPICIADICEYTNIFLK TTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGT
FGSGYVSGWGRVFHKG AACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTA
RSALVLQYLRVPLVDR CAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGA
ATCLRSTKFTIYNNMFC ACATACAGAGCAAAAGCGAAATGTGATTCGAATTATT
AGFHEGGRDSCQGDSG CCTCACCACAACTACAATGCAGCTATTAATAAGTACA
GPHVTEVEGTSFLTGIIS ACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTA
WGEECAMKGKYGIYT GTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGA
KVSRYVNWIKEKTKLT CAAGGAATACACGAACATCTTCCTCAAATTTGGATCTG
GGASPGTSSTGSPGSSP GCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGG
SASTGTGPGSSPSASTG GAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCAC
TGPGTPGSGTASSSPGS TTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC
STPSGATGSPGSNPSAS ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGA
TGTGPGASPGTSSTGSP AGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGA
GTPGSGTASSSPGSSTPS CCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAAC
GATGSPGTPGSGTASSS TGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAA
PGASPGTSSTGSPGASP GGCAAATATGGAATATATACCAAGGTATCCCGGTATG
GTSSTGSPGTPGSGTAS TCAACTGGATTAAGGAAAAAACAAAGCTCACTGGAGG
SSPGSSTPSGATGSPGA TGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTT
SPGTSSTGSPGTPGSGT CTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGGTTCT
ASSSPGSSTPSGATGSP AGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACCCC
GSNPSASTGTGPGSSPS GGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTA
ASTGTGPGSSTPSGATG CTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCT
SPGSSTPSGATGSPGAS TCTGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGG
PGTSSTGSPGASPGTSST CACCAGCTCTACTGGTTCTCCAGGTACCCCGGGCAGCG
GSPGASPGTSSTGSPGT GTACCGCATCTTCTTCTCCAGGTAGCTCTACTCCTTCTG
PGSGTASSSPGASPGTS GTGCAACTGGTTCTCCAGGTACTCCTGGCAGCGGTACC
STGSPGASPGTSSTGSP GCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCT
GASPGTSSTGSPGSSPS ACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTAC
ASTGTGPGTPGSGTASS TGGTTCTCCAGGTACCCCGGGTAGCGGTACTGCTTCTT
SPGASPGTSSTGSPGAS CCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGC
PGTSSTGSPGASPGTSST TCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTC
GSPGSSTPSGATGSPGS TCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTC
STPSGATGSPGASPGTS CAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCA
STGSPGTPGSGTASSSP GGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGG
GSSTPSGATGSPGSSTPS TTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTA
GATGSPGSSTPSGATGS GCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGC
PGSSPSASTGTGPGASP TCTACTCCTTCTGGTGCAACTGGCTCTCCAGGTGCATC
GTSSTGSPGASPGTSST TCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCATCCC
GSPGTPGSGTASSSPGA CTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCT
SPGTSSTGSPGASPGTSS GGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGGCAG
TGSPGASPGTSSTGSPG CGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTAC
ASPGTSSTGSPGTPGSG TAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTA
TASSSPGSSTPSGATGSP GCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGC
GTPGSGTASSSPGSSTPS TCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACT
GATGSPGTPGSGTASSS GGTACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTC
PGSSTPSGATGSPGSSTP TTCCTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTG
SGATGSPGSSPSASTGT GTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGT
GPGSSPSASTGTGPGAS TCTCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCT
PGTSSTGSPGTPGSGTA CCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCC
SSSPGSSTPSGATGSPGS AGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAG
SPSASTGTGPGSSPSAST GTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCAGGT
GTGPGASPGTSSTGSPG ACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAG
ASPGTSSTGSPGSSTPSG CTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCT
ATGSPGSSPSASTGTGP CTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCT
GASPGTSSTGSPGSSPS ACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCC
ASTGTGPGTPGSGTASS GTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGG
SPGSSTPSGATGSPGSST GCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGT
PSGATGSPGASPGTSST ACCAGCTCTACCGGTTCTCCAGGTACTCCTGGCAGCGG GSPG
TACTGCATCTTCCTCTCCAGGTGCTTCTCCGGGCACCA
GCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGC
TCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTC
TACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTA
CTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCT
TCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGG
TTCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTT
CTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCT
CCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCC
AGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAG
GTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGGT
TCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTC
TAGCCCGTCTGCATCTACTGGTACTGGTCCAGGTGCAT
CCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTCCT
GGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTAC
TCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTC
TGCATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTG
CTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACT
AGCTCTACTGGTTCTCCAGGTGCATCTCCTGGTACTAG
CTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTG
CAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTACC
GGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTAC
CGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACCGGTA
CCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTTCC
TCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTC
CCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCC
CAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA GGTTAA PC5, MGWGSRCCCPGRLDLL
atgggctgggggagccgctgctgctgcccgggacgtttggacctgctgtgcgtgctggc
pBC0037, CVLALLGGCLLPVCRT
gctgctcgggggctgcctgctccccgtgtgtcggacgcgcgtctacaccaaccactggg pBC0038
RVYTNHWAVKIAGGFP
cagtcaaaatcgccgggggcttcccggaggccaaccgtatcgccagcaagtacggattc
EANRIASKYGFINIGQIG
atcaacataggacagataggggccctgaaggactactaccacttctaccatagcaggacg
ALKDYYHFYHSRTIKRS
attaaaaggtcagttatctcgagcagagggacccacagtttcatttcaatggaaccaaaggt
VISSRGTHSFISMEPKVE
ggaatggatccaacagcaagtggtaaaaaagcggacaaagagggattatgacttcagtc
WIQQQVVKKRTKRDY
gtgcccagtctacctatttcaatgatcccaagtggcccagcatgtggtatatgcactgcagt
DFSRAQSTYFNDPKWP
gacaatacacatccctgccagtctgacatgaatatcgaaggagcctggaagagaggctac
SMWYMHCSDNTHPCQ
acgggaaagaacattgtggtcactatcctggatgacggaattgagagaacccatccagat
SDMNIEGAWKRGYTG
ctgatgcaaaactacgatgctctggcaagttgcgacgtgaatgggaatgacttggacccaa
KNIVVTILDDGIERTHP
tgcctcgttatgatgcaagcaacgagaacaagcatgggactcgctgtgctggagaagtgg
DLMQNYDALASCDVN
cagccgctgcaaacaattcgcactgcacagtcggaattgctttcaacgccaagatcggag
GNDLDPMPRYDASNEN
gagtgcgaatgctggacggagatgtcacggacatggttgaagcaaaatcagttagcttca
KHGTRCAGEVAAAAN
acccccagcacgtgcacatttacagcgccagctggggcccggatgatgatggcaagact
NSHCTVGIAFNAKIGGV
gtggacggaccagcccccctcacccggcaagcctttgaaaacggcgttagaatggggc
RMLDGDVTDMVEAKS
ggagaggcctcggctctgtgtttgtttgggcatctggaaatggtggaaggagcaaagacc
VSFNPQHVHIYSASWG
actgctcctgtgatggctacaccaacagcatctacaccatctccatcagcagcactgcaga
PDDDGKTVDGPAPLTR
aagcggaaagaaaccttggtacctggaagagtgttcatccacgctggccacaacctacag
QAFENGVRMGRRGLGS
cagcggggagtcctacgataagaaaatcatcactacagatctgaggcagcgttgcacgg
VFVWASGNGGRSKDH
acaaccacactgggacgtcagcctcagcccccatggctgcaggcatcattgcgctggcc
CSCDGYTNSIYTISISST
ctggaagccaatccgtttctgacctggagagacgtacagcatgttattgtcaggacttcccg
AESGKKPWYLEECSST
tgcgggacatttgaacgctaatgactggaaaaccaatgctgctggttttaaggtgagccatc
LATTYSSGESYDKKIITT
tttatggatttggactgatggacgcagaagccatggtgatggaggcagagaagtggacca
DLRQRCTDNHTGTSAS
ccgttccccggcagcacgtgtgtgtggagagcacagaccgacaaatcaagacaatccgc
APMAAGIIALALEANPF
cctaacagtgcagtgcgctccatctacaaagcttcaggctgctcggataaccccaaccgc
LTWRDVQHVIVRTSRA
catgtcaactacctggagcacgtcgttgtgcgcatcaccatcacccaccccaggagagga
GHLNANDWKTNAAGF
gacctggccatctacctgacctcgccctctggaactaggtctcagcttttggccaacaggct
KVSHLYGFGLMDAEA
atttgatcactccatggaaggattcaaaaactgggagttcatgaccattcattgctggggag
MVMEAEKWTTVPRQH
aaagagctgctggtgactgggtccttgaagtttatgatactccctctcagctaaggaacttta
VCVESTDRQIKTIRPNS
agactccaggtaaattgaaagaatggtctttggtcctctacggcACCTCCGTGCA
AVRSIYKASGCSDNPNR
GCCATATTCaccaaccaatgaatttccgaaagtggaacggttccgctatagccga
HVNYLEHVVVRITITHP
gttgaagaccccacagacgactatggcacagaggattatgcaggtccctgcgaccctga
RRGDLAIYLTSPSGTRS
gtgcagtgaggttggctgtgacgggccaggaccagaccactgcaatgactgtttgcacta
QLLANRLFDHSMEGFK
ctactacaagctgaaaaacaataccaggatctgtgtctccagctgcccccctggccactac
NWEFMTIHCWGERAA
cacgccgacaagaagcgctgcaggaagtgtgcccccaactgtgagtcctgctttgggag
GDWVLEVYDTPSQLRN
ccatggtgaccaatgcatgtcctgcaaatatggatactttctgaatgaagaaaccaacagct
FKTPGKLKEWSLVLYG
gtgttactcactgccctgatgggtcatatcaggataccaagaaaaatctttgccggaaatgc
TSVQPYSPTNEFPKVER
agtgaaaactgcaagacatgtactgaattccataactgtacagaatgtagggatgggttaa
FRYSRVEDPTDDYGTE
gcctgcagggatcccggtgctctgtctcctgtgaagatggacggtatttcaacggccagga
DYAGPCDPECSEVGCD
ctgccagccctgccaccgcttctgcgccacttgtgctggggcaggagctgatgggtgcatt
GPGPDHCNDCLHYYYK
aactgcacagagggctacttcatggaggatgggagatgcgtgcagagctgtagtatcagc
LKNNTRICVSSCPPGHY
tattactttgaccactcttcagagaatggatacaaatcctgcaaaaaatgtgatatcagt
HADKKRCRKCAPNCES
tgtttgacgtgcaatggcccaggattcaagaactgtacaagctgccctagtgggtat
CFGSHGDQCMSCKYGY
ctcttagacttaggaatgtgtcaaatgggagccatttgcaaggatgcaacggaaga
FLNEETNSCVTHCPDGS
gtcctgggcggaaggaggcttctgtatgcttgtgaaaaagaacaatctgtgccaacg
YQDTKKNLCRKCSENC gaaggttcttcaacaactttgctgcaaaacatgtacatttcaaggctga
KTCTEFHNCTECRDGLS LQGSRCSVSCEDGRYF NGQDCQPCHRFCATCA
GAGADGCINCTEGYFM EDGRCVQSCSISYYFDH SSENGYKSCKKCDISCL
TCNGPGFKNCTSCPSGY LLDLGMCQMGAICKDA TEESWAEGGFCMLVKK
NNLCQRKVLQQLCCKT CTFQG
Example 24
Construction of FIX-/FXI/-XTEN
[0383] Construction of FIX-TEV-XTEN_AE864 Genes and Vectors
[0384] The cloning vector containing the gene encoding FIX was
purchased from OriGene (SC126517). PCR reactions were performed to
abolish two BbsI restriction sites within the FIX coding region.
The resulting FIX coding region was then amplified using primers
that introduced NheI and SfiI-TEV-BsaI sequences on the 5' and 3'
end respectively. The digested FIX fragment was fused to
BsaI/HindIII digested XTEN_AE864 fragment and inserted into
NheI/HindIII digested pSecTag2C expression vector. The ligated DNA
mixture was electroporated into XL1-Blue bacterial cells.
Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The final construct is
pCW0648.001 which encodes the FIX-TEV-XTEN_AE864 protein (Table
25).
[0385] Construction of FIX-/FXI/-XTEN_AE864 Genes and Vectors
[0386] The TEV site was removed by digesting the pCW0648 expression
vector with SfiI and BsaI. Oligos containing sequences that encode
SfiI-KLTRAET-BsaI, SfiI-DFTRVVG-BsaI, or SfiI-/FXI/-BsaI were
annealed and ligated with the digested pCW0648 vector. The ligated
DNA mixture was electroporated into XL1-Blue bacterial cells.
Transformants were screened by DNA miniprep and the desired
constructs were confirmed by DNA sequencing. The resulting
expression vectors encode FIX-KLTRAET-XTEN_AE864 (pCW0735),
FIX-DFTRVVG-XTEN_AE864 (pCW0736) and FIX-/FXI/-XTEN_AE864
(pCW0737).
[0387] Construction of expression vectors encoding
FIX-/FXI/-XTEN_AE864 genes using Millipore Plasmids
[0388] Expression vector pCW0735 was digested with NheI and SalI.
The resulting 4181 bp fragment included nucleotides that encode the
FIX-KLTRAET-XTEN_AE864 protein. This fragment was ligated with
NheI/SalI digested CET1019-HD-puro (Millipore) or DC HD-puro
(Millipore). Both CET1019-HD-puro and DC-HD-puro feature dual
cassettes where a CMV promoter lies upstream of the gene insertion
site, CET1019-HD-puro also contains a UCOE element upstream of the
promoter. The ligated DNA mixture was electroporated into XL1-Blue
bacterial cells. Transformants were screened by DNA miniprep and
the desired constructs were confirmed by DNA sequencing. The
resulting expression vectors were pBC0033
(DC-HD-puro-FIX-KLTRAET-XTEN_AE864) and pBC0034
(CET1019-HD-puro-FIX-KLTRAET-XTEN_AE864) (Table 25).
TABLE-US-00031 TABLE 25 FIX-XTEN with cleavage sequence: amino acid
and nucleic acid sequences Amino Acid Name Sequence Nucleic Acid
Sequences FIX-TEV- MQRVNMIMAESPGLITI
atgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttag
XTEN_AE864 CLLGYLLSAECTVFLDH
gatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaa
ENANKILNRPKRYNSG
tcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagaga
KLEEFVQGNLERECME
gaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaa
EKCSFEEAREVFENTER
gaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaa
TTEFWKQYVDGDQCES
atggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaag
NPCLNGGSCKDDINSYE
gaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgt
CWCPFGFEGKNCELDV
aaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaa
TCNIKNGRCEQFCKNS
ccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttc
ADNKVVCSCTEGYRLA
taagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaa
ENQKSCEPAVPFPCGRV
accattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtg
SVSQTSKLTRAETVFPD
gagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgca
VDYVNSTEAETILDNIT
ttctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactg
QSTQSFNDFTRVVGGE
gtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagca
DAKPGQFPWQVVLNG
aaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaa
KVDAFCGGSIVNEKWI
ccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacaccta
VTAAHCVETGVKITVV
tttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtgg
AGEHNIEETEHTEQKRN
ctggggaagagtGttccacaaagggagatcagctttagttcttcagtaccttagagttccac
VIRIIPHHNYNAAINKY
ttgttgaccgagccacatgtctAcgatctacaaagttcaccatctataacaacatgttctgtg
NHDIALLELDEPLVLNS
ctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttac
YVTPICIADKEYTNIFLK
tgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatga
FGSGYVSGWGRVFHKG
aaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaac
RSALVLQYLRVPLVDR
aaagctcactGGCCCAGAAGGCCCAtccgaaaatctgtattttcagggtGG
ATCLRSTKFTIYNNMFC GTCTCCAGGTTCTCCAGCCGGGTCCCCAACTTCGACCG
AGFHEGGRDSCQGDSG AGGAAGGGACCTCCGAGTCAGCTACCCCGGAGTCCGG
GPHVTEVEGTSFLTGIIS TCCTGGCACCTCCACCGAACCATCGGAGGGCAGCGCC
WGEECAMKGKYGIYT CCTGGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAG
KVSRYVNWIKEKTKLT AGGGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCC
GPEGPSENLYFQGGSPG TGGTACTAGTACTGAACCATCCGAGGGGTCAGCTCCA
SPAGSPTSTEEGTSESAT GGCACGAGTGAGTCCGCTACCCCCGAGAGCGGACCGG
PESGPGTSTEPSEGSAP GCTCAGAGCCCGCCACGAGTGGCAGTGAAACTCCAGG
GSPAGSPTSTEEGTSTEP CTCAGAACCCGCCACTAGTGGGTCAGAGACTCCAGGC
SEGSAPGTSTEPSEGSA AGCCCTGCCGGATCCCCTACGTCCACCGAGGAGGGAA
PGTSESATPESGPGSEP CATCTGAGTCCGCAACACCCGAATCCGGTCCAGGCAC
ATSGSETPGSEPATSGS CTCCACGGAACCTAGTGAAGGCTCGGCACCAGGTACA
ETPGSPAGSPTSTEEGTS AGCACCGAACCTAGCGAGGGCAGCGCTCCCGGCAGCC
ESATPESGPGTSTEPSEG CTGCCGGCAGCCCAACCTCAACTGAGGAGGGCACCAG
SAPGTSTEPSEGSAPGSP TACTGAGCCCAGCGAGGGATCAGCACCTGGCACCAGC
AGSPTSTEEGTSTEPSE ACCGAACCTAGCGAGGGGAGCGCCCCTGGGACTAGCG
GSAPGTSTEPSEGSAPG AGTCAGCTACACCAGAGAGCGGGCCTGGAACTTCTAC
TSESATPESGPGTSTEPS CGAACCCAGTGAGGGATCCGCTCCAGGCACCTCCGAA
EGSAPGTSESATPESGP TCCGCAACCCCCGAATCCGGACCTGGCTCAGAGCCCG
GSEPATSGSETPGTSTEP CCACCAGCGGGAGCGAAACCCCTGGCACATCCACCGA
SEGSAPGTSTEPSEGSA GCCTAGCGAAGGGTCCGCACCCGGCACCAGTACAGAG
PGTSESATPESGPGTSES CCTAGCGAGGGATCAGCACCTGGCACCAGTGAATCTG
ATPESGPGSPAGSPTST CTACACCAGAGAGCGGCCCTGGAACCTCCGAGTCCGC
EEGTSESATPESGPGSEP TACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGCTCCC
ATSGSETPGTSESATPES CCACCTCAACAGAAGAGGGGACAAGCGAAAGCGCTAC
GPGTSTEPSEGSAPGTS GCCTGAGAGTGGCCCTGGCTCTGAGCCAGCCACCTCC
TEPSEGSAPGTSTEPSEG GGCTCTGAAACCCCTGGCACTAGTGAGTCTGCCACGCC
SAPGTSTEPSEGSAPGT TGAGTCCGGACCCGGGACCTCTACTGAGCCCTCGGAG
STEPSEGSAPGTSTEPSE GGGAGCGCTCCTGGCACGAGTACAGAACCTTCCGAAG
GSAPGSPAGSPTSTEEG GAAGTGCACCGGGCACAAGCACCGAGCCTTCCGAAGG
TSTEPSEGSAPGTSESAT CTCTGCTCCCGGAACCTCTACCGAACCCTCTGAAGGGT
PESGPGSEPATSGSETP CTGCACCCGGCACGAGCACCGAACCCAGCGAAGGGTC
GTSESATPESGPGSEPA AGCGCCTGGGACCTCAACAGAGCCCTCGGAAGGATCA
TSGSETPGTSESATPESG GCGCCTGGAAGCCCTGCAGGGAGTCCAACTTCCACGG
PGTSTEPSEGSAPGTSES AAGAAGGAACGTCTACAGAGCCATCAGAGGGGTCCGC
ATPESGPGSPAGSPTST ACCAGGTACCAGCGAATCCGCTACTCCCGAATCTGGC
EEGSPAGSPTSTEEGSP CCTGGGTCCGAACCTGCCACCTCCGGCTCTGAAACTCC
AGSPTSTEEGTSESATP AGGGACCTCCGAATCTGCCACACCCGAGAGCGGCCCT
ESGPGTSTEPSEGSAPG GGCTCCGAGCCCGCAACATCTGGCAGCGAGACACCTG
TSESATPESGPGSEPATS GCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGG
GSETPGTSESATPESGP CACCAGCACCGAGCCATCCGAGGGATCCGCCCCAGGC
GSEPATSGSETPGTSES ACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGGAT
ATPESGPGTSTEPSEGS CACCCGCTGGCTCCCCCACCAGTACCGAGGAGGGGTC
APGSPAGSPTSTEEGTS CCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCC
ESATPESGPGSEPATSG CCTGCCGGCTCCCCCACAAGTACCGAAGAGGGCACAA
SETPGTSESATPESGPGS GTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAG
PAGSPTSTEEGSPAGSP CACAGAGCCTTCCGAGGGGTCCGCACCAGGTACCTCA
TSTEEGTSTEPSEGSAP GAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGC
GTSESATPESGPGTSES CAGCCACCTCCGGGTCTGAGACACCCGGGACTTCCGA
ATPESGPGTSESATPES GAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCC
GPGSEPATSGSETPGSE GCCACTTCCGGCTCCGAAACTCCCGGCACAAGCGAGA
PATSGSETPGSPAGSPTS GCGCTACCCCAGAGTCAGGACCAGGAACATCTACAGA
TEEGTSTEPSEGSAPGT GCCCTCTGAAGGCTCCGCTCCAGGGTCCCCAGCCGGC
STEPSEGSAPGSEPATS AGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAGCG
GSETPGTSESATPESGP CCACACCCGAATCAGGGCCAGGGTCTGAGCCTGCTAC
GTSTEPSEGSAPGSSS CAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCGCC
ACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAGCC
CCACCTCCACTGAGGAGGGATCTCCTGCTGGCTCTCCA
ACATCTACTGAGGAAGGTACCTCAACCGAGCCATCCG
AGGGATCAGCTCCCGGCACCTCAGAGTCGGCAACCCC
GGAGTCTGGACCCGGAACTTCCGAAAGTGCCACACCA
GAGTCCGGTCCCGGGACTTCAGAATCAGCAACACCCG
AGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGTAG
TGAGACACCAGGATCAGAACCTGCTACCTCAGGGTCA
GAGACACCCGGATCTCCGGCAGGCTCACCAACCTCCA
CTGAGGAGGGCACCAGCACAGAACCAAGCGAGGGCTC
CGCACCCGGAACAAGCACTGAACCCAGTGAGGGTTCA
GCACCCGGCTCTGAGCCGGCCACAAGTGGCAGTGAGA
CACCCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGG
CCCAGGCACTAGTACCGAGCCCTCTGAAGGCAGTGCG CCAGGTTCGTCTTCATAA FIX-
MQRVNMIMAESPGLITI
atgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttag
KLTRAET- CLLGYLLSAECTVFLDH
gatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaa
XTEN_AE864 ENANKILNRPKRYNSG
tcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagaga
KLEEFVQGNLERECME
gaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaa
EKCSFEEAREVFENTER
gaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaa
TTEFWKQYVDGDQCES
atggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaag
NPCLNGGSCKDDINSYE
gaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgt
CWCPFGFEGKNCELDV
aaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaa
TCNIKNGRCEQFCKNS
ccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttc
ADNKVVCSCTEGYRLA
taagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaa
ENQKSCEPAVPFPCGRV
accattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtg
SVSQTSKLTRAETVFPD
gagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgca
VDYVNSTEAETILDNIT
ttctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactg
QSTQSFNDFTRVVGGE
gtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagca
DAKPGQFPWQVVLNG
aaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaa
KVDAFCGGSIVNEKWI
ccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacaccta
VTAAHCVETGVKITVV
tttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtgg
AGEHNIEETEHTEQKRN
ctggggaagagtGttccacaaagggagatcagctttagttcttcagtaccttagagttccac
VIRIIPHHNYNAAINKY
ttgttgaccgagccacatgtctAcgatctacaaagttcaccatctataacaacatgttctgtg
NHDIALLELDEPLVLNS
ctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttac
YVTPICIADKEYTNIFLK
tgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatga
FGSGYVSGWGRVFHKG
aaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaac
RSALVLQYLRVPLVDR aaagctcactGGCCCAGAAGGCCCAtccaagctAacGcgtgcGgagac
ATCLRSTKFTIYNNMFC AGGGTCTCCAGGTTCTCCAGCCGGGTCCCCAACTTCGA
AGFHEGGRDSCQGDSG CCGAGGAAGGGACCTCCGAGTCAGCTACCCCGGAGTC
GPHVTEVEGTSFLTGIIS CGGTCCTGGCACCTCCACCGAACCATCGGAGGGCAGC
WGEECAMKGKYGIYT GCCCCTGGGAGCCCTGCCGGGAGCCCTACAAGCACCG
KVSRYVNWIKEKTKLT AAGAGGGCACCAGTACAGAGCCAAGTGAGGGGAGCG
GPEGPSKLTRAETGSPG CCCCTGGTACTAGTACTGAACCATCCGAGGGGTCAGCT
SPAGSPTSTEEGTSESAT CCAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGAC
PESGPGTSTEPSEGSAP CGGGCTCAGAGCCCGCCACGAGTGGCAGTGAAACTCC
GSPAGSPTSTEEGTSTEP AGGCTCAGAACCCGCCACTAGTGGGTCAGAGACTCCA
SEGSAPGTSTEPSEGSA GGCAGCCCTGCCGGATCCCCTACGTCCACCGAGGAGG
PGTSESATPESGPGSEP GAACATCTGAGTCCGCAACACCCGAATCCGGTCCAGG
ATSGSETPGSEPATSGS CACCTCCACGGAACCTAGTGAAGGCTCGGCACCAGGT
ETPGSPAGSPTSTEEGTS ACAAGCACCGAACCTAGCGAGGGCAGCGCTCCCGGCA
ESATPESGPGTSTEPSEG GCCCTGCCGGCAGCCCAACCTCAACTGAGGAGGGCAC
SAPGTSTEPSEGSAPGSP CAGTACTGAGCCCAGCGAGGGATCAGCACCTGGCACC
AGSPTSTEEGTSTEPSE AGCACCGAACCTAGCGAGGGGAGCGCCCCTGGGACTA
GSAPGTSTEPSEGSAPG GCGAGTCAGCTACACCAGAGAGCGGGCCTGGAACTTC
TSESATPESGPGTSTEPS TACCGAACCCAGTGAGGGATCCGCTCCAGGCACCTCC
EGSAPGTSESATPESGP GAATCCGCAACCCCCGAATCCGGACCTGGCTCAGAGC
GSEPATSGSETPGTSTEP CCGCCACCAGCGGGAGCGAAACCCCTGGCACATCCAC
SEGSAPGTSTEPSEGSA CGAGCCTAGCGAAGGGTCCGCACCCGGCACCAGTACA
PGTSESATPESGPGTSES GAGCCTAGCGAGGGATCAGCACCTGGCACCAGTGAAT
ATPESGPGSPAGSPTST CTGCTACACCAGAGAGCGGCCCTGGAACCTCCGAGTC
EEGTSESATPESGPGSEP CGCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGCT
ATSGSETPGTSESATPES CCCCCACCTCAACAGAAGAGGGGACAAGCGAAAGCGC
GPGTSTEPSEGSAPGTS TACGCCTGAGAGTGGCCCTGGCTCTGAGCCAGCCACCT
TEPSEGSAPGTSTEPSEG CCGGCTCTGAAACCCCTGGCACTAGTGAGTCTGCCACG
SAPGTSTEPSEGSAPGT CCTGAGTCCGGACCCGGGACCTCTACTGAGCCCTCGG
STEPSEGSAPGTSTEPSE AGGGGAGCGCTCCTGGCACGAGTACAGAACCTTCCGA
GSAPGSPAGSPTSTEEG AGGAAGTGCACCGGGCACAAGCACCGAGCCTTCCGAA
TSTEPSEGSAPGTSESAT GGCTCTGCTCCCGGAACCTCTACCGAACCCTCTGAAGG
PESGPGSEPATSGSETP GTCTGCACCCGGCACGAGCACCGAACCCAGCGAAGGG
GTSESATPESGPGSEPA TCAGCGCCTGGGACCTCAACAGAGCCCTCGGAAGGAT
TSGSETPGTSESATPESG CAGCGCCTGGAAGCCCTGCAGGGAGTCCAACTTCCAC
PGTSTEPSEGSAPGTSES GGAAGAAGGAACGTCTACAGAGCCATCAGAGGGGTCC
ATPESGPGSPAGSPTST GCACCAGGTACCAGCGAATCCGCTACTCCCGAATCTG
EEGSPAGSPTSTEEGSP GCCCTGGGTCCGAACCTGCCACCTCCGGCTCTGAAACT
AGSPTSTEEGTSESATP CCAGGGACCTCCGAATCTGCCACACCCGAGAGCGGCC
ESGPGTSTEPSEGSAPG CTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC
TSESATPESGPGSEPATS TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCT
GSETPGTSESATPESGP GGCACCAGCACCGAGCCATCCGAGGGATCCGCCCCAG
GSEPATSGSETPGTSES GCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGG
ATPESGPGTSTEPSEGS ATCACCCGCTGGCTCCCCCACCAGTACCGAGGAGGGG
APGSPAGSPTSTEEGTS TCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGT
ESATPESGPGSEPATSG CCCCTGCCGGCTCCCCCACAAGTACCGAAGAGGGCAC
SETPGTSESATPESGPGS AAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACC
PAGSPTSTEEGSPAGSP AGCACAGAGCCTTCCGAGGGGTCCGCACCAGGTACCT
TSTEEGTSTEPSEGSAP CAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGA
GTSESATPESGPGTSES GCCAGCCACCTCCGGGTCTGAGACACCCGGGACTTCC
ATPESGPGTSESATPES GAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGC
GPGSEPATSGSETPGSE CCGCCACTTCCGGCTCCGAAACTCCCGGCACAAGCGA
PATSGSETPGSPAGSPTS GAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACA
TEEGTSTEPSEGSAPGT GAGCCCTCTGAAGGCTCCGCTCCAGGGTCCCCAGCCG
STEPSEGSAPGSEPATS GCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAG
GSETPGTSESATPESGP CGCCACACCCGAATCAGGGCCAGGGTCTGAGCCTGCT
GTSTEPSEGSAPGSSS ACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCG
CCACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAG
CCCCACCTCCACTGAGGAGGGATCTCCTGCTGGCTCTC
CAACATCTACTGAGGAAGGTACCTCAACCGAGCCATC
CGAGGGATCAGCTCCCGGCACCTCAGAGTCGGCAACC
CCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACAC
CAGAGTCCGGTCCCGGGACTTCAGAATCAGCAACACC
CGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGT
AGTGAGACACCAGGATCAGAACCTGCTACCTCAGGGT
CAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTC
CACTGAGGAGGGCACCAGCACAGAACCAAGCGAGGG
CTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGT
TCAGCACCCGGCTCTGAGCCGGCCACAAGTGGCAGTG
AGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAG
TGGCCCAGGCACTAGTACCGAGCCCTCTGAAGGCAGT GCGCCAGGTTCGTCTTCATAA FIX-
MQRVNMIMAESPGLITI
atgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttag
DFTRVVG- CLLGYLLSAECTVFLDH
gatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaa
XTEN_AE864 ENANKILNRPKRYNSG
tcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagaga
KLEEFVQGNLERECME
gaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaa
EKCSFEEAREVFENTER
gaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaa
TTEFWKQYVDGDQCES
atggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaag
NPCLNGGSCKDDINSYE
gaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgt
CWCPFGFEGKNCELDV
aaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaa
TCNIKNGRCEQFCKNS
ccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttc
ADNKVVCSCTEGYRLA
taagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaa
ENQKSCEPAVPFPCGRV
accattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtg
SVSQTSKLTRAETVFPD
gagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgca
VDYVNSTEAETILDNIT
ttctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactg
QSTQSFNDFTRVVGGE
gtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagca
DAKPGQFPWQVVLNG
aaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaa
KVDAFCGGSIVNEKWI
ccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacaccta
VTAAHCVETGVKITVV
tttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtgg
AGEHNIEETEHTEQKRN
ctggggaagagtGttccacaaagggagatcagctttagttcttcagtaccttagagttccac
VIRIIPHHNYNAAINKY
ttgttgaccgagccacatgtctAcgatctacaaagttcaccatctataacaacatgttctgtg
NHDIALLELDEPLVLNS
ctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttac
YVTPICIADKEYTNIFLK
tgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatga
FGSGYVSGWGRVFHKG
aaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaac
RSALVLQYLRVPLVDR aaagctcactGGCCCAGAAGGCCCAtccgacttcacAcgggtAgttggC
ATCLRSTKFTIYNNMFC GGGTCTCCAGGTTCTCCAGCCGGGTCCCCAACTTCGAC
AGFHEGGRDSCQGDSG CGAGGAAGGGACCTCCGAGTCAGCTACCCCGGAGTCC
GPHVTEVEGTSFLTGIIS GGTCCTGGCACCTCCACCGAACCATCGGAGGGCAGCG
WGEECAMKGKYGIYT CCCCTGGGAGCCCTGCCGGGAGCCCTACAAGCACCGA
KVSRYVNWIKEKTKLT AGAGGGCACCAGTACAGAGCCAAGTGAGGGGAGCGC
GPEGPSDFTRVVGGSPG CCCTGGTACTAGTACTGAACCATCCGAGGGGTCAGCTC
SPAGSPTSTEEGTSESAT CAGGCACGAGTGAGTCCGCTACCCCCGAGAGCGGACC
PESGPGTSTEPSEGSAP GGGCTCAGAGCCCGCCACGAGTGGCAGTGAAACTCCA
GSPAGSPTSTEEGTSTEP GGCTCAGAACCCGCCACTAGTGGGTCAGAGACTCCAG
SEGSAPGTSTEPSEGSA GCAGCCCTGCCGGATCCCCTACGTCCACCGAGGAGGG
PGTSESATPESGPGSEP AACATCTGAGTCCGCAACACCCGAATCCGGTCCAGGC
ATSGSETPGSEPATSGS ACCTCCACGGAACCTAGTGAAGGCTCGGCACCAGGTA
ETPGSPAGSPTSTEEGTS CAAGCACCGAACCTAGCGAGGGCAGCGCTCCCGGCAG
ESATPESGPGTSTEPSEG CCCTGCCGGCAGCCCAACCTCAACTGAGGAGGGCACC
SAPGTSTEPSEGSAPGSP AGTACTGAGCCCAGCGAGGGATCAGCACCTGGCACCA
AGSPTSTEEGTSTEPSE GCACCGAACCTAGCGAGGGGAGCGCCCCTGGGACTAG
GSAPGTSTEPSEGSAPG CGAGTCAGCTACACCAGAGAGCGGGCCTGGAACTTCT
TSESATPESGPGTSTEPS ACCGAACCCAGTGAGGGATCCGCTCCAGGCACCTCCG
EGSAPGTSESATPESGP AATCCGCAACCCCCGAATCCGGACCTGGCTCAGAGCC
GSEPATSGSETPGTSTEP CGCCACCAGCGGGAGCGAAACCCCTGGCACATCCACC
SEGSAPGTSTEPSEGSA GAGCCTAGCGAAGGGTCCGCACCCGGCACCAGTACAG
PGTSESATPESGPGTSES AGCCTAGCGAGGGATCAGCACCTGGCACCAGTGAATC
ATPESGPGSPAGSPTST TGCTACACCAGAGAGCGGCCCTGGAACCTCCGAGTCC
EEGTSESATPESGPGSEP GCTACCCCCGAGAGCGGGCCAGGTTCTCCTGCTGGCTC
ATSGSETPGTSESATPES CCCCACCTCAACAGAAGAGGGGACAAGCGAAAGCGCT
GPGTSTEPSEGSAPGTS ACGCCTGAGAGTGGCCCTGGCTCTGAGCCAGCCACCT
TEPSEGSAPGTSTEPSEG CCGGCTCTGAAACCCCTGGCACTAGTGAGTCTGCCACG
SAPGTSTEPSEGSAPGT CCTGAGTCCGGACCCGGGACCTCTACTGAGCCCTCGG
STEPSEGSAPGTSTEPSE AGGGGAGCGCTCCTGGCACGAGTACAGAACCTTCCGA
GSAPGSPAGSPTSTEEG AGGAAGTGCACCGGGCACAAGCACCGAGCCTTCCGAA
TSTEPSEGSAPGTSESAT GGCTCTGCTCCCGGAACCTCTACCGAACCCTCTGAAGG
PESGPGSEPATSGSETP GTCTGCACCCGGCACGAGCACCGAACCCAGCGAAGGG
GTSESATPESGPGSEPA TCAGCGCCTGGGACCTCAACAGAGCCCTCGGAAGGAT
TSGSETPGTSESATPESG CAGCGCCTGGAAGCCCTGCAGGGAGTCCAACTTCCAC
PGTSTEPSEGSAPGTSES GGAAGAAGGAACGTCTACAGAGCCATCAGAGGGGTCC
ATPESGPGSPAGSPTST GCACCAGGTACCAGCGAATCCGCTACTCCCGAATCTG
EEGSPAGSPTSTEEGSP GCCCTGGGTCCGAACCTGCCACCTCCGGCTCTGAAACT
AGSPTSTEEGTSESATP CCAGGGACCTCCGAATCTGCCACACCCGAGAGCGGCC
ESGPGTSTEPSEGSAPG CTGGCTCCGAGCCCGCAACATCTGGCAGCGAGACACC
TSESATPESGPGSEPATS TGGCACCTCCGAGAGCGCAACACCCGAGAGCGGCCCT
GSETPGTSESATPESGP GGCACCAGCACCGAGCCATCCGAGGGATCCGCCCCAG
GSEPATSGSETPGTSES GCACTTCTGAGTCAGCCACACCCGAAAGCGGACCAGG
ATPESGPGTSTEPSEGS ATCACCCGCTGGCTCCCCCACCAGTACCGAGGAGGGG
APGSPAGSPTSTEEGTS TCCCCCGCTGGAAGTCCAACAAGCACTGAGGAAGGGT
ESATPESGPGSEPATSG CCCCTGCCGGCTCCCCCACAAGTACCGAAGAGGGCAC
SETPGTSESATPESGPGS AAGTGAGAGCGCCACTCCCGAGTCCGGGCCTGGCACC
PAGSPTSTEEGSPAGSP AGCACAGAGCCTTCCGAGGGGTCCGCACCAGGTACCT
TSTEEGTSTEPSEGSAP CAGAGTCTGCTACCCCCGAGTCAGGGCCAGGATCAGA
GTSESATPESGPGTSES GCCAGCCACCTCCGGGTCTGAGACACCCGGGACTTCC
ATPESGPGTSESATPES GAGAGTGCCACCCCTGAGTCCGGACCCGGGTCCGAGC
GPGSEPATSGSETPGSE CCGCCACTTCCGGCTCCGAAACTCCCGGCACAAGCGA
PATSGSETPGSPAGSPTS GAGCGCTACCCCAGAGTCAGGACCAGGAACATCTACA
TEEGTSTEPSEGSAPGT GAGCCCTCTGAAGGCTCCGCTCCAGGGTCCCCAGCCG
STEPSEGSAPGSEPATS GCAGTCCCACTAGCACCGAGGAGGGAACCTCTGAAAG
GSETPGTSESATPESGP CGCCACACCCGAATCAGGGCCAGGGTCTGAGCCTGCT
GTSTEPSEGSAPGSSS ACCAGCGGCAGCGAGACACCAGGCACCTCTGAGTCCG
CCACACCAGAGTCCGGACCCGGATCTCCCGCTGGGAG
CCCCACCTCCACTGAGGAGGGATCTCCTGCTGGCTCTC
CAACATCTACTGAGGAAGGTACCTCAACCGAGCCATC
CGAGGGATCAGCTCCCGGCACCTCAGAGTCGGCAACC
CCGGAGTCTGGACCCGGAACTTCCGAAAGTGCCACAC
CAGAGTCCGGTCCCGGGACTTCAGAATCAGCAACACC
CGAGTCCGGCCCTGGGTCTGAACCCGCCACAAGTGGT
AGTGAGACACCAGGATCAGAACCTGCTACCTCAGGGT
CAGAGACACCCGGATCTCCGGCAGGCTCACCAACCTC
CACTGAGGAGGGCACCAGCACAGAACCAAGCGAGGG
CTCCGCACCCGGAACAAGCACTGAACCCAGTGAGGGT
TCAGCACCCGGCTCTGAGCCGGCCACAAGTGGCAGTG
AGACACCCGGCACTTCAGAGAGTGCCACCCCCGAGAG
TGGCCCAGGCACTAGTACCGAGCCCTCTGAAGGCAGT GCGCCAGGTTCGTCTTCATAA
FIX-/FXI/- MQRVNMIMAESPGLITI
atgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttag
XTEN_AE864 CLLGYLLSAECTVFLDH
gatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaa
ENANKILNRPKRYNSG
tcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagaga
KLEEFVQGNLERECME
gaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaa
EKCSFEEAREVFENTER
gaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaa
TTEFWKQYVDGDQCES
atggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaag
NPCLNGGSCKDDINSYE
gaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgt
CWCPFGFEGKNCELDV
aaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaa
TCNIKNGRCEQFCKNS
ccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttc
ADNKVVCSCTEGYRLA
taagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaa
ENQKSCEPAVPFPCGRV
accattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtg
SVSQTSKLTRAETVFPD
gagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgca
VDYVNSTEAETILDNIT
ttctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactg
QSTQSFNDFTRVVGGE
gtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagca
DAKPGQFPWQVVLNG
aaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaa
KVDAFCGGSIVNEKWI
ccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacaccta
VTAAHCVETGVKITVV
tttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtgg
AGEHNIEETEHTEQKRN
ctggggaagagtGttccacaaagggagatcagctttagttcttcagtaccttagagttccac
VIRIIPHHNYNAAINKY
ttgttgaccgagccacatgtctAcgatctacaaagttcaccatctataacaacatgttctgtg
NHDIALLELDEPLVLNS
ctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttac
YVTPICIADKEYTNIFLK
tgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatga
FGSGYVSGWGRVFHKG
aaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaac
RSALVLQYLRVPLVDR
aaagctcactGGCCCAGAAcaaacAtctaagctAacGcgtgcGgagacAgt
ATCLRSTKFTIYNNMFC AtttccaGGTTCTCCAGCCGGGTCCCCAACTTCGACCGAG
AGFHEGGRDSCQGDSG GAAGGGACCTCCGAGTCAGCTACCCCGGAGTCCGGTC
GPHVTEVEGTSFLTGIIS CTGGCACCTCCACCGAACCATCGGAGGGCAGCGCCCC
WGEECAMKGKYGIYT TGGGAGCCCTGCCGGGAGCCCTACAAGCACCGAAGAG
KVSRYVNWIKEKTKLT GGCACCAGTACAGAGCCAAGTGAGGGGAGCGCCCCTG
GPEQTSKLTRAETVFPG GTACTAGTACTGAACCATCCGAGGGGTCAGCTCCAGG
SPAGSPTSTEEGTSESAT CACGAGTGAGTCCGCTACCCCCGAGAGCGGACCGGGC
PESGPGTSTEPSEGSAP TCAGAGCCCGCCACGAGTGGCAGTGAAACTCCAGGCT
GSPAGSPTSTEEGTSTEP CAGAACCCGCCACTAGTGGGTCAGAGACTCCAGGCAG
SEGSAPGTSTEPSEGSA CCCTGCCGGATCCCCTACGTCCACCGAGGAGGGAACA
PGTSESATPESGPGSEP TCTGAGTCCGCAACACCCGAATCCGGTCCAGGCACCTC
ATSGSETPGSEPATSGS CACGGAACCTAGTGAAGGCTCGGCACCAGGTACAAGC
ETPGSPAGSPTSTEEGTS ACCGAACCTAGCGAGGGCAGCGCTCCCGGCAGCCCTG
ESATPESGPGTSTEPSEG CCGGCAGCCCAACCTCAACTGAGGAGGGCACCAGTAC
SAPGTSTEPSEGSAPGSP TGAGCCCAGCGAGGGATCAGCACCTGGCACCAGCACC
AGSPTSTEEGTSTEPSE GAACCTAGCGAGGGGAGCGCCCCTGGGACTAGCGAGT
GSAPGTSTEPSEGSAPG CAGCTACACCAGAGAGCGGGCCTGGAACTTCTACCGA
TSESATPESGPGTSTEPS ACCCAGTGAGGGATCCGCTCCAGGCACCTCCGAATCC
EGSAPGTSESATPESGP GCAACCCCCGAATCCGGACCTGGCTCAGAGCCCGCCA
GSEPATSGSETPGTSTEP CCAGCGGGAGCGAAACCCCTGGCACATCCACCGAGCC
SEGSAPGTSTEPSEGSA TAGCGAAGGGTCCGCACCCGGCACCAGTACAGAGCCT
PGTSESATPESGPGTSES AGCGAGGGATCAGCACCTGGCACCAGTGAATCTGCTA
ATPESGPGSPAGSPTST CACCAGAGAGCGGCCCTGGAACCTCCGAGTCCGCTAC
EEGTSESATPESGPGSEP CCCCGAGAGCGGGCCAGGTTCTCCTGCTGGCTCCCCCA
ATSGSETPGTSESATPES CCTCAACAGAAGAGGGGACAAGCGAAAGCGCTACGCC
GPGTSTEPSEGSAPGTS TGAGAGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCT
TEPSEGSAPGTSTEPSEG CTGAAACCCCTGGCACTAGTGAGTCTGCCACGCCTGA
SAPGTSTEPSEGSAPGT GTCCGGACCCGGGACCTCTACTGAGCCCTCGGAGGGG
STEPSEGSAPGTSTEPSE AGCGCTCCTGGCACGAGTACAGAACCTTCCGAAGGAA
GSAPGSPAGSPTSTEEG GTGCACCGGGCACAAGCACCGAGCCTTCCGAAGGCTC
TSTEPSEGSAPGTSESAT TGCTCCCGGAACCTCTACCGAACCCTCTGAAGGGTCTG
PESGPGSEPATSGSETP CACCCGGCACGAGCACCGAACCCAGCGAAGGGTCAGC
GTSESATPESGPGSEPA GCCTGGGACCTCAACAGAGCCCTCGGAAGGATCAGCG
TSGSETPGTSESATPESG CCTGGAAGCCCTGCAGGGAGTCCAACTTCCACGGAAG
PGTSTEPSEGSAPGTSES AAGGAACGTCTACAGAGCCATCAGAGGGGTCCGCACC
ATPESGPGSPAGSPTST AGGTACCAGCGAATCCGCTACTCCCGAATCTGGCCCTG
EEGSPAGSPTSTEEGSP GGTCCGAACCTGCCACCTCCGGCTCTGAAACTCCAGG
AGSPTSTEEGTSESATP GACCTCCGAATCTGCCACACCCGAGAGCGGCCCTGGC
ESGPGTSTEPSEGSAPG TCCGAGCCCGCAACATCTGGCAGCGAGACACCTGGCA
TSESATPESGPGSEPATS CCTCCGAGAGCGCAACACCCGAGAGCGGCCCTGGCAC
GSETPGTSESATPESGP CAGCACCGAGCCATCCGAGGGATCCGCCCCAGGCACT
GSEPATSGSETPGTSES TCTGAGTCAGCCACACCCGAAAGCGGACCAGGATCAC
ATPESGPGTSTEPSEGS CCGCTGGCTCCCCCACCAGTACCGAGGAGGGGTCCCC
APGSPAGSPTSTEEGTS CGCTGGAAGTCCAACAAGCACTGAGGAAGGGTCCCCT
ESATPESGPGSEPATSG GCCGGCTCCCCCACAAGTACCGAAGAGGGCACAAGTG
SETPGTSESATPESGPGS AGAGCGCCACTCCCGAGTCCGGGCCTGGCACCAGCAC
PAGSPTSTEEGSPAGSP AGAGCCTTCCGAGGGGTCCGCACCAGGTACCTCAGAG
TSTEEGTSTEPSEGSAP TCTGCTACCCCCGAGTCAGGGCCAGGATCAGAGCCAG
GTSESATPESGPGTSES CCACCTCCGGGTCTGAGACACCCGGGACTTCCGAGAG
ATPESGPGTSESATPES TGCCACCCCTGAGTCCGGACCCGGGTCCGAGCCCGCC
GPGSEPATSGSETPGSE ACTTCCGGCTCCGAAACTCCCGGCACAAGCGAGAGCG
PATSGSETPGSPAGSPTS CTACCCCAGAGTCAGGACCAGGAACATCTACAGAGCC
TEEGTSTEPSEGSAPGT CTCTGAAGGCTCCGCTCCAGGGTCCCCAGCCGGCAGTC
STEPSEGSAPGSEPATS CCACTAGCACCGAGGAGGGAACCTCTGAAAGCGCCAC
GSETPGTSESATPESGP ACCCGAATCAGGGCCAGGGTCTGAGCCTGCTACCAGC
GTSTEPSEGSAPGSSS GGCAGCGAGACACCAGGCACCTCTGAGTCCGCCACAC
CAGAGTCCGGACCCGGATCTCCCGCTGGGAGCCCCAC
CTCCACTGAGGAGGGATCTCCTGCTGGCTCTCCAACAT
CTACTGAGGAAGGTACCTCAACCGAGCCATCCGAGGG
ATCAGCTCCCGGCACCTCAGAGTCGGCAACCCCGGAG
TCTGGACCCGGAACTTCCGAAAGTGCCACACCAGAGT
CCGGTCCCGGGACTTCAGAATCAGCAACACCCGAGTC
CGGCCCTGGGTCTGAACCCGCCACAAGTGGTAGTGAG
ACACCAGGATCAGAACCTGCTACCTCAGGGTCAGAGA
CACCCGGATCTCCGGCAGGCTCACCAACCTCCACTGA
GGAGGGCACCAGCACAGAACCAAGCGAGGGCTCCGC
ACCCGGAACAAGCACTGAACCCAGTGAGGGTTCAGCA
CCCGGCTCTGAGCCGGCCACAAGTGGCAGTGAGACAC
CCGGCACTTCAGAGAGTGCCACCCCCGAGAGTGGCCC
AGGCACTAGTACCGAGCCCTCTGAAGGCAGTGCGCCA GGTTCGTCTTCATAA
Example 25
Expression of FVII-XTEN and FIX-XTEN
[0389] Transient Transfection of Mammalian Cells
[0390] Mammalian cells, including CHO-K1, BHK, COS-7, and HEK293,
were found to express FVII-XTEN or FIX-XTEN using different XTEN
lengths when transfected. The following are details for methods
used to express the various FVII-XTEN and FIX-XTEN fusion protein
constructs by transient transfection.
[0391] HEK293 cells were plated the day before transfection,
1.times.10.sup.5 per well in 12-well plate in 1 ml medium
containing 10% FBS, 1.times. Pen/Strep, and 5 mg/ml vitamin K. For
transfection the day after plating the cells, plasmid DNA (0.6
.mu.g) diluted in OptiMEM (total 25 .mu.l) was mixed with diluted
FuGENE6 (2.1 .mu.l FuGENE6 in 22.9 .mu.l OptiMEM) and incubated for
30 min at room temperature before adding to the cells. On day 3 or
4 after transfection the culture medium was collected, centrifuged
at 500.times.g for 5 min at room temperature, and then the
supernatant filtered using 0.2 .mu.m filter before testing for
expression of FVII-XTEN or FIX-XTEN in ELISA and performance in a
clotting assay (PT for FVII activity and aPTT for FIX activity).
The results are presented in Table 26.
[0392] It should be noted that the titer measured for FVII-XTEN by
PT assay (active FVII protein) was higher than the titer measured
by ELISA (total FVII protein), and while the exact cause for this
remained to be clarified, it could be due to (1) underestimation of
FVII in the context of FVII-XTEN due to epitope shielding by XTEN,
(2) overestimation of clotting activity by the PT assay, or a
combination of both (1) and (2). It should also be noted that the
titer measured for FIX by aPTT assay (active FIX protein) was
significantly lower than the titer measured by ELISA (total FIX
protein), only about 20%, due to unknown reasons, but one of which
could be insufficient propeptide processing, a phenomenon that has
been reported for recombinant FIX produced in CHO or other
mammalian cells. The titer of FIX-XTEN by aPTT was even lower
proportionally than ELISA compared to FIX alone, suggesting the
activity of FIX could be reduced by fusing to XTEN, an assumption
confirmed by analyzing the activity and ELISA titers of protein
after TEV treatment for materials produced from cells transfected
with plasmids constructs encoding a FVII-XTEN but with TEV cleavage
site inserted in between.
TABLE-US-00032 TABLE 26 Expression of FVII, FVII-XTEN_AE864, FIX,
and FIX-XTEN_AE864 FVII FVII-XTEN FIX FIX-XTEN ELISA Clotting ELISA
Clotting ELISA Clotting ELISA Clotting ng/ml 391.0 397.7 34.8 176.6
309.8 60.0 13.7 0.4
[0393] Generation of CHO-K1 Stable Pools and Cell Lines That
Produce FVII-XTEN
[0394] Cells: CHO-K1 cells purchased from ATCC (Cat. CCL-61, Lot
58078551) were revived in Complete Medium (F-12K, 10% FBS and
1.times.P/S, Appendix 1) and passaged for four generations before
multiple vials were frozen in the Complete Medium with 5% DMSO. One
vial was revived in medium similar to the Complete Medium but with
5% FBS and passaged one more time before transfection.
[0395] Generation of Stable Pool: Construction of plasmids pBC0014,
pBC0016, and pBC0018 encoding FVII-AE864, FVII-AE864, and
FVII-AE288, respectively, has been described in the Examples above.
Two of the plasmids pBC0016 and pBC0018 also carry UCOE. The
plasmids were first linearized with PvuII and then transfected with
FuGENE6 transfection reagent into separate T25 flasks of CHO-K1
cells from above, 3.6 .mu.g plasmid DNA for 6.5.times.10.sup.5
cells per flask. Two days later the cells were transferred to T75
and cultured in Selection Medium (Complete Medium with 10 .mu.g/ml
puromycin and 5 .mu.g/ml vitamin K). The flasks were changed to
fresh Selection Medium every 2-3 days. Two weeks after
transfection, cells from T75 flasks were frozen as stable pool.
[0396] Selection of clones: For primary screening, frozen stable
pool cells were revived and seeded in 6 96-well plates at a target
density of 0.5 cell/well. About 1 week after seeding spent medium
from wells with single cell cluster as observed under microscope
were tested for expression of FVII by ELISA. The number of clones
tested in the primary screening by the ELISA was: 154 for pBC0014,
210 for pBC0016, and 135 for pBC0018. Significant numbers of clones
expressed no or non-detectable levels of FVII (FIG. 9, black bars,
expressed as ng/ml), but 15-20% of the clones expressed FVII of 3-8
fold higher, these clones were then selected for further screening
and selection, 20 for pBC0014, 30 for pBC0016, and 20 for pBC0018.
The size of the cell clusters in these wells was scored 1-10 with 1
being the smallest cluster and 10 the largest cluster; the results
are shown as gray bars in FIG. 9. The distribution of the cell
cluster size of these clones was similar to that of all the clones
for the same variant, suggesting they were selected not just
because they were the fastest growers.
[0397] For additional rounds of screening, normalized numbers of
cells were seeded in multi-well plates. Spent medium were harvested
2-3 days after seeding and were tested for FVII concentration by
ELISA and clotting activity by PT; Cells were also harvested from
the plates and counted using Vi-Cell. Clones were ranked by (1)
FVII titers according to ELISA and clotting; (2) Ratios of ELISA
titer/cell count and clotting titer/cell count; and (3) Integrity
and homogeneity of products produced by the clones as measured by
Western blots. Selection of clones for each of the construct
pBC0014, pBC0016, and pBC0018 was described separately in the
following.
[0398] pBC0014: For the second round of screening, cells in 96-well
plates for the top 20 clones selected from primary screening were
first expanded in T25 flasks and then seeded in duplicate 24-well
plates, one cultured for 2 days and the other one for 3 days. Spent
medium were collected from the plates for FVII ELISA and cells
harvested and counted by Vi-Cell. Fourteen clones were selected
according to titers by ELISA and clotting, ELISA titer/cell and
clotting titer/cell count ratios and further screened. Frozen vials
were prepared for 9 clones, 1F10, 2F7, 6H4, 1A3, 6F10, 5C2, 5F1,
3H2, 4C8. Out of the 14 clones, 1F10, 1F4, 2F7, 4C8, 6H4, and 6G1
were again screened and ranked according to titers by ELISA and
clotting, ratios of ELISA titer/cell count and clotting titer/cell
count, and product integrity and homogeneity by Western blot (FIGS.
10-12). Clone 6G1 expressed a product that is significantly smaller
than the full-length product (FIG. 12) and was discarded.
Additional frozen vials were prepared for clones 1F10, 2F7, 6H4,
and 4C8. Clone 4C8 was tested for production of FVII-AE864 in
roller bottles.
[0399] pBC0016: For the second round of screening, cells in 96-well
plates for the top 30 clones selected from primary screening were
transferred to 12-well plates and then T25 and ranked by titers
according to ELISA and clotting assays, ratios of ELISA titer/cell
count and clotting titer/cell count. For the third round of
screening, fifteen clones including 1D4, 1G2, 1G6, 2C11, 2H6, 3A2,
3B1, 3C7, 3F2, 3H1, 3H6, 3H10, 4G8, 5E12, 6F11 were tested, ranked
according to above criteria plus Western blot (FIG. 13) and frozen
cells were prepared for all of the 15 clones, except 3H6. Eight
clones including 1G2, 2C11, 3B1, 3C7, 3F2, 3H10, 4G8, 5E12 were
selected as the top clones and additional frozen vials were
prepared for them. Clone 3H10 was selected for scale-up production
in roller bottles.
[0400] pBC0018: For the second round of screening, cells in 96-well
plates for the top 20 clones selected from primary screening were
first expanded in T25 flasks and then seeded in 24-well plates.
Spent medium were collected from the plates for FVII ELISA and
cells harvested and counted by Vi-Cell. Twelve clones were selected
according to titers by ELISA and clotting, ELISA titer/cell and
clotting titer/cell count ratios and further screened. Frozen vials
were prepared for 9 clones, 2C3, 2D5, 3B2, 3B10, 3G2, 3G12, 5A12,
6A3, and 6E7. Out of the 9 clones, 2D5, 3B2, 3G2, 3G12, 5A12, 6A3,
and 6E7 were again screened and ranked according to titers by ELISA
and clotting, ratios of ELISA titer/cell count and clotting
titer/cell count, and product integrity and homogeneity by Western
blot (FIG. 14). Clone 3B2 expressed products that displayed
multiple bands on Western blot and it was discarded. Additional
frozen vials were prepared for clones 2D5, 3G2, 3G12, 5A12, 6A3,
6E7. Clones 3G12 and 6E7 were used for production of FVII-AE288 in
roller bottles.
[0401] Production of FVII-XTEN Secreted in Cell Culture Medium in
Roller Bottles
[0402] CHO-K1 cell stable pools or clones were expanded in T175, 35
ml Selection Medium per flask. Cells were harvested from by
trypsinization and used to seed roller bottles (1700 cm.sup.2
surface area per bottle) on Day 0, 300 ml Selection Medium with
cells from 1-2 T175 flasks for every roller bottle. The
spent/conditioned medium was removed on Day 3 (or 4) and refilled
with 300 ml fresh Selection Medium. On Day 5 (or 6) the
spent/conditioned medium was removed and discarded (or harvested if
XTEN fusion proteins in this medium can be purified) and 300 ml
Transition Medium (UltraCHO containing 1% FBS, 0.1% Ex-Cyte, 5
mg/ml vitamin K, and 1.times. Pen/Step) was added to each roller
bottle. On Day 7 (or 8) the spent medium was removed and discarded
(or harvested if XTEN fusion proteins can be purified from this
medium) and Expression Medium (OptiMEM containing 0.1% Ex-Cyte, 1%
ITS-A, 5 mg/ml vitamin K, and 1.times. Pen/Strep) was added, 300 ml
per bottle or other volumes depending on results from optimization.
Conditioned medium could be harvested once everyday, or once every
2, or 3, or 4 days depending on product titer and quality desired.
To harvest, the conditioned medium was poured into centrifuge
bottles, and fresh Expression Medium was added, 300 ml per bottle
or other volumes depending on results from optimization. This
production of harvesting spent medium and refilling with fresh
medium could last for 2-4 weeks until titer or/and product quality
are considered too low, when the roller bottles are terminated. The
conditioned medium was then centrifuged, 3500 rpm in a bench-top
centrifuge, for 10 min, at 4-8.degree. C. The supernatant was then
filtered using a 0.2 mm filter. The filtrate was either processed
immediately or was stored in -80.degree. C. freezer before
processing by tangential flow filtration (TFF) for
purification.
Example 26
Purification and Characterization of FVII-XTEN Constructs
[0403] Concentration and Buffer Exchange of FVII-XTEN_AE864 by
Tangential Flow Filtration and Diafiltration
[0404] Supernatant batches S279, S281, 5282 and S287, totaling 10.7
L in volume, from stable CHO cells lines expressing FVII-AE864
(AC404) were filtered using a Cuno ZetaPlus Biocap filter and a
Cuno BioAssure capsule. They were subsequently concentrated
approximately 20 fold by tangential flow filtration using a
Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using
the same tangential flow filtration cartridge the sample was
diafiltered with 10 mM tris pH 7.5, 1 mM EDTA with 5 volumes worth
of buffer exchange. Samples were divided into 50 ml aliquots and
frozen at -80.degree. C. No FVII activity was detectable in the
permeate fractions from the filtration and .about.75% recovery of
the activity was seen in the concentrated, buffer exchanged
material.
[0405] Purification of FVII-XTEN_AE864 by BaSO.sub.4Adsorption
[0406] FVII-AE864 (AC404) containing supernatant was concentrated
and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA.
Subsequently, 5 ml of this sample was diluted 10 fold in PBS,
additional NaCl was added to 50 mM, and then BaSO.sub.4 was added
to 20 mg/ml. The sample was bound on a nutator at room temperature
for 30 minutes. The sample was then centrifuged at 3000 rpm for 5
minutes to pellet the BaSO.sub.4. The supernatant was discarded and
the pellet resuspended in 5 ml if 200 mM sodium acetate and nutated
for 30 minutes at room temperature. This was repeated two more
times. After the third wash the pellet was resuspended in 0.8 ml of
100 mM trisodium citrate pH 7.0 and nutated for 30 minutes at room
temperature. This was repeated once. A Bradford assay was performed
to determine the total amount of protein in the sample and FVII
activity was assayed in a PT based factor assay with Innovin as the
activating thromboplastin (Table 27). The ratio of activity to
total protein demonstrated a net purification of .about.12 fold
from this purification step.
TABLE-US-00033 TABLE 27 Purification Table of FVII-AE864 by BaSO4
Absorption Total Total Volume Activity Protein Specific Purity
Fraction (ml) (U) (ug) Activity Purification (%) Feed 5 17.3 6300
0.003 1.0 0.1% FT 1 5 4.0 4687 0.001 0.3 0.0% Wash 1 5 NA 57 NA NA
NA Wash 2 5 NA 8 NA NA NA Wash 3 5 NA 8 NA NA NA Elution 1 0.8 3.2
85 0.038 13.8 1.9% Elution 2 0.8 0.4 12 0.030 11.0 1.5%
[0407] Purification of FVII-XTEN_AE864 by aGla Affinity
Chromatography
[0408] A monoclonal antibody (clone ID CaFVII-22) which binds the
GLA domain of FVII in a calcium dependent manner was coupled to
Ultralink beads from Pierce. Coupling was performed by adding 10 mg
of antibody in PBS to 1.25 of resin and bringing the final volume
to 15 ml with coupling buffer (100 mM MOPS, 700 mM sodium citrate,
pH 8.0). This produced 10 ml of resin slurry and a 1 mg to 1 ml
ratio of antibody mass to bead slurry volume. The slurry was
incubated for 2 hours at room temperature and then the beads were
washed with coupling buffer. A BCA assay indicated .about.70% of
the antibody was coupled to the beads. The beads are then quenched
with 1M tris pH 8.0 for 2 hours at room temperature. The beads were
equilibrated into 10 mM tris pH 7.5 and 10 mM CaCl.sub.2 and 5.5 ml
of beads was mixed with 50 ml of concentrated, buffer exchanged
FVII-AE864 (AC404) supernatant in 10 mM tris pH 7.5 and .about.10
mM CaCl.sub.2. The sample was incubated at 4.degree. C. overnight
on a nutator to bind the FVII-XTEN to the resin. The following day
the beads were washed three times with 45 ml 10 mM tris, 500 mM
NaCl, 10 mM CaCl.sub.2, pH 7.5 and then eluted with 20 ml of 10 mM
tris, 100 mM EDTA, pH 7.5. SDS-PAGE analysis indicates that the
purity is in excess of 90% (FIG. 15).
[0409] Activation of FVII-XTEN_AE864 and FVII-XTEN_AE288
[0410] Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398)
were activated to FVIIa-AE864 and FVIIa-AE288 by addition of FXa.
The FVII-XTEN proteins were buffer exchanged into 10 mM Tris, 10 mM
CaCl.sub.2, pH7.5 via repeat rounds of concentration in an Amicon
ultra 10,000 Da MWCO concentrator and subsequent dilution. The
final volume was 1 ml at .about.0.4 mg/ml. FXa from Novagen was
added to a final concentration of 10 units/ml and the sample
incubated overnight at 4.degree. C. Reducing SDS-PAGE indicated a
complete conversion of FVII-XTEN proteins to FVIIa-XTEN proteins by
the downward shift in the top band with DTT compared to the
non-reduced sample which represents the loss of the light chain
from the molecule, which can only occur upon activation (FIG. 16).
Additionally, the light chain can be seen appearing lower on the
gel and running at the same position as the light chain of control
FVIIa, further confirming the transition of the FVII domain from
FVII to FVIIa. Under similar buffer conditions FVII-XTEN fusions
are activated to FVIIa-XTEN by the addition of thrombin, FIXa,
FXIIa or any other protease capable of selectively cutting the
peptide bond between R152 and 1153.
[0411] Autoactivation of FVII-XTEN_AE864 and FVII-XTEN_AE288
[0412] Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398)
were activated to FVIIa-AE864 and FVIIa-AE288 by incubating the
sample at 4.degree. C. for 1 week. The FVII-XTEN proteins were
buffer exchanged into 10 mM Tris, 10 mM CaCl2, pH7.5 via repeat
rounds of concentration in an Amicon ultra 10,000 Da MWCO
concentrator and subsequent dilution. After the incubation the
protein was assayed by SDS-PAGE and the top band displays the
characteristic downward shift in the top band with DTT compared to
the non-reduced sample which represents the loss of the light chain
from the molecule, which can only occur upon activation (FIG. 17).
Additionally, the light chain can bee seen appearing lower on the
gel at the same point as the two lots of FXa activated material,
further corroborating the conclusion that the proteins
auto-activated to FVIIa-XTEN.
[0413] Purification of FVII-XTEN_AE864 by Anion Exchange
Chromatography
[0414] A sample of FVII-AE864 (AC404) containing supernatant was
concentrated and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA
and then adjusted to a final concentration of .about.5 mM
CaCl.sub.2 with the addition of 1M CaCl.sub.2. The sample was
loaded onto a 2 ml macrocap Q column equilibrated on an Akta
chromatography system. The protein was eluted with a linear
gradient of 0-100% buffer B over 20 column volumes. Buffer A was
comprised of 20 mM MES, 5 mM CaCl.sub.2pH 6.0 and buffer B was
comprised of 20 mM MES, 5 mM CaCl.sub.2pH 6.0 and 500 mM NaCl.
Fractions were assayed for FVII activity using a PT based factor
assay with Innovin as the activating thromboplastin. A single tight
peak of activity was seen eluting between 47.9 and 52.4 ml, or 23.2
to 27.8 mS/cm (FIG. 19). A Bradford assay was performed to
determine the total amount of protein in the load and elution
fractions. The ratio of the activity to the total protein
demonstrated an .about.5 fold net purification from the column.
[0415] Purification of FVII-XTEN_AE864 by Hydrophobic Interaction
Chromatography
[0416] A sample of FVII-AE864 (AC404) containing supernatant was
concentrated and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA
and then adjusted to a final concentration of .about.5 mM
CaCl.sub.2 with the addition of 1M CaCl.sub.2. The sample was
loaded onto a 2 ml toyopearl phenyl column equilibrated on an Akta
chromatography system. The protein was eluted with a linear
gradient of 0-100% buffer B over 20 column volumes. Buffer A was
comprised of 10 mM Tris, 5 mM CaCl.sub.2, 3M NaCl, pH 7.5 and
buffer B was comprised of 10 mM Tris, 5 mM CaCl.sub.2, pH 7.5.
Fractions were assayed for FVII activity using a PT based factor
assay with Innovin as the activating thromboplastin. A single peak
of activity was seen eluting between 1M and 2M NaCl (FIG. 20). A
Bradford assay was performed to determine the total amount of
protein in the load and elution fractions. The ratio of the
activity to the total protein demonstrated an .about.2 fold net
purification from the column.
[0417] Removal of Aggregated Protein from Monomeric FVII-AE864 with
Anion Exchange Chromatography
[0418] Affinity purified FVII-AE864 (AC404) was loaded was adjusted
to pH 6.0 by addition of 200 mM MES, 210 mM CaCl2 pH 6.0 at a ratio
of 1 ml buffer to 10 ml sample. Using an Akta FPLC system the
sample was purified using a 2 ml macrocap Q column. The column was
equilibrated into buffer A (20 mM MES, 1 mM CaCl.sub.2, pH 6.0) and
the sample loaded. The sample was eluted using a linear gradient of
30% to 80% buffer B (20 mM MES, 1 mM CaCl.sub.2, pH 6.0+500 mM
NaCl) over 20 column volumes. The 215 nm chromatogram showed two
peaks in the elution profile (FIG. 21A). The fractions
corresponding to the early peak and the late peak were pooled and
analyzed via size exclusion chromatography (SEC) with 60 cm BioSep
G4000 column. The early peak contained a monodispersed population
with a characteristic hydrodynamic radius of a monomeric AE864
protein (10.1 nm or apparent MW of 1.9 MDa) (FIG. 21B). The late
peak contained two populations, the smaller monomeric peak
demonstrating the absence of aggregates in the early peak. and an
earlier eluting, larger peak at the void volume of the column (22
ml) characteristic of aggregated protein.
[0419] SEC Analysis of FVII-AE864 and FVII-AE288
[0420] FVII-AE864 (AC404) and FVII-AE288 (AC398) were purified by
affinity and anion exchange chromatography and characterized. Size
exclusion chromatography with 60 cm BioSep G4000 column indicated a
monodispersed population with a characteristic hydrodynamic radius
for either a monomeric AE864 protein (10.1 nm or apparent MW of 1.9
MDa for an apparent molecular weight factor of 15.2) or a monomeric
AE288 protein (8.2 nm or apparent MW of 650 kDa for an apparent
molecular weight factor of 9.0) (FIG. 18). Minimal aggregation was
seen in either sample. SDS-PAGE showed a >90% pure protein with
minimal host cell protein contamination.
[0421] Lipidated Tissue Factor Initiated Clotting Activity Analysis
of FVII-AE864 and FVII-AE288
[0422] Activity was assayed by a PT based factor VII assay as
follows: A standard curve was prepared by diluting normal plasma
ten fold with FVII deficient plasma and then conducting 4, 5 fold
serial dilutions again with factor VII deficient plasma. This
created a standard curve with points at 100, 20, 4, 0.8 and 0.16
mUnits/ml of activity, where one unit of activity is defined as the
amount of FVII activity in 1 ml of normal human plasma. A
FVII-deficient plasma was also included to determine the background
level of activity in the null plasma. The sample was prepared by
adding FVII-XTEN to FVII deficient plasma at a ratio of 1:10 by
volume. The samples were incubated at 37 C in a molecular devices
plate reader spectrophotometer for 3 minutes at which point the
clotting reaction was initiated by the addition of 2 volumes of
thromboplastin (Dade Innovin, B4212-50) per one volume of sample.
The turbidity was monitored at 405 nm for 5 minutes to create
reaction profiles. The PT time, or time to onset of clotting
activity, was defined as the first time where OD405 nm increased by
0.06 over baseline. A log-linear standard curve was created with
the log of activity relating linearly to the PT time. From this the
activity of the sample in the plate well was determined and then
the activity in the sample determined by multiplying by 11 to
account for the dilution into the FVII deficient plasma. Based upon
quadruplicate measurements the activity of the FVII-AE864 (AC404)
fusion was 30 Units/ml and the FVII-AE288 (AC398) was 15 U/ml.
Additionally, this lapidated tissue factor activation of clotting
is used to assay the activity of FVII-XTEN fusions in clotting
assays with more sophisticated readouts like thrombin generation
assays, TEG assays, rotem assays and other in vitro/ex vivo which
involve the detection of clotting enzyme function by substrate
turnover, mechanical clot formation or photo-optical clot
detection.
[0423] Soluble Tissue Factor Initiated Clotting Activity Analysis
of FVII-AE864 and FVII-AE288
[0424] After activation of FVII-AE288 (AC398) to FVIIa-AE288 the
activity was measured by soluble tissue factor (sTF) induced
clotting. This is performed using the Stago STA-Clot FVIIa activity
assays kit. Briefly, the samples were incubated with sTF, which
binds and enhances FVIIa activity, but does not convert FVII to
FVIIa, The time to induce a clot in FVII null plasma was defined as
the first time where OD405 nm increased by 0.06 over baseline when
monitored in the molecular devices plate reader. This time is
compared to a standard curve comprised of known FVIIa amounts added
into FVII null plasma, and an activity number calculated. The
FVIIa-AE288 sample contained an activity equivalent to 112 U/ml of
FVIIa activity. Additionally, this soluble tissue factor activation
of clotting is used to assay the activity of FVII-XTEN fusions in
clotting assays with more sophisticated readouts like thrombin
generation assays, TEG assays, rotem assays and other in vitro/ex
vivo which involve the detection of clotting enzyme function by
substrate turnover, mechanical clot formation or photo-optical clot
detection.
[0425] ELISA Based Concentration Determination of FVII-AE864 and
FVII-AE288
[0426] FVII-XTEN fusion concentrations were determined using and
ELISA assay with an affinity purified polyclonal sheep anti-human
FVII antibody, where an unmodified form of the antibody is used to
capture the protein and the HRP conjugated form was used to detect
the protein. The capture antibody was coated at 4 C overnight on to
a high binding 96 well assay plate (Corning 3690). The plate was
blocked with 3% BSA in PBS for 1 hour at room temperature. The
plate was washed 6 times in PBST with a plate washer. Samples or
standards, diluted in PBST, were then bound into the appropriate
wells for 2 hours at room temperature. The standard curve ranged
from 10 ng/ml to <1 pg/ml and was prepared by serially diluting
commercial plasma derived FVII of a known concentration (Abcam Cat#
ab62386) in PBST. The plate was again washed 6 times with PBST
using a plate washer. The FVII-XTEN was then detected using the
detection antibody which was bound for 1 hour at 37 C. The plate
was again washed 6 times with PBST using a plate washer and washed
one further time with water. Signal was then developed with TMB
substrate and quantified by reading at 405 nm on a molecular
devices plate reader spectrophotometer. A four parameter fit is
then performed on the standards and the concentration of the
samples determined by comparison to the standard curve.
[0427] Assessment of FVII-AE864 and FVII-AE288 Activity via Direct
Turnover of a Fluorogenic Substrate
[0428] FVII-XTEN fusion activity is determined by monitoring the
cleavage of a peptide bond in the substrate
D-FPR-6-amino-1-naphthalenesulfonamide (D-FPR-ANSN) where the DFPR
moiety is a peptide chain linked to a secondary amine in the ANSH
moiety. When the bond between the arginine residue and the ANSH
moiety is cleaved by the serine protease activity of the FVII
catalytic domain the ANSH is released and becomes an intense
fluorophore. FVII-XTEN activity is measured at enzyme
concentrations ranging from 50 .mu.M to 1 .mu.M with substrate
concentrations ranging from 50 .mu.M to 100 .mu.M in 20 mM tris pH
8.0, 135 mM NaCl. By monitoring the change in ANSN fluorescence
(excitation 352 nm, emission 470 nm) over time the activity of the
FVIIa catalytic domain can be determined. This activity can be
compared to a standard curve derived from FVIIa to determine the
amount of FVIIa equivalents in the sample, or kinetic properties
such as kcat and Km for can be determined.
[0429] Assessment of FVII-AE864 and FVII-AE288 Activity via a FXa
Coupled Chromogenic Substrate Assay
[0430] When complexed to Tissue Factor (TF), in presence of
phospholipids and Calcium, FVII and FVII-XTEN activate factor X to
factor Xa. Biophen Factor VII is a chromogenic assay for testing
factor VII activity. Factor VII forms an enzymatic complex with
Tissue Factor, provided by rabbit Thromboplastin. It then activates
factor X, present in the assay at a constant concentration and in
excess, to factor Xa. The concentration of FXa is exactly measured
by its activity on a specific factor Xa chromogenic substrate
(SXa-11). Factor Xa cleaves the substrate and generates pNA. The
amount of pNA generated is directly proportional to the factor Xa
activity. Finally, there is a direct relationship between the
amount of factor VII activity in the assayed sample and the factor
Xa activity generated, measured by the amount of pNA released,
determined by color development at 405 nm. By comparing the signal
from an unknown sample to the signal from a standard curve of know
FVII activity, it is possible to calculate the amount of FVII
activity in an unknown sample.
Example 27
ELISA Assays for FIX-XTEN Concentration Determination
[0431] FIX-XTEN concentrations were determined using and ELISA
assay with a specific matched pair of antibodies, where the
detection antibody was conjugated to HRP to simplify detection
(Affinity Biologicals cat# FIX-EIA). The capture antibody was
coated at 4 C overnight on to a high binding 96 well assay plate
(Corning 3690). The plate was blocked with 3% BSA in PBS for 1 hour
at room temperature. The plate was washed 6 times in PBST with a
plate washer. Samples or standards, diluted in PBST, were then
bound into the appropriate wells for 2 hours at room temperature.
The standard curve ranged from 25 ng/ml to <1 pg/ml and was
prepared by serially diluting commercial plasma derived FIX of a
known concentration (Abeam Cat# ab62544) in PBST. The plate was
again washed 6 times with PBST using a plate washer. The FIX was
then detected using the detection antibody which was bound for 1
hour at 37 C. The plate was again washed 6 times with PBST using a
plate washer and washed one further time with water. Signal was
then developed with TMB substrate and quantified by reading at 405
nm on a molecular devices plate reader spectrophotometer. A four
parameter fit is then performed on the standards and the
concentration of the samples determined by comparison to the
standard curve.
Example 28
aPTT Based Assays for FIX-XTEN Activity Determination
[0432] FIX-XTEN would act to replace FIX in the intrinsic or
contact activated coagulation pathway. The activity of this
coagulation pathway is assessed using an activated partial
thromboplastin time assay (aPTT). FIX activity specifically was
measured as follows, a standard curve was prepared by diluting
normal control plasma (Pacific Hemostasis cat#100595) two fold with
FIX deficient plasma (cat#100900) and then conducting 6, 4 fold
serial dilutions again with factor IX deficient plasma. This
created a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5
and 0.1 mUnits/ml of activity, where one unit of activity is
defined as the amount of FIX activity in 1 ml of normal human
plasma. A FIX-deficient plasma was also included to determine the
background level of activity in the null plasma. The sample was
prepared by adding FIX-XTEN to FIX deficient plasma at a ratio of
1:10 by volume. The samples were tested using an aPTT assay as
follows. The samples were incubated at 37 C in a molecular devices
plate reader spectrophotometer for 2 minutes at which point an
equal volume of aPTT reagent (Pacific Hemostasis cat#100402) was
added and an additional 3 minute 37 C incubation performed. After
the incubation the assay was activated by adding one volume of
calcium chloride (Pacific Hemostasis cat#100304). The turbidity was
monitored at 450 nm for 5 minutes to create reaction profiles. The
aPTT time, or time to onset of clotting activity, was defined as
the first time where OD405 nm increased by 0.06 over baseline. A
log-linear standard curve was created with the log of activity
relating linearly to the aPTT time. From this the activity of the
sample in the plate well was determined and then the activity in
the sample determined by multiplying by 11 to account for the
dilution into the FIX deficient plasma.
Example 29
FIX/cFM/XTEN has Enhanced Activity Compared to FIX-XTEN
[0433] FIX (pCW0596), FIX-XTEN (pCW0597), FIX/cFXI1/XTEN (pCW0735),
FIX/cFXI2/XTEN (pCW0736) and FIX/cFXI3/XTEN (pCW0737) were
transiently expressed in CHO-K1 cells. Transient transfection
supernatants were concentrated in 30,000 MWCO concentrators by
approximately 15-fold. The concentration of the concentrated and
unconcentrated samples was determined by ELISA. The clotting
activity of the concentrated samples was then determined using an
aPTT based factor assay. For the XTEN containing the activity was
drastically altered by the presence any of the FXIc cleavage sites.
In all three cases the presence of a FXI cleavage site enhanced the
clotting activity by greater than 30-fold (see FIG. 22 and Table
28). The relatively consistent ELISA measurement indicates that
this is an enhancement of the specific activity, rather than a
change in titer. Additionally, the ratio of the activity
measurement to the ELISA concentration for the FXI cleavage site
constructs was now similar to the ratios for FIX, indicating that
the FIX-FXIc-XTEN contained a FIX domain of similar properties to
the FIX domain expressed in the absence of XTEN.
TABLE-US-00034 TABLE 28 Activity of FIX/cFXI/XTEN Constructs
Concentration by Concentration by Fraction Construct ELISA (ng/ml)
Activity (ng/ml) Active FIX/cTEV/XTEN NA NA 4% FIX 197 30 15%
FIX-XTEN 10 0 2% FIX/cFXI1/XTEN 23 7 30% FIX/cFXI2/XTEN 26 9 34%
FIX/cFXI3/XTEN 29 11 40%
Example 30
Pharmacokinetic Analysis of CFXTEN Fusion Polypeptide in
Rats--FVII-XTEN_AE864
[0434] The pharmacokinetics of the CFXTEN FVII-XTEN_AE864, compared
to FVII alone, were tested in Sprague-Dawley. FVII-XTEN_AE864 and
FVII were administered to female Sprague-Dawley rats (n=3) IV
through a jugular vein catheter at 3 .mu.g/rat. Blood samples (0.2
mL) were collected into prechilled heparinized tubes at predose,
0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and processed
into plasma. Quantitation of the test articles was performed by
ELISA assay using an anti-FVII antibody for both capture and
detection. A non-compartmental analysis was performed in WinNonLin
with all time points included in the fit to determine the PK
parameters.
[0435] The pharmacokinetic results are summarized in Table 29 and
FIG. 23. The data show XTEN can greatly extend the half-life of
FVII as a CFXTEN fusion protein as compared to FVII alone;
FVII-XTEN has a half life of approximately 38 hours as compared to
1 hour for FVII. In addition FVII-XTEN was confined to the
bloodstream, with a calculated volume of distribution of 50.8 mL in
rats, indicating little extravasation into the extracellular
space.
TABLE-US-00035 TABLE 29 Half-life of FVII test articles in rats
Test Article (Construct) T 1/2 (hrs) FVII-XTEN(AP315) 37.9 FVII
(P318) 1.0
Example 31
Pharmacokinetic Analysis of CF XTEN Fusion Polypeptide in
Rats--FIX-XTEN_AE864
[0436] The pharmacokinetics of macrocap Q purified FIX-XTEN_AE864
were tested in Sprague-Dawley rats (n=3) and compared to unpurified
FIX-XTEN, FIX-XTEN TEV cleaved (a preparation in which the XTEN is
removed from the fusion protein by use of TEV protease), and
commercially-available FIX Benefix. Compounds were administered to
female Sprague-Dawley rats IV through jugular vein catheter at 3
.mu.g/rat. Blood samples (0.2 mL) were collected into prechilled
heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72
hour time points, and processed into plasma. Quantitation of the
test articles was performed by ELISA assay using an anti-FIX
antibody for both capture and detection. A non-compartmental
analysis was performed in WinNonLin with all time points included
in the fit to determine the PK parameters.
[0437] The pharmacokinetic results are summarized in Table 30 and
FIG. 24. The data show XTEN can greatly extend the half life of FIX
as a CFXTEN fusion protein as compared to either FIX-XTEN TEV
cleaved or FIX Benefix; FIX-XTEN has a half life of 34.7 hours as
compared to 4.6 hour for FIX Benefix. In addition FIX-XTEN was
confined to the bloodstream with a calculated volume of
distribution of 38 mL in rats, indicating little extravasation into
the extracellular space.
TABLE-US-00036 TABLE 30 Half-life of FIX test articles in rats Test
Article (Construct) T 1/2 (hrs) FIX-XTEN macro cap Q 34.7 (AP316a)
FIX-XTEN (AP316) 33.1 FIX-XTEN TEV (AP316b) 1.5 FIX Benefix 3.3
Example 32
Pharmacodynamic Evaluation of FVIIa-XTEN_AE864 in Animal Models
[0438] The in vivo pharmacologic activity of FVIIa-XTEN constructs
is assessed using a variety of preclinical models of bleeding
including but not limited to those of hemophilia, surgery, trauma,
thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced
bleeding and hydrodynamic injection. These models can be developed
in multiple species including mice, rat, rabbits, and dogs using
methods equivalent to those used and published for other FVIIa
approaches. FVIIa-XTEN compositions are provided in an aqueous
buffer compatible with in vivo administration (for example:
phosphate-buffered saline or Tris-buffered saline). The
compositions are administered at appropriate doses, dosing
frequency, dosing schedule and route of administration as optimized
for the particular model. Efficacy determinations include
measurement of FVIIa activity, prothrombin time (PT), activated
partial prothrombin time (aPTT), bleeding time, whole blood
clotting time (WBCT), thrombelastography (TEG or ROTEM), among
others.
[0439] In one example of a PD model, FVIIa-XTEN and FVII are
administered to genetically-deficient or experimentally-induced
HemA or HemB mice. At various time points post-administration,
levels of FVIIa and FVIIa-XTEN are measured by ELISA, activity of
FVIIa and FVIIa-XTEN are measured by commercially-available FVIIa
activity kits and clotting time is measured by PT assay. Overall,
the results can indicate that the FVIIa-XTEN constructs may be more
efficacious at inhibiting bleeding as compared to FVIIa and/or
equivalent in potency to comparable dosage of FVIIa with less
frequent or more convenient dosing intervals.
[0440] In a mouse bleeding challenge PD model FVIIa-XTEN and FVIIa
are administered to genetically-deficient or experimentally-induced
HemA or HemB mice and effect on hemostatic challenge is measured.
Hemostatic challenge can include tail transaction challenge,
hemarthropthy challenge, joint bleeding or saphenous vein challenge
among others. At various time points post-administration levels of
FVII and FVIIa-XTEN are measured by ELISA, activity of FVII and
FVIIa-XTEN are measured by commercially available FVIIa activity
kit, bleeding time is measured and clotting time is measured by PT
assay. Overall the results can indicate that the VIIa-XTEN
constructs may be more efficacious at inhibiting bleeding as
compared to FVIIa and/or equivalent in potency to comparable dosage
of FVIIa with less frequent or more convenient dosing
intervals.
[0441] In a dog PD model, FVIIa-XTEN and FVII are administered to
genetically-deficient hemophiliac dogs. At various time points post
administration, levels of FVIIa and FVIIa-XTEN are measured by
ELISA, activity of FVIIa and FVIIa-XTEN are measured by
commercially available FVIIa activity kit and clotting time is
measured by PT assay. Overall the results can indicate that the
FVIIa-XTEN constructs may be more efficacious at inhibiting
bleeding as compared to FVIIa and/or equivalent in potency to
comparable dosage of FVIIa with less frequent or more convenient
dosing.
[0442] In a dog bleeding challenge PD model FVIIa-XTEN and FVIIa
are administered to genetically deficient hemophiliac dogs and
effect on hemostatic challenge is measured. Hemostatic challenge
can include cuticle bleeding time among others. At various time
points post administration levels of FVII and FVIIa-XTEN are
measured by ELISA, activity of FVII and FVIIa-XTEN are measured by
commercially available FVIIa activity kit, bleeding time is
measured and clotting time is measured by PT assay. Overall the
results can indicate that the VIIa-XTEN constructs may be more
efficacious at inhibiting bleeding as compared to FVIIa and/or
equivalent in potency to comparable dosage of FVIIa with less
frequent or more convenient dosing intervals.
[0443] Additional preclinical models of bleeding include but are
not limited to those of hemophilia, surgery, trauma,
thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced
bleeding and hydrodynamic injection. These models can developed in
multiple species including mice, rat, rabbits, and dogs using
methods equivalent to those used and published for other FVIIa
approaches. Overall the results can indicate that the FVIIa-XTEN
constructs may be more efficacious at inhibiting bleeding as
compared to FVIIa and/or equivalent in potency to comparable dosage
of FVIIa with less frequent or more convenient dosing
intervals.
Example 33
Pharmacodynamic Evaluation of FIX-XTEN_AE864 in Animal Models
[0444] The in vivo pharmacologic activity of FIX-XTEN constructs is
assessed using a variety of preclinical models of bleeding
including, but not limited to, those of hemophilia, surgery,
trauma, thrombocytopenia/platelet dysfunction,
clopidogrel/heparin-induced bleeding and hydrodynamic injection.
These models can be developed in multiple species including mice,
rat, rabbits, and dogs using methods equivalent to those used and
published for other FIX approaches. FIX-XTEN compositions are
provided in an aqueous buffer compatible with in vivo
administration (for example: phosphate-buffered saline or
Tris-buffered saline). The compositions are administered at
appropriate doses, dosing frequency, dosing schedule and route of
administration as optimized for the particular model. Efficacy
readouts include measurement of FIX activity, PT, aPTT, bleeding
time, whole blood clotting time (WBCT), thrombelastography (TEG or
ROTEM), among others.
[0445] In one example of a PD model, FIX-XTEN and FIX are
administered to genetically-deficient or experimentally-induced
HemA or HemB mice. At various time points post-administration,
levels of FIX and FIX-XTEN are measured by ELISA, activity of FIX
and FIX-XTEN are measured by commercially available FIX activity
kit and clotting time is measured by aPTT assay. Overall the
results can indicate that the FIX-XTEN constructs may be more
efficacious at inhibiting bleeding as compared to FIX and/or
equivalent in potency to comparable dosage of FIX with less
frequent or more convenient dosing intervals.
[0446] In a mouse bleeding challenge PD model FIX-XTEN and FIX are
administered to genetically deficient or experimentally induced
HemA or Hem B mice and effect on hemostatic challenge is measured.
Hemostatic challenge can include tail transaction challenge,
hemarthropthy challenge, joint bleeding or saphenous vein challenge
among others. At various time points post administration levels of
FIX and FIX-XTEN are measured by ELISA, activity of FIX and
FIX-XTEN are measured by commercially available FIX activity kit,
bleeding time is measured and clotting time is measured by aPTT
assay. Overall the results can indicate that the FIX-XTEN
constructs may be more efficacious at inhibiting bleeding as
compared to FIX and/or equivalent in potency to comparable dosage
of FIX with less frequent or more convenient dosing intervals.
[0447] In a dog PD model, FIX-XTEN and FIX are administered to
genetically-deficient hemophiliac dogs. At various time points
post-administration, levels of FIX and FIX-XTEN are measured by
ELISA, activity of FIX and FIX-XTEN are measured by commercially
available FIX activity kit and clotting time is measured by aPTT
assay. Overall, the results can indicate that the FIX-XTEN
constructs may be more efficacious at inhibiting bleeding as
compared to FIX and/or equivalent in potency to comparable dosage
of FIX with less frequent or more convenient dosing intervals.
[0448] In a dog bleeding challenge PD model FIX a-XTEN and FIX are
administered to genetically-deficient hemophiliac dogs and effect
on hemostatic challenge is measured. Hemostatic challenge can
include cuticle bleeding time, amongst other assays. At various
time points post-administration levels of FIX and FIX-XTEN are
measured by ELISA, activity of FIX and FIX-XTEN are measured by
commercially available FIX activity kit, bleeding time is measured
and clotting time is measured by aPTT assay. Overall, the results
can indicate that the FIX-XTEN constructs may be more efficacious
at inhibiting bleeding as compared to FIX and/or equivalent in
potency to comparable dosage of FIX with less frequent or more
convenient, dosing intervals.
[0449] Additional preclinical models of bleeding include, but are
not limited to, those of hemophilia, surgery, trauma,
thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced
bleeding and hydrodynamic injection. These models can be developed
in multiple species, including mice, rat, rabbits, and dogs using
methods equivalent to those used and published for other FIX
approaches. Overall the results can indicate that the FIX-XTEN
constructs may be more efficacious at inhibiting bleeding as
compared to FIX and/or equivalent in potency to comparable dosage
of FIX with less frequent or more convenient dosing intervals.
Example 34
CFXTEN with Cleavage Sequences
[0450] C-terminal XTEN Releasable by FXIa
[0451] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site cleavage
sequence can be incorporated into the FIX-XTEN that contains an
amino acid sequence that is recognized and cleaved by the FXIa
protease (EC 3.4.21.27, Uniprot P03951). Specifically the amino
acid sequence KLTRAET is cut after the arginine of the sequence by
FXIa protease. FXI is the pro-coagulant protease located
immediately before FIX in the intrinsic or contact activated
coagulation pathway. Active FXIa is produced from FXI by
proteolytic cleavage of the zymogen by FXIIa. Once activated, its
natural role in coagulation is to activate FIX by excising a
peptide from zymogen by cutting the protein at positions R191 and
R226 of FIX, which then perpetuates the coagulation pathway.
Production of FXIa is tightly controlled and only occurs when
coagulation is necessary for proper hemostasis. Therefore, by
incorporation of the KLTRAET cleavage sequence, the XTEN domain
would only be removed from FIX concurrent with activation of the
intrinsic coagulation pathway and when coagulation is required
physiologically. This creates a situation where the FIX-XTEN fusion
protein is processed in one additional manner during the activation
of the intrinsic pathway. In addition to the natural cleavages that
occur at R191 and R226 of the FIX domain by FXIa, a third cleavage
would occur at the XTEN release site which would decouple the now
activated FIXa from the XTEN protein. In a desirable feature of the
inventive composition, this creates a situation where FIX-XTEN
would remain intact as a pro-drug until activation of coagulation,
at which time the molecule is processed to produce free FIXa which
reconstitutes or augments clotting function in a subject in need
thereof.
[0452] C-Terminal XTEN Releasable by FXIIa
[0453] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the XTEN release site sequence
can contain an amino acid sequence that is recognized and cleaved
by the FXIIa protease (EC 3.4.21.38, Uniprot P00748). Specifically
the sequence TMTRIVGG is cut after the arginine at position 4 of
the sequence. FXII is a pro-coagulant protease located before FIX
in the intrinsic or contact activated coagulation pathway. Active
FXIIa is produced from FXII by contact with non-self surfaces and
by cleavage by kallikrein. Once activated its natural role in
coagulation is to activate FXI (FIG. 3) by proteolytic cleavage of
the zymogen, which then in turn, perpetuates the coagulation
pathway. Production of FXIIa is tightly controlled and only occurs
when coagulation is necessary for proper hemostasis. Therefore, by
incorporation of the TMTRIVGG cleavage sequence, the XTEN domain
would only be removed from FIX concurrent with activation of the
intrinsic coagulation pathway and when coagulation is required
physiologically. This creates a situation where FIX-XTEN fusion is
processed in one additional manner during the activation of the
intrinsic pathway. In addition to the natural cleavages that occur
at R191 and R226 of the FIX domain by FXIa, a third cleavage would
occur at the XTEN release site that would decouple the now
activated FIXa from the XTEN protein. In a desirable feature of the
inventive composition, this creates a situation where FIX-XTEN
would remain intact as a pro-drug until activation of coagulation,
at which time the molecule is processed to produce free FIXa which
reconstitutes or augments clotting function in a subject in need
thereof.
[0454] C-Terminal XTEN Releasable by Kallikrein
[0455] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the XTEN release site sequence
can an amino acid sequence that is recognized and cleaved by the
kallikrein protease (EC 3.4.21.34, Uniprot P03952). Specifically
the sequence SPFRVVGG [Rawlings N. D., et al. (2008) Nucleic Acids
Res., 36: D320], is cut after the arginine at position 4 of the
sequence. Kallikrein is a pro-coagulant protease located before FIX
in the intrinsic or contact activated coagulation pathway. Active
Kallikrein is produced from Plasma Kallirien by contact with
non-self surfaces. Once activated its natural role in coagulation
is to activate FXII (FIG. 3) by proteolytic cleavage of the
zymogen, which then in turn, perpetuates the coagulation pathway.
Production of kallikrien is tightly controlled and only occurs when
coagulation is necessary for proper hemostasis. Therefore, by
incorporation of the SPFRVVGG cleavage sequence the XTEN domain
would only be removed from FIX concurrent with activation of the
intrinsic coagulation pathway and when coagulation is required
physiologically. This creates a situation where FIX-XTEN fusion is
processed in one additional manner during the activation of the
intrinsic pathway. In addition to the natural cleavages that occur
at R191 and R226 of the FIX domain by FXIa, a third cleavage would
occur at the XTEN release site that would decouple the now
activated FIXa from the XTEN protein. In a desirable feature of the
inventive composition, this creates a situation where FIX-XTEN
would remain intact as a pro-drug until activation of coagulation,
at which time the molecule is processed to produce free FIXa which
reconstitutes or augments clotting function in a subject in need
thereof.
[0456] C-Terminal XTEN Releasable by FVIIa
[0457] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site sequence
contains an amino acid sequence that is recognized and cleaved by
the FVIIa protease (EC 3.4.21.21, Uniprot P08709). Specifically the
sequence LQVRIVGG [Rawlings N. D., et al. (2008) Nucleic Acids
Res., 36: D320], is cut after the arginine at position 4 in the
sequence. FVIIa is a pro-coagulant protease located before FIX in
the extrinsic or cellular injury activated coagulation pathway.
Active FVIIa is produced from FVII in an autocatalytic process
aided by binding to tissue factor, phospholipids and calcium. Once
activated its natural role in coagulation is to activate FIX and FX
(FIG. 3) by proteolytic cleavage of the zymogens, which then in
turn, perpetuate the coagulation pathway. FVIIa activity is tightly
controlled and only occurs when coagulation is necessary for proper
hemostasis. Therefore, by incorporation of the LQVRIVGG cleavage
sequence the XTEN domain would only be removed from FIX concurrent
with activation of the intrinsic coagulation pathway and when
coagulation is required physiologically. This creates a situation
where FIX-XTEN fusion is processed in one additional manner during
the activation of the intrinsic pathway. In addition to the natural
cleavages that would occur at R191 and R226 of the FIX domain by
FVIIa, a third cleavage would occur at the XTEN release site which
would decouple the now activated FIXa from the XTEN protein. In a
desirable feature of the inventive composition, this creates a
situation where FIX-XTEN would remain intact as a pro-drug until
activation of coagulation, at which time the molecule is processed
to produce free FIXa which reconstitutes or augments clotting
function in a subject in need thereof.
[0458] C-Terminal XTEN Releasable by FIXa
[0459] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site cleavage
sequence contains an amino acid sequence that is recognized and
cleaved by the FIXa protease (EC 3.4.21.22, Uniprot P00740).
Specifically the sequence PLGRIVGG [Rawlings N. D., et al. (2008)
Nucleic Acids Res., 36: D320], is cut after the arginine at
position 4 of the sequence. Active FIXa is produced by cleavage of
FIX by either FXIa or FVIIa in the presence of phospholipids and
calcium. Once activated its natural role in coagulation is to
activate FX (FIG. 3) by proteolytic cleavage of the zymogen, which
then in turn, perpetuates the coagulation pathway. FIXa activity is
tightly controlled and only occurs when coagulation is necessary
for proper hemostasis. Therefore, by incorporation of the PLGRIVGG
sequence, the XTEN domain would only be removed from FIX concurrent
with activation of either the extrinsic or intrinsic coagulation
pathways, and when coagulation is required physiologically. This
creates a situation where FIX-XTEN fusion is processed in one
additional manner during the activation of the intrinsic pathway.
In addition to the natural cleavages that would occur at R191 and
R226 of the FIX domain by FVIIa or FXIa, a third cleavage would
occur at the XTEN release site which would decouple the now
activated FIXa from the XTEN protein. In a desirable feature of the
inventive composition, this creates a situation where FIX-XTEN
would remain intact as a pro-drug until activation of coagulation,
at which time the molecule is processed to produce free FIXa which
reconstitutes or augments clotting function in a subject in need
thereof.
[0460] C-Terminal XTEN Releasable by FXa
[0461] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the FXa
protease (EC 3.4.21.6, Uniprot P00742). Specifically the sequence
IEGRTVGG [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after the arginine at position 4 in the sequence.
Active FXa is produced by cleavage of FX by FIXa in the presence of
phospholipids and calcium and is the step immediately down stream
from factor IX in the coagulation pathway. Once activated its
natural role in coagulation is to activate FII (FIG. 3) by
proteolytic cleavage of the zymogen, which then in turn,
perpetuates the coagulation pathway. FXa activity is tightly
controlled and only occurs when coagulation is necessary for proper
hemostasis. Therefore, by incorporation of the IEGRTVGG sequence,
the XTEN domain would only be removed from FIX concurrent with
activation of either the extrinsic or intrinsic coagulation
pathways, and when coagulation is required physiologically. This
creates a situation where FIX-XTEN fusion is processed in one
additional manner during the activation of clotting. In addition to
the natural cleavages that would occur at R191 and R226 of the FIX
domain by FVIIa or FXIa, a third cleavage would occur at the XTEN
release site which would decouple the now activated FIXa from the
XTEN protein. In a desirable feature of the inventive composition,
this creates a situation where FIX-XTEN would remain intact as a
pro-drug until activation of coagulation, at which time the
molecule is processed to produce free FIXa which reconstitutes or
augments clotting function in a subject in need thereof.
[0462] C-terminal XTEN Releasable by FIIa (Thrombin)
[0463] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the FIIa
protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequence
LTPRSLLV [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after the arginine at position 4 in the sequence.
Active FIIa is produced by cleavage of F11 by FXa in the presence
of phospholipids and calcium and is down stream from factor IX in
the coagulation pathway. Once activated its natural role in
coagulation is to cleave fibrinogin (FIG. 3), which then in turn,
begins clot formation. FIIa activity is tightly controlled and only
occurs when coagulation is necessary for proper hemostasis.
Therefore, by incorporation of the LTPRSLLV sequence, the XTEN
domain would only be removed from FIX concurrent with activation of
either the extrinsic or intrinsic coagulation pathways, and when
coagulation is required physiologically. This creates a situation
where FIX-XTEN fusion is processed in one additional manner during
the activation of coagulation. In addition to the natural cleavages
that would occur at R191 and R226 of the FIX domain by FVIIa or
FXIa, a third cleavage would occur at the XTEN release site which
would decouple the now activated FIXa from the XTEN protein. In a
desirable feature of the inventive composition, this creates a
situation where FIX-XTEN would remain intact as a pro-drug until
activation of coagulation, at which time the molecule is processed
to produce free FIXa which reconstitutes or augments clotting
function in a subject in need thereof.
[0464] C-Terminal XTEN Releasable by Elastase-2
[0465] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the
elastase-2 protease (EC 3.4.21.37, Uniprot P08246). Specifically
the sequence LGPVSGVP [Rawlings N. D., et al. (2008) Nucleic Acids
Res., 36: D320], is cut after position 4 in the sequence. Elastase
is constitutively expressed by neutrophils and is present at all
times in the circulation. Its activity is tightly controlled by
serpins and is therefore minimally active most of the time.
Therefore as the long lived FIX-XTEN circulates, a fraction of it
is cleaved, creating a pool of shorter-lived FIX to be used in
coagulation. In a desirable feature of the inventive composition,
this creates a circulating pro-drug depot that constantly releases
a prophylactic amount of FIX.
[0466] C-Terminal XTEN Releasable by MMP-12
[0467] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the MMP-12
protease (EC 3.4.24.65, Uniprot P39900). Specifically the sequence
GPAGLGGA [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4 of the sequence. MMP-12 is
constitutively expressed in whole blood. Therefore as the long
lived FIX-XTEN circulates, a fraction of it is cleaved, creating a
pool of shorter-lived FIX to be used in coagulation. In a desirable
feature of the inventive composition, this creates a circulating
pro-drug depot that constantly releases a prophylactic amount of
FIX.
[0468] C-Terminal XTEN Releasable by MMP-13
[0469] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the MMP-13
protease (EC 3.4.24.-, Uniprot P45452). Specifically the sequence
GPAGLRGA [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4. MMP-13 is constitutively expressed
in whole blood. Therefore as the long lived FIX-XTEN circulates, a
fraction of it is cleaved, creating a pool of shorter-lived FIX to
be used in coagulation. In a desirable feature of the inventive
composition, this creates a circulating pro-drug depot that
constantly releases a prophylactic amount of FIX.
[0470] C-Terminal XTEN Releasable by MMP-17
[0471] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the MMP-20
protease (EC.3.4.24.-, Uniprot Q9ULZ9). Specifically the sequence
APLGLRLR [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4 in the sequence. MMP-17 is
constitutively expressed in whole blood. Therefore as the long
lived FIX-XTEN circulates, a fraction of it is cleaved, creating a
pool of shorter-lived FIX to be used in coagulation. In a desirable
feature of the inventive composition, this creates a circulating
pro-drug depot that constantly releases a prophylactic amount of
FIX.
[0472] C-Terminal XTEN Releasable by MMP-20
[0473] An FIX-XTEN fusion protein consisting of an XTEN protein
fused to the C-terminus of FIX can be created with a XTEN release
site cleavage sequence placed in between the FIX and XTEN
components, as depicted in FIG. 2F. Exemplary sequences are
provided in Table 42. In this case, the release site contains an
amino acid sequence that is recognized and cleaved by the MMP-20
protease (EC.3.4.24.-, Uniprot 060882). Specifically the sequence
PALPLVAQ [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36:
D320], is cut after position 4 (depicted by the arrow). MMP-20 is
constitutively expressed in whole blood. Therefore as the long
lived FIX-XTEN circulates, a fraction of it is cleaved, creating a
pool of shorter-lived FIX to be used in coagulation. In a desirable
feature of the inventive composition, this creates a circulating
pro-drug depot that constantly releases a prophylactic amount of
FIX.
[0474] Optimization of the Release Rate of C-Terminal XTEN
[0475] Variants of the foregoing Examples can be created in which
the release rate of C-terminal XTEN is altered. As the rate of XTEN
release by an XTEN release protease is dependent on the sequence of
the XTEN release site, by varying the amino acid sequence in the
XTEN release site one can control the rate of XTEN release. The
sequence specificity of many proteases is well known in the art,
and is documented in several data bases. In this case, the amino
acid specificity of proteases is mapped using combinatorial
libraries of substrates [Harris, J. L., et al. (2000) Proc Natl
Acad Sci USA, 97: 7754] or by following the cleavage of substrate
mixtures as illustrated in [Schellenberger, V., et al. (1993)
Biochemistry, 32: 4344]. An alternative is the identification of
optimal protease cleavage sequences by phage display [Matthews, D.,
et al. (1993) Science, 260: 1113]. Constructs is made with variant
sequences and assayed for XTEN release using standard assays for
detection of the XTEN polypeptides.
Example 35
Integration of XTEN Internal to the CF Sequence
[0476] Internal XTEN Fusion into the KNSADK Loop
[0477] An FIX-XTEN fusion protein consisting of an XTEN protein
inserted into a loop of FIX can be created, as depicted in FIG. 2F.
Specifically, the XTEN sequence is inserted as a fusion into the
KNSADNK loop of the EGF2 domain (residues 146-152), which has no
known hemophilia B mutations and is not highly structured in the
FIX crystal structure. In this case, the insertion is made by
dividing the native sequence at the SA bond of the loop sequence
and fusing the XTEN sequence into the gap. This would give rise to
a loop sequence KNS-XTEN-ADNK. In a desirable feature of the
inventive composition, this creates a situation where FIX-XTEN
would remain intact as a pro-drug until activation of coagulation,
at which time the molecule is processed to produce FIXa-XTEN, which
reconstitutes or augments clotting function in a subject in need
thereof.
[0478] Internal XTEN Fusion into the LAEN Loop
[0479] An FIX-XTEN fusion protein consisting of an XTEN protein
inserted into a loop of FIX can be created, as depicted in FIG. 2F.
Specifically, the XTEN sequence is inserted as a fusion into the
LAEN loop of the EGF2 domain (residues 163-166), which has no known
hemophilia B mutations and is not highly structured in the FIX
crystal structure. In this case, the insertion is made by dividing
the native sequence at the AE bond of the sequence and fusing the
XTEN sequence into the gap. This would give rise to a loop sequence
LA-XTEN-EN. In a desirable feature of the inventive composition,
this creates a situation where FIX-XTEN would remain intact as a
pro-drug until activation of coagulation, at which time the
molecule is processed to produce FIXa-XTEN, which reconstitutes or
augments clotting function in a subject in need thereof.
[0480] Internal XTEN Fusion into the Activation Peptide
[0481] An FIX-XTEN fusion protein consisting of an XTEN protein
inserted into a loop of FIX can be created, as depicted in FIG. 2D.
Specifically, the XTEN fusion is placed into the activation peptide
(residues 192-226) such that neither of the two native FXIa
cleavage sites is disrupted. The insertion is made by dividing the
native sequence at the T209-1210 bond of the sequence and fusing
the XTEN sequence into the gap. This gives rise to a sequence
starting at residue 188 of
KLTRAETVFPDVDYVNSTEAET-XTEN-ILDNITQSTQSFNDFTRVVGGE. FXI is the
pro-coagulant protease located immediately before FIX in the
intrinsic or contact activated coagulation pathway. Active FXIa is
produced from FXI by proteolytic cleavage of the zymogen by FXIIa.
Once activated its natural role in coagulation is to activate FIX
(FIG. 4) by excising the activation peptide from the FIX zymogen by
cutting the protein at positions R191 and R226. These cuts sites
are depicted by arrows and the sequences are designed to leave the
P4-P4' sites unaltered to allow for natural cleavage activity
during the coagulation cascade. Therefore the XTEN domain would
only be removed from FIX as part of the normal activation process
within the intrinsic coagulation pathway.
[0482] Internal XTEN Fusion in Between the FIX EGF Domains
[0483] An FIX-XTEN fusion protein consisting of an XTEN protein
inserted into a loop of FIX can be created, as depicted in FIG. 2C.
Specifically, the XTEN fusion is placed in between the two EGF like
domains of FIX (junction is between residues 129 and 130). The
insertion is made by dividing the native sequence at the E129-L130
bond and fusing the XTEN sequence into the gap. This would give
rise to a sequence starting at residue 121 of
FGFEGKNCE-XTEN-LDVTCNIKNGR. Practically, this creates a situation
where FIX-XTEN would circulate intact until activation of
coagulation, at which time the molecule is processed to produce
FIXa-XTEN, which reconstitutes or augments clotting function in an
individual.
Example 36
Human Clinical Trial Designs for Evaluating CFXTEN Comprising
FVIIa
[0484] NovoSeven.RTM. is recombinant human coagulation factor VIIa
(rFVIIa), intended for promoting hemostasis by activating the
extrinsic pathway of the coagulation cascade. Due to its short
half-life, NovoSeven is dosed intravenously from every 2 to every 6
hours until hemostasis is achieved. Fusion of XTEN to FVII improves
the half-life of the protein, thus enabling a reduced dosing
frequency using such FVII-containing fusion protein
compositions.
[0485] Clinical trials can be designed such that the efficacy and
advantages of FVIIa-XTEN, relative to NovoSeven, can be verified in
humans. For example, the FVIIa-XTEN, can be used in clinical trials
for treatment of bleeding as performed for NovoSeven. Such studies
would comprise three phases. First, a Phase I safety and
pharmacokinetics study in adult patients is conducted to determine
the maximum tolerated dose and pharmacokinetics and
pharmacodynamics in humans (either normal subjects or patients with
hemophilia), as well as to define potential toxicities and adverse
events to be tracked in future studies. The study is conducted in
which single rising doses of FVIIa-XTEN compositions is
administered and biochemical, PK, and clinical parameters is
measured. This would permit the determination of the maximum
tolerated dose and establish the threshold and maximum
concentrations in dosage and circulating drug that constitute the
therapeutic window for the respective components. Thereafter,
clinical trials is conducted in patients with the disease, disorder
or condition.
[0486] Clinical trials could be conducted in patients suffering
from any disease in which NovoSeven may be expected to provide
clinical benefit. For example, such indications include bleeding
episodes in hemophilia A or B, patients with inhibitors to factor
VIII or factor IX, and in patients with acquired hemophilia,
prevention of bleeding in surgical interventions or invasive
procedures in hemophilia A or B patients with inhibitors to factor
VIII or factor IX and in patients with acquired hemophilia,
treatment of bleeding episodes in patients with congenital FVII
deficiency, and prevention of bleeding in surgical interventions or
invasive procedures in patients with congenital FVII deficiency.
FVIIa-XTEN may also be indicated for use in additional patient
populations. Parameters and clinical endpoints are measured as a
function of the dosing of the fusion proteins compositions,
yielding dose-ranging information on doses that is appropriate for
a subsequent Phase III trial, in addition to collecting safety data
related to adverse events. The PK parameters are correlated to the
physiologic, clinical and safety parameter data to establish the
therapeutic window and the therapeutic dose regimen for the
FVII-XTEN composition, permitting the clinician to establish the
appropriate dose ranges for the composition. Finally, a phase III
efficacy study is conducted wherein patients is administered the
FVII-XTEN composition at the dose regimen, and a positive control
(such as a commercially-available NovoSeven), or a placebo is
administered using a dosing schedule deemed appropriate given the
pharmacokinetic and pharmacodynamic properties of the respective
compositions, with all agents administered for an appropriately
extended period of time to achieve the study endpoints. Parameters
that are monitored include PT assay, bleeding time assay, control
of bleeding episodes, or the occurrence of spontaneous bleeding
episodes; parameters that is tracked relative to the placebo or
positive control groups. Efficacy outcomes is determined using
standard statistical methods. Toxicity and adverse event markers
are also be followed in this study to verify that the compound is
safe when used in the manner described.
Example 37
Human Clinical Trial Designs for Evaluating CFXTEN Comprising
FIX
[0487] BeneFIX.RTM., Coagulation Factor IX (Recombinant), is
indicated for the control and prevention of hemorrhagic episodes in
patients with hemophilia B (congenital factor IX deficiency or
Christmas disease), including control and prevention of bleeding in
surgical settings. Dosage and duration of treatment for all factor
a products depend on the severity of the factor IX deficiency, the
location and extent of bleeding, and the patient's clinical
condition, age and recovery of factor IX. Fusion of XTEN to FIX
improves the half-life of the protein, thus enabling a reduced
dosing frequency.
[0488] Clinical trials can be designed such that the efficacy and
advantages of FIX-XTEN, relative to other factor IX clinical
products, can be verified in humans. For example, the FIX-XTEN, can
be used in clinical trials for treatment of hemorrhagic episodes as
performed for Benefix. Such studies would comprise three phases.
First, a Phase I safety and pharmacokinetics study in adult
patients is conducted to determine the maximum tolerated dose and
pharmacokinetics and pharmacodynamics in humans (either normal
subjects or patients with hemophilia), as well as to define
potential toxicities and adverse events to be tracked in future
studies. The study is conducted in which single rising doses of
FIX-XTEN compositions is administered and biochemical, PK, and
clinical parameters is measured. This would permit the
determination of the maximum tolerated dose and establish the
threshold and maximum concentrations in dosage and circulating drug
that constitute the therapeutic window for the respective
components. Thereafter, clinical trials is conducted in patients
with the disease, disorder or condition.
[0489] Clinical trials could be conducted in patients suffering
from any disease in which factor IX may be expected to provide
clinical benefit. For example, such indications include the control
and prevention of hemorrhagic episodes in patients with hemophilia
B (congenital factor IX deficiency or Christmas disease), including
control and prevention of bleeding in surgical settings. FIX-XTEN
may also be indicated for use in additional patient populations.
Parameters and clinical endpoints are measured as a function of the
dosing of the fusion proteins compositions, yielding dose-ranging
information on doses that is appropriate for a subsequent Phase III
trial, in addition to collecting safety data related to adverse
events. The PK parameters are correlated to the physiologic,
clinical and safety parameter data to establish the therapeutic
window and the therapeutic dose regimen for the FIX-XTEN
composition, permitting the clinician to establish the appropriate
dose ranges for the composition. Finally, a phase III efficacy
study is conducted wherein patients is administered the FIX-XTEN
composition at the dose regimen, and a positive control (such as a
commercially-available BeneFIX), or a placebo is administered using
a dosing schedule deemed appropriate given the pharmacokinetic and
pharmacodynamic properties of the respective compositions, with all
agents administered for an appropriately extended period of time to
achieve the study endpoints. Parameters that are monitored include
aPTT assay, bleeding time assay, control of bleeding episodes, or
the occurrence of spontaneous bleeding episodes; parameters that is
tracked relative to the placebo or positive control groups.
Efficacy outcomes is determined using standard statistical methods.
Toxicity and adverse event markers are also be followed in this
study to verify that the compound is safe when used in the manner
described.
Example 38
Analytical Size Exclusion Chromatography of XTEN Fusion Proteins
with Diverse Payloads
[0490] Size exclusion chromatography analyses were performed on
fusion proteins containing various therapeutic proteins and
unstructured recombinant proteins of increasing length. An
exemplary assay used a TSKGel-G4000 SWXL (7.8 mm.times.30 cm)
column in which 40 pig of purified glucagon fusion protein at a
concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min
in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profiles were
monitored using OD214 nm and OD280 nm. Column calibration for all
assays were performed using a size exclusion calibration standard
from BioRad; the markers include thyroglobulin (670 kDa), bovine
gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine
myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative
chromatographic profiles of Glucagon-Y288, Glucagon-Y144,
Glucagon-Y72, Glucagon-Y36 are shown as an overlay in FIG. 35. The
data show that the apparent molecular weight of each compound is
proportional to the length of the attached XTEN sequence. However,
the data also show that the apparent molecular weight of each
construct is significantly larger than that expected for a globular
protein (as shown by comparison to the standard proteins run in the
same assay). Based on the SEC analyses for all constructs
evaluated, including a CFXTEN composition, the apparent molecular
weights, the apparent molecular weight factor (expressed as the
ratio of apparent molecular weight to the calculated molecular
weight) and the hydrodynamic radius (R.sub.Hin nm) are shown in
Table 31. The results indicate that incorporation of different
XTENs of 576 amino acids or greater confers an apparent molecular
weight for the fusion protein of approximately 339 kDa to 760, and
that XTEN of 864 amino acids or greater confers an apparent
molecular weight greater than approximately 800 kDA. The results of
proportional increases in apparent molecular weight to actual
molecular weight were consistent for fusion proteins created with
XTEN from several different motif families; i.e., AD, AE, AF, AG,
and AM, with increases of at least four-fold and ratios as high as
about 17-fold. Additionally, the incorporation of XTEN fusion
partners with 576 amino acids or more into fusion proteins with the
various payloads (and 288 residues in the case of glucagon fused to
Y288) resulted with a hydrodynamic radius of 7 nm or greater; well
beyond the glomerular pore size of approximately 3-5 nm.
Accordingly, it is expected that fusion proteins comprising growth
and XTEN have reduced renal clearance, contributing to increased
terminal half-life and improving the therapeutic or biologic effect
relative to a corresponding un-fused biologic payload protein.
TABLE-US-00037 TABLE 31 SEC analysis of various polypeptides
Apparent XTEN or Actual Apparent Molecular Construct fusion
Therapeutic MW MW Weight R.sub.H Name partner Protein (kDa) (kDa)
Factor (nm) AC14 Y288 Glucagon 28.7 370 12.9 7.0 AC28 Y144 Glucagon
16.1 117 7.3 5.0 AC34 Y72 Glucagon 9.9 58.6 5.9 3.8 AC33 Y36
Glucagon 6.8 29.4 4.3 2.6 AC89 AF120 Glucagon 14.1 76.4 5.4 4.3
AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.2
5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1
7.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6
AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7
10.1 AC85 AE864 Exendin-4 83.6 938 11.2 8.9 AC114 AM875 Exendin-4
82.4 1344 16.3 9.4 AC143 AM875 CF 100.6 846 8.4 8.7 AC227 AM875
IL-1ra 95.4 1103 11.6 9.2 AC228 AM1318 IL-1ra 134.8 2286 17.0
10.5
Example 39
Pharmacokinetics of Extended Polypeptides Fused to GFP in
Cynomolgus Monkeys
[0491] The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576,
GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys
to determine the effect of composition and length of the
unstructured polypeptides on PK parameters. Blood samples were
analyzed at various times after injection and the concentration of
GFP in plasma was measured by ELISA using a polyclonal antibody
against GFP for capture and a biotinylated preparation of the same
polyclonal antibody for detection. Results are summarized in FIG.
25. They show a surprising increase of half-life with increasing
length of the XTEN sequence. For example, a half-life of 10 h was
determined for GFP-XTEN_L288 (with 288 amino acid residues in the
XTEN). Doubling the length of the unstructured polypeptide fusion
partner to 576 amino acids increased the half-life to 20-22 h for
multiple fusion protein constructs; i.e., GFP-XTEN_L576,
GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the
unstructured polypeptide fusion partner length to 836 residues
resulted in a half-life of 72-75 h for XTEN_AD836-GFP. Thus,
increasing the polymer length by 288 residues from 288 to 576
residues increased in vivo half-life by about 10 h. However,
increasing the polypeptide length by 260 residues from 576 residues
to 836 residues increased half-life by more than 50 h. These
results show that there is a surprising threshold of unstructured
polypeptide length that results in a greater than proportional gain
in in vivo half-life. Thus, fusion proteins comprising extended,
unstructured polypeptides are expected to have the property of
enhanced pharmacokinetics compared to polypeptides of shorter
lengths.
Example 40
Serum Stability of XTEN
[0492] A fusion protein containing XTEN_AE864 fused to the
N-terminus of GFP was incubated in monkey plasma and rat kidney
lysate for up to 7 days at 37.degree. C. Samples were withdrawn at
time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by
detection using Western analysis and detection with antibodies
against GFP as shown in FIG. 26. The sequence of XTEN_AE864 showed
negligible signs of degradation over 7 days in plasma. However,
XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days.
The in vivo stability of the fusion protein was tested in plasma
samples wherein the GFP_AE864 was immunoprecipitated and analyzed
by SDS PAGE as described above. Samples that were withdrawn up to 7
days after injection showed very few signs of degradation. The
results demonstrate the resistance of CFXTEN to degradation due to
serum proteases; a factor in the enhancement of pharmacokinetic
properties of the CFXTEN fusion proteins.
Example 41
Increasing Solubility and Stability of a Peptide Payload by Linking
to XTEN
[0493] In order to evaluate the ability of XTEN to enhance the
physical/chemical properties of solubility and stability, fusion
proteins of glucagon plus shorter-length XTEN were prepared and
evaluated. The test articles were prepared in Tris-buffered saline
at neutral pH and characterization of the Gcg-XTEN solution was by
reverse-phase HPLC and size exclusion chromatography to affirm that
the protein was homogeneous and non-aggregated in solution. The
data are presented in Table 32. For comparative purposes, the
solubility limit of unmodified glucagon in the same buffer was
measured at 60 .mu.M (0.2 mg/mL), and the result demonstrate that
for all lengths of XTEN added, a substantial increase in solubility
was attained. Importantly, in most cases the glucagon-XTEN fusion
proteins were prepared to achieve target concentrations and were
not evaluated to determine the maximum solubility limits for the
given construct. However, in the case of glucagon linked to the
AF-144 XTEN, the limit of solubility was determined, with the
result that a 60-fold increase in solubility was achieved, compared
to glucagon not linked to XTEN. In addition, the glucagon-AF144
CFXTEN was evaluated for stability, and was found to be stable in
liquid formulation for at least 6 months under refrigerated
conditions and for approximately one month at 37.degree. C. (data
not shown).
[0494] The data support the conclusion that the linking of
short-length XTEN polypeptides to a biologically active protein
such as glucagon can markedly enhance the solubility properties of
the protein by the resulting fusion protein, as well as confer
stability at the higher protein concentrations.
TABLE-US-00038 TABLE 32 Solubility of Glucagon-XTEN constructs Test
Article Solubility Glucagon 60 .mu.M Glucagon-Y36 >370 .mu.M
Glucagon-Y72 >293 .mu.M Glucagon-AF108 >145 .mu.M
Glucagon-AF120 >160 .mu.M Glucagon-Y144 >497 .mu.M
Glucagon-AE144 >467 .mu.M Glucagon-AF144 >3600 .mu.M
Glucagon-Y288 >163 .mu.M
Example 42
Analysis of Sequences for Secondary Structure by Prediction
Algorithms
[0495] Amino acid sequences can be assessed for secondary structure
via certain computer programs or algorithms, such as the well-known
Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:
222-45) and the Garnier-Osguthorpe-Robson, or "GOR" method (Garnier
J, Gibrat J F, Robson B. (1996). GOR method for predicting protein
secondary structure from amino acid sequence. Methods Enzymol
266:540-553). For a given sequence, the algorithms can predict
whether there exists some or no secondary structure at all,
expressed as total and/or percentage of residues of the sequence
that form, for example, alpha-helices or beta-sheets or the
percentage of residues of the sequence predicted to result in
random coil formation.
[0496] Several representative sequences from XTEN "families" have
been assessed using two algorithm tools for the Chou-Fasman and GOR
methods to assess the degree of secondary structure in these
sequences. The Chou-Fasman tool was provided by William R. Pearson
and the University of Virginia, at the "Biosupport" interne site,
URL located on the World Wide Web at
lasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it
existed on Jun. 19, 2009. The GOR tool was provided by Pole
Informatique Lyonnais at the Network Protein Sequence Analysis
internet site, URL located on the World Wide Web at
.npsa-pbilibcp.fr/cgi-bin/secpred_gor4.pl as it existed on Jun. 19,
2008.
[0497] As a first step in the analyses, a single XTEN sequence was
analyzed by the two algorithms. The AE864 composition is a XTEN
with 864 amino acid residues created from multiple copies of four
12 amino acid sequence motifs consisting of the amino acids G, S,
T, E, P, and A. The sequence motifs are characterized by the fact
that there is limited repetitiveness within the motifs and within
the overall sequence in that the sequence of any two consecutive
amino acids is not repeated more than twice in any one 12 amino
acid motif, and that no three contiguous amino acids of full-length
the XTEN are identical. Successively longer portions of the AF 864
sequence from the N-terminus were analyzed by the Chou-Fasman and
GOR algorithms (the latter requires a minimum length of 17 amino
acids). The sequences were analyzed by entering the FASTA format
sequences into the prediction tools and running the analysis. The
results from the analyses are presented in Table 33.
[0498] The results indicate that, by the Chou-Fasman calculations,
the four motifs of the AE family (Table 1) have no alpha-helices or
beta sheets. The sequence up to 288 residues was similarly found to
have no alpha-helices or beta sheets. The 432 residue sequence is
predicted to have a small amount of secondary structure, with only
2 amino acids contributing to an alpha-helix for an overall
percentage of 0.5%. The full-length AF864 polypeptide has the same
two amino acids contributing to an alpha-helix, for an overall
percentage of 0.2%. Calculations for random coil formation revealed
that with increasing length, the percentage of random coil
formation increased. The first 24 amino acids of the sequence had
91% random coil formation, which increased with increasing length
up to the 99.77% value for the full-length sequence.
[0499] Numerous XTEN sequences of 500 amino acids or longer from
the other motif families were also analyzed and revealed that the
majority had greater than 95% random coil formation. The exceptions
were those sequences with one or more instances of three contiguous
serine residues, which resulted in predicted beta-sheet formation.
However, even these sequences still had approximately 99% random
coil formation.
[0500] In contrast, a polypeptide sequence of 84 residues limited
to A, S, and P amino acids was assessed by the Chou-Fasman
algorithm, which predicted a high degree of predicted
alpha-helices. The sequence, which had multiple repeat "AA" and
"AAA" sequences, had an overall predicted percentage of alpha-helix
structure of 69%. The GOR algorithm predicted 78.57% random coil
formation; far less than any sequence consisting of 12 amino acid
sequence motifs consisting of the amino acids G, S, T, E, P,
analyzed in the present Example.
[0501] The analysis supports the conclusion that: 1) XTEN created
from multiple sequence motifs of G, S, T, E, P, and A that have
limited repetitiveness as to contiguous amino acids are predicted
to have very low amounts of alpha-helices and beta-sheets; 2) that
increasing the length of the XTEN does not appreciably increase the
probability of alpha-helix or beta-sheet formation; and 3) that
progressively increasing the length of the XTEN sequence by
addition of non-repetitive 12-mers consisting of the amino acids G,
S, T, E, P, and A results in increased percentage of random coil
formation. In contrast, polypeptides created from amino acids
limited to A, S and P that have a higher degree of internal
repetitiveness are predicted to have a high percentage of
alpha-helices, as determined by the Chou-Fasman algorithm, as well
as random coil formation. Based on the numerous sequences evaluated
by these methods, it is concluded that XTEN created from sequence
motifs of G, S, T, E, P, and A that have limited repetitiveness
(defined as no more than two identical contiguous amino acids in
any one motif) greater than about 400 amino acid residues in length
are expected to have very limited secondary structure. With the
exception of motifs containing three contiguous serines, it is
believed that any order or combination of sequence motifs from
Table 3 can be used to create an XTEN polypeptide of a length
greater than about 400 residues that will result in an XTEN
sequence that is substantially devoid of secondary structure. Such
sequences are expected to have the characteristics described in the
CFXTEN embodiments of the invention disclosed herein.
TABLE-US-00039 TABLE 33 CHOU-FASMAN and GOR prediction calculations
of polypeptide sequences SEQ No. Chou-Fasman GOR NAME Sequence
Residues Calculation Calculation GSTSESPSGTAP 12 Residue totals*:
H: 0 E: 0 Not percent: H: 0.0 E: 0.0 Determined GTS TPESGSASP 12
Residue totals: H: 0 E: 0 Not percent: H: 0.0 E: 0.0 Determined
GTSPSGESSTAP 12 Residue totals: H: 0 E: 0 Not percent: H: 0.0 E:
0.0 Determined GSTSSTAESPGP 12 Residue totals: H: 0 E: 0 Not
percent: H: 0.0 E: 0.0 Determined GSPAGSPTSTEEGTSESATPESGP 24
Residue totals: H: 0 E: 0 91.67% percent: H: 0.0 E: 0.0
GSPAGSPTSTEEGTSESATPESGPG 36 Residue totals: H: 0 E: 0 94.44%
TSTEPSEGSAP percent: H: 0.0 E: 0.0 GSPAGSPTSTEEGTSESATPESGPG 48
Residue totals: H: 0 E: 0 93.75% TSTEPSEGSAPGSPAGSPTSTEE percent:
H: 0.0 E: 0.0 GSPAGSPTSTEEGTSESATPESGPG 60 Residue totals: H: 0 E:
0 96.67% TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.0 E: 0.0
TEPSEGSAP GSPAGSPTSTEEGTSESATPESGPG 108 Residue totals: H: 0 E: 0
97.22% TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.0 E: 0.0
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSE TPGSEPATSGSETP
GSPAGSPTSTEEGTSESATPESGPG 216 Residue totals: H: 0 E: 0 99.07%
TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.0 E: 0.0
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGP GTSTEPSEGSAP
GSPAGSPTSTEEGTSESATPESGPG 432 Residue totals: H: 2 E: 3 99.54%
TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.5 E: 0.7
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAP AE864 GSPAGSPTSTEEGTSESATPESGPG 864
Residue totals: H: 2 E: 3 99.77% TSTEPSEGSAPGSPAGSPTSTEEGTS
percent: H: 0.2 E: 0.3 TEPSEGSAPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSA
PGTSTEPSEGSAPGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSES
ATPESGPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPG
TSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSES
ATPESGPGTSTEPSEGSAPGTSESA TPESGPGSPAGSPTSTEEGSPAGSPT
STEEGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGTSES
ATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATP
ESGPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGSEPATSGSET
PGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPG
TSESATPESGPGTSTEPSEGSAP AD 576 GSSESGSSEGGPGSGGEPSESGSSG 576
Residue totals: H: 7 E: 0 99.65% SSESGSSEGGPGSSESGSSEGGPGSS
percent: H: 1.2 E: 0.0 ESGSSEGGPGSSESGSSEGGPGSSE
SGSSEGGPGESPGGSSGSESGSEGS SGPGESSGSSESGSSEGGPGSSESGS
SEGGPGSSESGSSEGGPGSGGEPSE SGSSGESPGGSSGSESGESPGGSSG
SESGSGGEPSESGSSGSSESGSSEG GPGSGGEPSESGSSGSGGEPSESGS
SGSEGSSGPGESSGESPGGSSGSES GSGGEPSESGSSGSGGEPSESGSSG
SGGEPSESGSSGSSESGSSEGGPGE SPGGSSGSESGESPGGSSGSESGESP
GGSSGSESGESPGGSSGSESGESPG GSSGSESGSSESGSSEGGPGSGGEP
SESGSSGSEGSSGPGESSGSSESGSS EGGPGSGGEPSESGSSGSSESGSSE
GGPGSGGEPSESGSSGESPGGSSGS ESGESPGGSSGSESGSSESGSSEGG
PGSGGEPSESGSSGSSESGSSEGGP GSGGEPSESGSSGSGGEPSESGSSG
ESPGGSSGSESGSEGSSGPGESSGSS ESGSSEGGPGSEGSSGPGESS AE576
GSPAGSPTSTEEGTSESATPESGPG 576 Residue totals: H: 2 E: 0 99.65%
TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.4 E: 0.0
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAP AF540 GSTSSTAESPGPGSTSSTAESPGPGS 540 Residue totals:
H: 2 E: 0 99.65 TSESPSGTAPGSTSSTAESPGPGSTS percent: H: 0.4 E: 0.0
STAESPGPGTSTPESGSASPGSTSES PSGTAPGTSPSGESSTAPGSTSESPS
GTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGSTSESPSGTAP
GTSPSGESSTAPGSTSESPSGTAPGS TSESPSGTAPGSTSESPSGTAPGTST
PESGSASPGSTSESPSGTAPGTSTPE SGSASPGSTSSTAESPGPGSTSSTAE
SPGPGTSTPESGSASPGTSTPESGSA SPGSTSESPSGTAPGTSTPESGSASP
GTSTPESGSASPGSTSESPSGTAPGS TSESPSGTAPGSTSESPSGTAPGSTS
STAESPGPGTSTPESGSASPGTSTPE SGSASPGSTSESPSGTAPGSTSESPS
GTAPGTSTPESGSASPGSTSESPSGT APGSTSESPSGTAPGTSTPESGSASP
GTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGSTSSTAESPGPGTST
PESGSASPGSTSESPSGTAP AF504 GASPGTSSTGSPGSSPSASTGTGPG 504 Residue
totals: H: 0 E: 0 94.44% SSPSASTGTGPGTPGSGTASSSPGSS percent: H: 0.0
E: 0.0 TPSGATGSPGSNPSASTGTGPGASP GTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGTPGSGTASSSPGASPGTS STGSPGASPGTSSTGSPGTPGSGTA
SSSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSP
GSNPSASTGTGPGSSPSASTGTGPG SSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGASPG
TSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATG
SPGSSTPSGATGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPG
SSTPSGATGSPGSSTPSGATGSPGSS PSASTGTGPGASPGTSSTGSP AE864
GSPAGSPTSTEEGTSESATPESGPG 864 Residue totals: H: 2 E: 3 99.77%
TSTEPSEGSAPGSPAGSPTSTEEGTS percent: H: 0.2 E: 0.4
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPAT
SGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTS
ESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSET PGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAP AF864
GSTSESPSGTAPGTSPSGESSTAPGS 875 Residue totals: H: 2 E: 0 95.20%
TSESPSGTAPGSTSESPSGTAPGTST percent: H: 0.2 E: 0.0
PESGSASPGTSTPESGSASPGSTSES PSGTAPGSTSESPSGTAPGTSPSGES
STAPGSTSESPSGTAPGTSPSGESST APGTSPSGESSTAPGSTSSTAESPGP
GTSPSGESSTAPGTSPSGESSTAPGS TSSTAESPGPGTSTPESGSASPGTST
PESGSASPGSTSESPSGTAPGSTSES PSGTAPGTSTPESGSASPGSTSSTAE
SPGPGTSTPESGSASPGSTSESPSGT APGTSPSGESSTAPGSTSSTAESPGP
GTSPSGESSTAPGTSTPESGSASPGS TSSTAESPGPGSTSSTAESPGPGSTS
STAESPGPGSTSSTAESPGPGTSPSG ESSTAPGSTSESPSGTAPGSTSESPS
GTAPGTSTPESGPXXXGASASGAP STXXXXSESPSGTAPGSTSESPSGT
APGSTSESPSGTAPGSTSESPSGTAP GSTSESPSGTAPGSTSESPSGTAPGT
STPESGSASPGTSPSGESSTAPGTSP SGESSTAPGSTSSTAESPGPGTSPSG
ESSTAPGTSTPESGSASPGSTSESPS GTAPGSTSESPSGTAPGTSPSGESST
APGSTSESPSGTAPGTSTPESGSASP GTSTPESGSASPGSTSESPSGTAPGT
STPESGSASPGSTSSTAESPGPGSTS ESPSGTAPGSTSESPSGTAPGTSPSG
ESSTAPGSTSSTAESPGPGTSPSGES STAPGTSTPESGSASPGTSPSGESST
APGTSPSGESSTAPGTSPSGESSTAP GSTSSTAESPGPGSTSSTAESPGPGT
SPSGESSTAPGSSPSASTGTGPGSST PSGATGSPGSSTPSGATGSP AG864
GGSPGASPGTSSTGSPGSSPSASTG 868 Residue totals: H: 0 E: 0 94.70%
TGPGSSPSASTGTGPGTPGSGTASS percent: H: 0.0 E: 0.0
SPGSSTPSGATGSPGSNPSASTGTG PGASPGTSSTGSPGTPGSGTASSSP
GSSTPSGATGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTP
SGATGSPGSNPSASTGTGPGSSPSA STGTGPGSSTPSGATGSPGSSTPSG
ATGSPGASPGTSSTGSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTAS
SSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSPSASTGTGP
GTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPS
GATGSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSS
TGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGS
PGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPG
TPGSGTASSSPGSSTPSGATGSPGT PGSGTASSSPGSSTPSGATGSPGSST
PSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGTPGSG
TASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSST
GSPGASPGTSSTGSPGSSTPSGATG SPGSSPSASTGTGPGASPGTSSTGSP
GSSPSASTGTGPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGA SPGTSSTGSP
AM875 GTSTEPSEGSAPGSEPATSGSETPG 875 Residue totals: H: 7 E: 3
98.63% SPAGSPTSTEEGSTSSTAESPGPGTS percent: H: 0.8 E: 0.3
TPESGSASPGSTSESPSGTAPGSTSE SPSGTAPGTSTPESGSASPGTSTPES
GSASPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTS
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGSEPAT
SGSETPGSPAGSPTSTEEGSSTPSGA TGSPGTPGSGTASSSPGSSTPSGAT
GSPGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAP GASASGAPSTGGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSTS STAESPGPGSTSESPSGTAPGTSPSG
ESSTAPGTPGSGTASSSPGSSTPSG ATGSPGSSPSASTGTGPGSEPATSG
SETPGTSESATPESGPGSEPATSGSE TPGSTSSTAESPGPGSTSSTAESPGP
GTSPSGESSTAPGSEPATSGSETPGS EPATSGSETPGTSTEPSEGSAPGSTS
STAESPGPGTSTPESGSASPGSTSES PSGTAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSSTPSGAT GSPGSSPSASTGTGPGASPGTSSTG
SPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGT SESATPESGPGTSTEPSEGSAPGTST EPSEGSAP
AM1318 GTSTEPSEGSAPGSEPATSGSETPG 1318 Residue totals: H: 7 E: 0
99.17% SPAGSPTSTEEGSTSSTAESPGPGTS percent: H: 0.7 E: 0.0
TPESGSASPGSTSESPSGTAPGSTSE SPSGTAPGTSTPESGSASPGTSTPES
GSASPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGTSTEPSEGS
APGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGTS
TEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGSEPAT
SGSETPGSPAGSPTSTEEGSSTPSGA TGSPGTPGSGTASSSPGSSTPSGAT
GSPGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAP GPEPTGPAPSGGSEPATSGSETPGT
SESATPESGPGSPAGSPTSTEEGTSE SATPESGPGSPAGSPTSTEEGSPAG
SPTSTEEGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSTSSTAES
PGPGSTSESPSGTAPGTSPSGESSTA PGSTSESPSGTAPGSTSESPSGTAPG
TSPSGESSTAPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSES ATPESGPGTSTEPSEGSAPGTSESA
TPESGPGTSTEPSEGSAPGTSPSGES STAPGTSPSGESSTAPGTSPSGESST
APGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGSSPSASTGTGP
GSSTPSGATGSPGSSTPSGATGSPG SSTPSGATGSPGSSTPSGATGSPGA
SPGTSSTGSPGASASGAPSTGGTSP SGESSTAPGSTSSTAESPGPGTSPSG
ESSTAPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSSPSASTG
TGPGSSTPSGATGSPGASPGTSSTG SPGTSTPESGSASPGTSPSGESSTAP
GTSPSGESSTAPGTSESATPESGPGS EPATSGSETPGTSTEPSEGSAPGSTS
ESPSGTAPGSTSESPSGTAPGTSTPE SGSASPGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETP
GSSTPSGATGSPGASPGTSSTGSPG SSTPSGATGSPGSTSESPSGTAPGTS
PSGESSTAPGSTSSTAESPGPGSSTP SGATGSPGASPGTSSTGSPGTPGSG
TASSSPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAP AM923
MAEPAGSPTSTEEGASPGTSSTGSP 924 Residue totals: H: 4 E: 3 98.70%
GSSTPSGATGSPGSSTPSGATGSPG percent: H: 0.4 E: 0.3
TSTEPSEGSAPGSEPATSGSETPGSP AGSPTSTEEGSTSSTAESPGPGTSTP
ESGSASPGSTSESPSGTAPGSTSESP SGTAPGTSTPESGSASPGTSTPESGS
ASPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEG
TSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSTE
PSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGSEPATS
GSETPGSPAGSPTSTEEGSSTPSGA TGSPGTPGSGTASSSPGSSTPSGAT
GSPGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAP GASASGAPSTGGTSESATPESGPGS
PAGSPTSTEEGSPAGSPTSTEEGSTS STAESPGPGSTSESPSGTAPGTSPSG
ESSTAPGTPGSGTASSSPGSSTPSG ATGSPGSSPSASTGTGPGSEPATSG
SETPGTSESATPESGPGSEPATSGSE TPGSTSSTAESPGPGSTSSTAESPGP
GTSPSGESSTAPGSEPATSGSETPGS EPATSGSETPGTSTEPSEGSAPGSTS
STAESPGPGTSTPESGSASPGSTSES PSGTAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSSTPSGAT GSPGSSPSASTGTGPGASPGTSSTG
SPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSSTPSGATGSPG
SSPSASTGTGPGASPGTSSTGSPGT SESATPESGPGTSTEPSEGSAPGTST EPSEGSAP AE912
MAEPAGSPTSTEEGTPGSGTASSSP 913 Residue totals: H: 8 E: 3 99.45%
GSSTPSGATGSPGASPGTSSTGSPG percent: H: 0.9 E: 0.3
SPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGS
APGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSES ATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAP
GSPAGSPTSTEEGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTS
ESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTS
ESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGTSESATPES
GPGSEPATSGSETPGSEPATSGSET PGSPAGSPTSTEEGTSTEPSEGSAP
GTSTEPSEGSAPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAP BC 864
GTSTEPSEPGSAGTSTEPSEPGSAG Residue totals: H: 0 E: 0 99.77%
SEPATSGTEPSGSGASEPTSTEPGSE percent: H: 0 E: 0
PATSGTEPSGSEPATSGTEPSGSEP ATSGTEPSGSGASEPTSTEPGTSTEP
SEPGSAGSEPATSGTEPSGTSTEPSE PGSAGSEPATSGTEPSGSEPATSGT
EPSGTSTEPSEPGSAGTSTEPSEPGS AGSEPATSGTEPSGSEPATSGTEPS
GTSEPSTSEPGAGSGASEPTSTEPG TSEPSTSEPGAGSEPATSGTEPSGSE
PATSGTEPSGTSTEPSEPGSAGTSTE PSEPGSAGSGASEPTSTEPGSEPATS
GTEPSGSEPATSGTEPSGSEPATSG TEPSGSEPATSGTEPSGTSTEPSEPG
SAGSEPATSGTEPSGSGASEPTSTE PGTSTEPSEPGSAGSEPATSGTEPS
GSGASEPTSTEPGTSTEPSEPGSAG SGASEPTSTEPGSEPATSGTEPSGS
GASEPTSTEPGSEPATSGTEPSGSG ASEPTSTEPGTSTEPSEPGSAGSEPA
TSGTEPSGSGASEPTSTEPGTSTEPS EPGSAGSEPATSGTEPSGTSTEPSEP
GSAGSEPATSGTEPSGTSTEPSEPG SAGTSTEPSEPGSAGTSTEPSEPGS
AGTSTEPSEPGSAGTSTEPSEPGSA GTSTEPSEPGSAGTSEPSTSEPGAG
SGASEPTSTEPGTSTEPSEPGSAGTS TEPSEPGSAGTSTEPSEPGSAGSEP
ATSGTEPSGSGASEPTSTEPGSEPA TSGTEPSGSEPATSGTEPSGSEPATS
GTEPSGSEPATSGTEPSGTSEPSTSE PGAGSEPATSGTEPSGSGASEPTST
EPGTSTEPSEPGSAGSEPATSGTEPS GSGASEPTSTEPGTSTEPSEPGSA
ASPAAPAPASPAAPAPSAPAAAPA 84 Residue totals: H: 58 E: 0 78.57%
SPAPAAPSAPAPAAPSAASPAAPSA percent: H: 69.0 E: 0.0
PPAAASPAAPSAPPAASAAAPAAA SAAASAPSAAA
*H: alpha-helix E: beta-sheet
Example 43
Analysis of Polypeptide Sequences for Repetitiveness
[0502] Polypeptide amino acid sequences can be assessed for
repetitiveness by quantifying the number of times a shorter
subsequence appears within the overall polypeptide. For example, a
polypeptide of 200 amino acid residues has 192 overlapping 9-amino
acid subsequences (or 9-mer "frames"), but the number of unique
9-mer subsequences will depend on the amount of repetitiveness
within the sequence. In the present analysis, different sequences
were assessed for repetitiveness by summing the occurrence of all
unique 3-mer subsequences for each 3-amino acid frame across the
first 200 amino acids of the polymer portion divided by the
absolute number of unique 3-mer subsequences within the 200 amino
acid sequence. The resulting subsequence score is a reflection of
the degree of repetitiveness within the polypeptide.
[0503] The results, shown in Table 34, indicate that the
unstructured polypeptides consisting of 2 or 3 amino acid types
have high subsequence scores, while those of consisting of 12 amino
acids motifs of the six amino acids G, S, T, E, P, and A with a low
degree of internal repetitiveness, have subsequence scores of less
than 10, and in some cases, less than 5. For example, the L288
sequence has two amino acid types and has short, highly repetitive
sequences, resulting in a subsequence score of 50.0. The
polypeptide J288 has three amino acid types but also has short,
repetitive sequences, resulting in a subsequence score of 33.3.
Y576 also has three amino acid types, but is not made of internal
repeats, reflected in the subsequence score of 15.7 over the first
200 amino acids. W576 consists of four types of amino acids, but
has a higher degree of internal repetitiveness, e.g., "GGSG",
resulting in a subsequence score of 23.4. The AD576 consists of
four types of 12 amino acid motifs, each consisting of four types
of amino acids. Because of the low degree of internal
repetitiveness of the individual motifs, the overall subsequence
score over the first 200 amino acids is 13.6. In contrast, XTEN's
consisting of four motifs contains six types of amino acids, each
with a low degree of internal repetitiveness have lower subsequence
scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5).
CONCLUSIONS
[0504] The results indicate that the combination of 12 amino acid
subsequence motifs, each consisting of four to six amino acid types
that are essentially non-repetitive, into a longer XTEN polypeptide
results in an overall sequence that is non-repetitive. This is
despite the fact that each subsequence motif may be used multiple
times across the sequence. In contrast, polymers created from
smaller numbers of amino acid types resulted in higher subsequence
scores, although the actual sequence can be tailored to reduce the
degree of repetitiveness to result in lower subsequence scores.
TABLE-US-00040 TABLE 34 Subsequence score calculations of
polypeptide sequences Seq Name Amino Acid Sequence Score J288
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE 33.3
GGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSG
GEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGS
GGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE
GGSGGEGGSGGEGGSGGEGGSGGEG K288
GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGG 46.9
EGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEG
GGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEG
EGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGG
EGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE
GGEGEGGGEGGEGEGGGEGGEGEGGGEG L288
SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSS 50.0
ESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSES
SESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSE
SSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESS
SSESSSESSESSSSESSSESSESSSSESSSESSESSSSES Y288
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEG 26.8
EGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGE
GGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGE
GSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGE
GSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGE
GSEGSGEGEGSEGSGE Q576
GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGGGEGGKPEGGKPE 18.5
GEGKPGGGEGKPGGKPEGGGGKPEGEGKPGGGGGKPGGKPEGEGKPGGGEGG
KPEGKPGEGGEGKPGGKPEGGGEGKPGGGKPGEGGKPGEGKPGGGEGGKPEGG
KPEGEGKPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGEGGEGKPGGGKPEGE
GKPGGGKPGGGEGGKPEGEGKPGGKPEGGGEGKPGGKPEGGGKPEGGGEGKP
GGGKPGEGGKPGEGEGKPGGKPEGEGKPGGEGGGKPEGKPGGGEGGKPEGGKP
GEGGKPEGGKPGEGGEGKPGGGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGG
KPEGGKPEGGGEGKPGGGKPEGEGKPGGGEGKPGGKPEGGGGKPGEGGKPEGG
KPGGEGGGKPEGEGKPGGKPGEGGGGKPGGKPEGEGKPGEGGEGKPGGKPEGG
GEGKPGGKPEGGGEGKPGGGKPGEGGKPEGGGKPGEGGKPGEGGKPEGEGKPG
GGEGKPGGKPGEGGKPEGGGEGKPGGKPGGEGGGKPEGGKPGEGGKPEG U576
GEGKPGGKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKPGGKPGEGGKPEG 18.1
GSGGKPGGGGKPGGKPGGEGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGG
KPGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPEGGSGGKPGGK
PEGGSGGKPGGSGKPGGKPGEGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPGG
KPGEGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEGSGKPGGKPGSGGEGKPG
GKPEGGSGGKPGGGKPGGEGKPGSGGKPGEGGKPGSGGGKPGGKPGGEGEGKP
GGKPGEGGKPGGEGSGKPGGGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPE
GGGGKPEGSGKPGGGGKPEGSGKPGGGKPEGGSGGKPGGSGKPGGKPGEGGG
KPEGSGKPGGGSGKPGGKPEGGGKPEGGSGGKPGGKPEGGSGGKPGGKPGGEG
SGKPGGKPGSGEGGKPGGKPGEGSGGKPGGKPEGGSGGKPGGSGKPGGKPEGG
GSGKPGGKPGEGGKPGGEGSGKPGGSGKPG W576
GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGSGKP 23.4
GSGKPGGGGKPGSGSGKPGGGKPGGSGGKPGGGSGKPGKPGSGGSGKPGSGKP
GGGSGGKPGKPGSGGSGGKPGKPGSGGGSGKPGKPGSGGSGGKPGKPGSGGSG
GKPGKPGSGGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGKPGSG
KPGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGGKPGSGSGKPGG
GKPGSGSGKPGGGKPGGSGGKPGGSGGKPGKPGSGGGSGKPGKPGSGGGSGKP
GKPGGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGGKPGKPGS
GGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGSGK
PGSGKPGGGSGGKPGKPGSGGSGKPGSGKPGSGGSGKPGKPGGSGSGKPGSGKP
GGGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGSG
GKPGKPGSGGSGGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGG Y576
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGE 15.7
GEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGE
GEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEG
EGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEGE
GGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGE
GSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEG
GSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGS
EGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGS
EGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEGSE
GSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEGGE
GSGEGEGGGEGSEGEGSEGSGEGEGSGEGSE AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSE 13.6
GGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESG
SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGES
PGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSS
GSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSES
GSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGG
SSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSE
SGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESG
ESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESG
SSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSG PGESS
AE576 AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE 6.1
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP
AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AF540
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESP 8.8
GPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS
GTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSES
PSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST
PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGS
TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAP
GSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGT
APGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
SASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPE
SGSASPGSTSESPSGTAP AF504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT 7.0
GSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG
TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTGTGP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA
STGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS 6.1
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP
ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAP
AF864 GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA
7.5 SPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPS
GTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSG
ESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS
ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGT
SPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGP
GSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGT
APGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPS
GTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPE
SGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTST
PESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGT
STPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGP
GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESST
APGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAE
SPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSP
AG868 GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP 7.5
SGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS
PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSAST
GTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPG
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
PSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP
GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGT
SSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGAS
PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
TGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA
STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGAS
PGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
GASPGTSSTGSP AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA 4.5
SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSG
SETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSP
GSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSG
TASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEP
ATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGS
EPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGT
GPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSG
ATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTST EPSEGSAP
AM1318 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA
4.5 SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSG
SETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT
STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSP
GSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATP
ESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESA
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP
SGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGT
SESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGP
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESST
APGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSAST
GTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPG
TSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTS
ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSP
GASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPES
GPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
SASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTS
ESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGT
PGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
Example 44
Calculation of TEPITOPE Scores
[0505] TEPITOPE scores of 9 mer peptide sequence can be calculated
by adding pocket potentials as described by Sturniolo [Sturniolo,
T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example,
separate Tepitope scores were calculated for individual HLA
alleles. Table 35 shows as an example the pocket potentials for
HLA*0101B, which occurs in high frequency in the Caucasian
population. To calculate the TEPITOPE score of a peptide with
sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual
pocket potentials in Table 35 were added. The HLA*0101B score of a
9 mer peptide with the sequence FDKLPRTSG is the sum of 0, -1.3, 0,
0.9, 0, -1.8, 0.09, 0, 0.
[0506] To evaluate the TEPITOPE scores for long peptides one can
repeat the process for all 9 mer subsequences of the sequences.
This process can be repeated for the proteins encoded by other HLA
alleles. Tables 36-39 give pocket potentials for the protein
products of HLA alleles that occur with high frequency in the
Caucasian population.
[0507] TEPITOPE scores calculated by this method range from
approximately -10 to +10. However, 9 mer peptides that lack a
hydrophobic amino acid (FKLMVWY) in P1 position have calculated
TEPITOPE scores in the range of -1009 to -989. This value is
biologically meaningless and reflects the fact that a hydrophobic
amino acid serves as an anchor residue for HLA binding and peptides
lacking a hydrophobic residue in P1 are considered non binders to
HLA. Because most XTEN sequences lack hydrophobic residues, all
combinations of 9 mer subsequences will have TEPITOPEs in the range
in the range of -1009 to -989. This method confirms that XTEN
polypeptides may have few or no predicted T-cell epitopes.
TABLE-US-00041 TABLE 35 Pocket potential for HLA*0101B allele.
Amino Acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -2.4 -- -2.7 -2 -- -1.9 E
-999 0.1 -1.2 -0.4 -- -2.4 -0.6 -- -1.9 F 0 0.8 0.8 0.08 -- -2.1
0.3 -- -0.4 G -999 0.5 0.2 -0.7 -- -0.3 -1.1 -- -0.8 H -999 0.8 0.2
-0.7 -- -2.2 0.1 -- -1.1 I -1 1.1 1.5 0.5 -- -1.9 0.6 -- 0.7 K -999
1.1 0 -2.1 -- -2 -0.2 -- -1.7 L -1 1 1 0.9 -- -2 0.3 -- 0.5 M -1
1.1 1.4 0.8 -- -1.8 0.09 -- 0.08 N -999 0.8 0.5 0.04 -- -1.1 0.1 --
-1.2 P -999 -0.5 0.3 -1.9 -- -0.2 0.07 -- -1.1 Q -999 1.2 0 0.1 --
-1.8 0.2 -- -1.6 R -999 2.2 0.7 -2.1 -- -1.8 0.09 -- -1 S -999 -0.3
0.2 -0.7 -- -0.6 -0.2 -- -0.3 T -999 0 0 -1 -- -1.2 0.09 -- -0.2 V
-1 2.1 0.5 -0.1 -- -1.1 0.7 -- 0.3 W 0 -0.1 0 -1.8 -- -2.4 -0.1 --
-1.4 Y 0 0.9 0.8 -1.1 -- -2 0.5 -- -0.9
TABLE-US-00042 TABLE 36 Pocket potential for HLA*0301B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 2.3 -- -2.4 -0.6 -- -0.6 E
-999 0.1 -1.2 -1 -- -1.4 -0.2 -- -0.3 F -1 0.8 0.8 -1 -- -1.4 0.5
-- 0.9 G -999 0.5 0.2 0.5 -- -0.7 0.1 -- 0.4 H -999 0.8 0.2 0 --
-0.1 -0.8 -- -0.5 I 0 1.1 1.5 0.5 -- 0.7 0.4 -- 0.6 K -999 1.1 0 -1
-- 1.3 -0.9 -- -0.2 L 0 1 1 0 -- 0.2 0.2 -- -0 M 0 1.1 1.4 0 --
-0.9 1.1 -- 1.1 N -999 0.8 0.5 0.2 -- -0.6 -0.1 -- -0.6 P -999 -0.5
0.3 -1 -- 0.5 0.7 -- -0.3 Q -999 1.2 0 0 -- -0.3 -0.1 -- -0.2 R
-999 2.2 0.7 -1 -- 1 -0.9 -- 0.5 S -999 -0.3 0.2 0.7 -- -0.1 0.07
-- 1.1 T -999 0 0 -1 -- 0.8 -0.1 -- -0.5 V 0 2.1 0.5 0 -- 1.2 0.2
-- 0.3 W -1 -0.1 0 -1 -- -1.4 -0.6 -- -1 Y -1 0.9 0.8 -1 -- -1.4
-0.1 -- 0.3
TABLE-US-00043 TABLE 37 Pocket potential for HLA*0401B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 1.4 -- -1.1 -0.3 -- -1.7 E
-999 0.1 -1.2 1.5 -- -2.4 0.2 -- -1.7 F 0 0.8 0.8 -0.9 -- -1.1 -1
-- -1 G -999 0.5 0.2 -1.6 -- -1.5 -1.3 -- -1 H -999 0.8 0.2 1.1 --
-1.4 0 -- 0.08 I -1 1.1 1.5 0.8 -- -0.1 0.08 -- -0.3 K -999 1.1 0
-1.7 -- -2.4 -0.3 -- -0.3 L -1 1 1 0.8 -- -1.1 0.7 -- -1 M -1 1.1
1.4 0.9 -- -1.1 0.8 -- -0.4 N -999 0.8 0.5 0.9 -- 1.3 0.6 -- -1.4 P
-999 -0.5 0.3 -1.6 -- 0 -0.7 -- -1.3 Q -999 1.2 0 0.8 -- -1.5 0 --
0.5 R -999 2.2 0.7 -1.9 -- -2.4 -1.2 -- -1 S -999 -0.3 0.2 0.8 -- 1
-0.2 -- 0.7 T -999 0 0 0.7 -- 1.9 -0.1 -- -1.2 V -1 2.1 0.5 -0.9 --
0.9 0.08 -- -0.7 W 0 -0.1 0 -1.2 -- -1 -1.4 -- -1 Y 0 0.9 0.8 -1.6
-- -1.5 -1.2 -- -1
TABLE-US-00044 TABLE 38 Pocket potential for HLA*0701B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -1.6 -- -2.5 -1.3 -- -1.2 E
-999 0.1 -1.2 -1.4 -- -2.5 0.9 -- -0.3 F 0 0.8 0.8 0.2 -- -0.8 2.1
-- 2.1 G -999 0.5 0.2 -1.1 -- -0.6 0 -- -0.6 H -999 0.8 0.2 0.1 --
-0.8 0.9 -- -0.2 I -1 1.1 1.5 1.1 -- -0.5 2.4 -- 3.4 K -999 1.1 0
-1.3 -- -1.1 0.5 -- -1.1 L -1 1 1 -0.8 -- -0.9 2.2 -- 3.4 M -1 1.1
1.4 -0.4 -- -0.8 1.8 -- 2 N -999 0.8 0.5 -1.1 -- -0.6 1.4 -- -0.5 P
-999 -0.5 0.3 -1.2 -- -0.5 -0.2 -- -0.6 Q -999 1.2 0 -1.5 -- -1.1
1.1 -- -0.9 R -999 2.2 0.7 -1.1 -- -1.1 0.7 -- -0.8 S -999 -0.3 0.2
1.5 -- 0.6 0.4 -- -0.3 T -999 0 0 1.4 -- -0.1 0.9 -- 0.4 V -1 2.1
0.5 0.9 -- 0.1 1.6 -- 2 W 0 -0.1 0 -1.1 -- -0.9 1.4 -- 0.8 Y 0 0.9
0.8 -0.9 -- -1 1.7 -- 1.1
TABLE-US-00045 TABLE 39 Pocket potential for HLA*1501B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 0 0 0 -- 0 0 -- 0 C
-999 0 0 0 -- 0 0 -- 0 D -999 -1.3 -1.3 -0.4 -- -0.4 -0.7 -- -1.9 E
-999 0.1 -1.2 -0.6 -- -1 -0.7 -- -1.9 F -1 0.8 0.8 2.4 -- -0.3 1.4
-- -0.4 G -999 0.5 0.2 0 -- 0.5 0 -- -0.8 H -999 0.8 0.2 1.1 --
-0.5 0.6 -- -1.1 I 0 1.1 1.5 0.6 -- 0.05 1.5 -- 0.7 K -999 1.1 0
-0.7 -- -0.3 -0.3 -- -1.7 L 0 1 1 0.5 -- 0.2 1.9 -- 0.5 M 0 1.1 1.4
1 -- 0.1 1.7 -- 0.08 N -999 0.8 0.5 -0.2 -- 0.7 0.7 -- -1.2 P -999
-0.5 0.3 -0.3 -- -0.2 0.3 -- -1.1 Q -999 1.2 0 -0.8 -- -0.8 -0.3 --
-1.6 R -999 2.2 0.7 0.2 -- 1 -0.5 -- -1 S -999 -0.3 0.2 -0.3 -- 0.6
0.3 -- -0.3 T -999 0 0 -0.3 -- -0 0.2 -- -0.2 V 0 2.1 0.5 0.2 --
-0.3 0.3 -- 0.3 W -1 -0.1 0 0.4 -- -0.4 0.6 -- -1.4 Y -1 0.9 0.8
2.5 -- 0.4 0.7 -- -0.9
TABLE-US-00046 TABLE 40 Exemplary Biological Activity, Exemplary
Assays and Preferred Indications Biologically Exemplary Active
Biological Activity Preferred Protein Activity Assay Indication:
Factor IX Coagulation Factor IX Hemophilia (Coagulation factor IX
is a clotting B; bleeding; factor IX vitamin K- activity: Factor IX
(human); dependent Valder R. deficiency; Factor IX factor that et
al., 2001 Christmas Complex; circulates in "Posttranslational
disease; Christmas factor; the blood as modifications of bleeding
plasma an inactive recombinant episodes in thromboplastin zymogen.
myotube- patients with component Factor IX is synthesized factor
VIII (PTC); converted to human factor IX" inhibitor or prothrombin
an active form Blood 97:130- Factor VII complex by factor Xla, 138.
Activated deficiency concentrate which excises partial (PCC); the
activation thromboplastin Nonacog alpha; peptide and time: Rao LV,
MONONINE; thus generates Activation of ALPHANINE- a heavy chain
human factor SD; and a light VII during BEBULIN; chain held
clotting in vitro PROPLEX- together by Blood. 1985; T; KONYNE; one
or more 65(1):218-26; PROFILNINE disulfide Park CH, A SD; bonds. In
the diagnostic BeneFIX; blood challenge: mild IMMUNINE coagulation
hemophilia B VH) cascade, with normal activated activated partial
factor IX thromboplastin activates time. Blood factor X to Coagul
its active Fibrinolysis. form through 2010 Jun; interactions
21(4):368-71. with Ca+2 ions, membrane phospholipids, and factor
VIII. Alterations of this gene, including point mutations,
insertions and deletions, cause factor IX deficiency, which is a
recessive X- linked disorder, also called hemophilia B or Christmas
disease. Factor VII Coagulation Coagulation Bleeding (Coagulation
factor VII is Assay using Disorders; Factor VII; a vitamin K-
Prothrombin Coronary Active-site dependent Clotting Time
Restenosis; inactivated factor factor (Belaaouaj AA Hemophilia VII
essential for et al., J. Biol. A and B; (DEGR- hemostasis. Chem.
275: Liver VIIa/FFR- This factor 27123-8, 2000; Disorders; Vila);
Eptacog circulates in Diaz-Collier JA Thrombosis; alfa; the blood
in et al., Thromb Vascular Coagulation a zymogen Haemost 71:
Restenosis; Factor form, and is 339-46,1994). Surgery- Vila;
Novoseven; converted to related NiaStase; an active form
hemorrhagic Novostase; by either factor episodes MONOCLATE- IXa,
factor Xa, P) factor XIIa, or thrombin by minor proteolysis. Upon
activation of the factor VII, a heavy chain containing a catalytic
domain and a light chain containing 2 EGF-like domains are
generated, and two chains are held together by a disulfide bond. In
the presence of factor III and calcium ions, the activated factor
then further activates the coagulation cascade by converting factor
IX to factor IXa and/or factor X to factor Xa. Defects in this gene
can cause coagulopathy.
TABLE-US-00047 TABLE 41 Exemplary CFXTEN comprising CF and single
XTEN CFXTEN Name* Amino Acid Sequence DNA Nucleotide Sequence FVII-
ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AE288
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GTSESATPESGPGS
CCCGGTGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCC EPATSGSETPGTSE
CAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAG SATPESGPGSEPAT
GTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTA SGSETPGTSESATP
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC ESGPGTSTEPSEGS
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT APGSPAGSPTSTEE
GAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGC GTSESATPESGPGS
TCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCA EPATSGSETPGTSE
ACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCC SATPESGPGSPAGS
GGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTG PTSTEEGSPAGSPT
AGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCAC STEEGTSTEPSEGS
CGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGA APGTSESATPESGP
AGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACC GTSESATPESGPGT
AGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGG SESATPESGPGSEP
TACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACT ATSGSETPGSEPAT
TCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAA SGSETPGSPAGSPT
CCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCG STEEGTSTEPSEGS
GCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCT APGTSTEPSEGSAP
CTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTC GSEPATSGSETPGT
CGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGA SESATPESGPGTST
GGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTC EPSEGSAP
TGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCT
GGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCA CCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AE864 RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GSPAGSPTSTEEGT
CCCGGTGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGG SESATPESGPGTST
AAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGG EPSEGSAPGSPAGS
TACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAG PTSTEEGTSTEPSE
CCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCT GSAPGTSTEPSEGS
ACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT APGTSESATPESGP
GAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGC GSEPATSGSETPGS
GCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT EPATSGSETPGSPA
TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCG GSPTSTEEGTSESA
GTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGACCTC TPESGPGTSTEPSE
TACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTC GSAPGTSTEPSEGS
CGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGC APGSPAGSPTSTEE
ACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACC GTSTEPSEGSAPGT
AGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGG STEPSEGSAPGTSE
TACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC SATPESGPGTSTEP
CTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCT SEGSAPGTSESATP
GAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT ESGPGSEPATSGSE
GAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGC TPGTSTEPSEGSAP
GCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT GTSTEPSEGSAPGT
TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCG SESATPESGPGTSE
AAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTGAAG SATPESGPGSPAGS
GTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAAT PTSTEEGTSESATP
CCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG ESGPGSEPATSGSE
GCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAG TPGTSESATPESGP
AAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCAG GTSTEPSEGSAPGT
GTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTA STEPSEGSAPGTST
CCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTC EPSEGSAPGTSTEP
TACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACT SEGSAPGTSTEPSE
GAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAA GSAPGTSTEPSEGS
CCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTT APGSPAGSPTSTEE
CCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTG GTSTEPSEGSAPGT
AAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGG SESATPESGPGSEP
GTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCAC ATSGSETPGTSESA
CGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGC TPESGPGSEPATSG
ACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCC SETPGTSESATPES
AGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGG GPGTSTEPSEGSAP
TACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAG GTSESATPESGPGS
CGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT PAGSPTSTEEGSPA
GAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTG GSPTSTEEGSPAGS
AACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCG PTSTEEGTSESATP
CTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCC ESGPGTSTEPSEGS
GACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAAC APGTSESATPESGP
TTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGACCTCT GSEPATSGSETPGT
ACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCC SESATPESGPGSEP
GGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCA ATSGSETPGTSESA
CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA TPESGPGTSTEPSE
GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGT GSAPGSPAGSPTST
ACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGC EEGTSESATPESGP
GAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTG GSEPATSGSETPGT
AAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTGA SESATPESGPGSPA
ACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTC GSPTSTEEGSPAGS
TCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAAC PTSTEEGTSTEPSE
CCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGG GSAPGTSESATPES
TTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCCTGAG GPGTSESATPESGP
TCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCG GTSESATPESGPGS
AGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAG EPATSGSETPGSEP
AAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAG ATSGSETPGSPAGS
GTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTAC PTSTEEGTSTEPSE
TTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCT GSAPGTSTEPSEGS
GAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCG APGSEPATSGSETP
GCTACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTA GTSESATPESGPGT
CCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTCTCC STEPSEGSAP
GACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAA
GGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGC
AGCGCTCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAA
ACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCC
CAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AF864 RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GSTSESPSGTAPGT
CCCGGTGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTC SPSGESSTAPGSTS
CAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG ESPSGTAPGSTSES
TTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCT PSGTAPGTSTPESG
ACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTA SASPGTSTPESGSA
CTCCTGAAAGCGGTTCCGCTTCTCCAGGTACCTCTACTCC SPGSTSESPSGTAP
GGAAAGCGGTTCTGCATCTCCAGGTTCTACCAGCGAATCT GSTSESPSGTAPGT
CCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGT SPSGESSTAPGSTS
CTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTC ESPSGTAPGTSPSG
TACCGCACCAGGTTCTACTAGCGAATCTCCGTCTGGCACT ESSTAPGTSPSGES
GCTCCAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTC STAPGSTSSTAESP
CAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCTCCAGG GPGTSPSGESSTAP
TTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACC GTSPSGESSTAPGS
TCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTC TSSTAESPGPGTST
CGAGCGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTC PESGSASPGTSTPE
TACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAA SGSASPGSTSESPS
AGCGGCTCTGCATCTCCAGGTACTTCTACCCCTGAAAGCG GTAPGSTSESPSGT
GTTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGG APGTSTPESGSASP
CACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACT GSTSSTAESPGPGT
GCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTC STPESGSASPGSTS
CAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGG ESPSGTAPGTSPSG
TACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCT ESSTAPGSTSSTAE
ACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTC SPGPGTSPSGESST
CGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTC APGTSTPESGSASP
TACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGT GSTSSTAESPGPGS
GAATCTTCTACTGCTCCAGGTACCTCTACTCCTGAAAGCG TSSTAESPGPGSTS
GTTCTGCATCTCCAGGTTCCACTAGCTCTACCGCAGAATC STAESPGPGSTSST
TCCGGGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCT AESPGPGTSPSGES
GGCCCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTC STAPGSTSESPSGT
CAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG APGSTSESPSGTAP
TACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCT GTSTPESGPXXXG
ACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCA ASASGAPSTXXXX
GCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTACCCC SESPSGTAPGSTSE
TGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGCAAGCG SPSGTAPGSTSESP
GCGCGCCAAGCACGGGAXXXXXXXXTAGCGAATCTCCTT SGTAPGSTSESPSG
CTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGG TAPGSTSESPSGTA
TACTGCTCCAGGTTCTACCAGCGAATCTCCTTCTGGTACT PGSTSESPSGTAPG
GCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTC TSTPESGSASPGTS
CAGGTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG PSGESSTAPGTSPS
TTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTACT GESSTAPGSTSSTA
TCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTC ESPGPGTSPSGESS
CTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAG TAPGTSTPESGSAS
CGGCGAATCTTCTACTGCTCCAGGTTCTACCAGCTCTACT PGSTSESPSGTAPG
GCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGGTGAAT STSESPSGTAPGTS
CTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTC PSGESSTAPGSTSE
CGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACC SPSGTAPGTSTPES
GCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCAC GSASPGTSTPESGS
CAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGG ASPGSTSESPSGTA
TTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACT PGTSTPESGSASPG
TCTACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTA STSSTAESPGPGST
CCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCG SESPSGTAPGSTSE
AATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGA SPSGTAPGTSPSGE
AAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCT SSTAPGSTSSTAES
GAATCTCCGGGTCCAGGTTCTACCAGCGAATCTCCTTCTG PGPGTSPSGESSTA
GCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTAC PGTSTPESGSASPG
CGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCA TSPSGESSTAPGTS
CCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG PSGESSTAPGTSPS
GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTAC GESSTAPGSTSSTA
TTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTACCTCC ESPGPGSTSSTAES
CCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTA PGPGTSPSGESSTA
GCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGG
PGSSPSASTGTGPG TGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACTGCT
SSTPSGATGSPGSS GAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAAT TPSGATGSP
CTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTAC
TGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGC
CCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAG
GTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AG864 RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GASPGTSSTGSPGS
CCCGGTGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTC SPSASTGTGPGSSP
CAGGTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGG SASTGTGPGTPGS
TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGGTACC GTASSSPGSSTPSG
CCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTA ATGSPGSNPSASTG
CTCCGTCTGGTGCTACCGGCTCTCCAGGTTCTAACCCTTC TGPGASPGTSSTGS
TGCATCCACCGGTACCGGCCCAGGTGCTTCTCCGGGCACC PGTPGSGTASSSPG
AGCTCTACTGGTTCTCCAGGTACCCCGGGCAGCGGTACCG SSTPSGATGSPGTP
CATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCAACT GSGTASSSPGASPG
GGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTT TSSTGSPGASPGTS
CTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCC STGSPGTPGSGTAS
AGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGT SSPGSSTPSGATGS
ACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCT PGASPGTSSTGSPG
CTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCC TPGSGTASSSPGSS
GGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGTAG TPSGATGSPGSNPS
CGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTG ASTGTGPGSSPSAS
GTGCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCAC TGTGPGSSTPSGAT
CGGTACCGGCCCAGGTTCTAGCCCTTCTGCTTCCACCGGT GSPGSSTPSGATGS
ACTGGCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGGCT PGASPGTSSTGSPG
CCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCC ASPGTSSTGSPGAS
AGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGT PGTSSTGSPGTPGS
GCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTT GTASSSPGASPGTS
CTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGG STGSPGASPGTSST
CAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGT GSPGASPGTSSTGS
ACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCACTA PGSSPSASTGTGPG
GCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACTAGCTC TPGSGTASSSPGAS
TACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGT PGTSSTGSPGASPG
ACTGGCCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCT TSSTGSPGASPGTS
CTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCC STGSPGSSTPSGAT
AGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGT GSPGSSTPSGATGS
GCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTAGCT PGASPGTSSTGSPG
CTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTAC TPGSGTASSSPGSS
TCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGC TPSGATGSPGSSTP
ACCAGCTCTACCGGTTCTCCAGGTACCCCGGGCAGCGGT SGATGSPGSSTPSG
ACCGCATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTG ATGSPGSSPSASTG
CTACCGGTTCCCCAGGTAGCTCTACCCCGTCTGGTGCAAC TGPGASPGTSSTGS
CGGCTCCCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGC PGASPGTSSTGSPG
TCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCC TPGSGTASSSPGAS
CAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGG PGTSSTGSPGASPG
TGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACT TSSTGSPGASPGTS
CCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTCTC STGSPGASPGTSST
CGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGG GSPGTPGSGTASSS
CACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACT PGSSTPSGATGSPG
AGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCT TPGSGTASSSPGSS
CTACTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTC TPSGATGSPGTPGS
TTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGT GTASSSPGSSTPSG
TCTCCAGGTACCCCGGGTAGCGGTACCGCATCTTCTTCTC ATGSPGSSTPSGAT
CAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGG GSPGSSPSASTGTG
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGC PGSSPSASTGTGPG
TCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTAGCTCTA ASPGTSSTGSPGTP
CCCCGTCTGGTGCTACTGGCTCCCCAGGTTCTAGCCCTTC GSGTASSSPGSSTP
TGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGCA SGATGSPGSSPSAS
TCTACTGGTACTGGTCCAGGTGCATCCCCGGGCACTAGCT TGTGPGSSPSASTG
CTACCGGTTCTCCAGGTACTCCTGGTAGCGGTACTGCTTC TGPGASPGTSSTGS
TTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGTT PGASPGTSSTGSPG
CTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCC SSTPSGATGSPGSS
AGGTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGT PSASTGTGPGASPG
GCTTCTCCGGGTACTAGCTCTACTGGTTCTCCAGGTGCAT TSSTGSPGSSPSAS
CTCCTGGTACTAGCTCTACTGGTTCTCCAGGTAGCTCTAC TGTGPGTPGSGTA
TCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCT SSSPGSSTPSGATG
GCATCTACCGGTACTGGTCCAGGTGCATCCCCTGGTACCA SPGSSTPSGATGSP
GCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCTTCTACC GASPGTSSTGSP
GGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCATCTT
CCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTC
CCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCA
GGTGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AM875 RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GTSTEPSEGSAPGS
CCCGGTGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA EPATSGSETPGSPA
CCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA GSPTSTEEGSTSST
GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTT AESPGPGTSTPESG
CTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTC SASPGSTSESPSGT
TACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGC APGSTSESPSGTAP
GAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAAT GTSTPESGSASPGT
CCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAG STPESGSASPGSEP
CGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGT ATSGSETPGTSESA
TCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTG TPESGPGSPAGSPT
AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCG STEEGTSTEPSEGS
GCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGG APGTSESATPESGP
AAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG GTSTEPSEGSAPGT
GTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTA STEPSEGSAPGSPA
CTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC GSPTSTEEGTSTEP
TACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGC SEGSAPGTSTEPSE
AGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAA GSAPGTSESATPES
CCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTT GPGTSESATPESGP
CCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC GTSTEPSEGSAPGT
CTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGA STEPSEGSAPGTSE
ATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGC SATPESGPGTSTEP
GCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCA SEGSAPGSEPATSG
CCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA SETPGSPAGSPTST
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTA EEGSSTPSGATGSP
GCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCC GTPGSGTASSSPGS
GGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTAC STPSGATGSPGTST
CCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGC EPSEGSAPGTSTEP
GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGG SEGSAPGSEPATSG
TGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAG SETPGSPAGSPTST
GGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGT EEGSPAGSPTSTEE
AGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA GTSTEPSEGSAPGA
ACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGG SASGAPSTGGTSES
AAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAG ATPESGPGSPAGSP
GTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGC TSTEEGSPAGSPTS
AAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGA TEEGSTSSTAESPG
AAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGG PGSTSESPSGTAPG
CTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCT TSPSGESSTAPGTP
CCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTG GSGTASSSPGSSTP
AATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGG SGATGSPGSSPSAS
CACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACT TGTGPGSEPATSGS
GCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTC ETPGTSESATPESG
CAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG PGSEPATSGSETPG
TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCAGGTAGC STSSTAESPGPGST
GAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTG SSTAESPGPGTSPS
AAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGG GESSTAPGSEPATS
CTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTAC GSETPGSEPATSGS
TGCAGAATCTCCGGGCCCAGGTTCTACTAGCTCTACTGCA ETPGTSTEPSEGSA
GAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTT PGSTSSTAESPGPG
CTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTG TSTPESGSASPGST
AAACTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAA SESPSGTAPGTSTE
CCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGCGCACC PSEGSAPGTSTEPS
AGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGT EGSAPGTSTEPSEG
ACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTA SAPGSSTPSGATGS
CTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTAC PGSSPSASTGTGPG
CGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGA ASPGTSSTGSPGSE
ACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCT PATSGSETPGTSES
TCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTG ATPESGPGSPAGSP
CAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGG TSTEEGSSTPSGAT
TACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGT GSPGSSPSASTGTG
TCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCC PGASPGTSSTGSPG
CAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAG TSESATPESGPGTS
GTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAAGGTAG TEPSEGSAPGTSTE
CTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGC PSEGSAP
CCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGG
GCACCAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGC
TACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCT
GAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAA GGTAGCGCACCA FVII-
ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AM1318
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT GTSTEPSEGSAPGS
CCCGGTGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA EPATSGSETPGSPA
CCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA GSPTSTEEGSTSST
GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTT AESPGPGTSTPESG
CTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTC SASPGSTSESPSGT
TACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGC APGSTSESPSGTAP
GAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAAT GTSTPESGSASPGT
CCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAG STPESGSASPGSEP
CGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGT ATSGSETPGTSESA
TCTGCATCTCCAGGTAGCGAACCGGCAACCTCCGGCTCTG TPESGPGSPAGSPT
AAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCG STEEGTSTEPSEGS
GCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGG APGTSESATPESGP
AAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG GTSTEPSEGSAPGT
GTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTA STEPSEGSAPGSPA
CTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTC GSPTSTEEGTSTEP
TACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGC SEGSAPGTSTEPSE
AGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAA GSAPGTSESATPES
CCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTT
GPGTSESATPESGP CCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCC
GTSTEPSEGSAPGT CTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGA
STEPSEGSAPGTSE ATCCGGTCCAGGTACCTCTACTGAACCTTCCGAAGGCAGC
SATPESGPGTSTEP GCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCA
SEGSAPGSEPATSG CCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA
SETPGSPAGSPTST GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTA
EEGSSTPSGATGSP GCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCC
GTPGSGTASSSPGS GGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTAC
STPSGATGSPGTST CCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGC
EPSEGSAPGTSTEP GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGG
SEGSAPGSEPATSG TGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCGAG
SETPGSPAGSPTST GGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGT
EEGSPAGSPTSTEE AGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA
GTSTEPSEGSAPGP ACTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGG
EPTGPAPSGGSEPA AAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAG
TSGSETPGTSESAT GTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTCC
PESGPGSPAGSPTS AGAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCGAAC
TEEGTSESATPESG CGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAA
PGSPAGSPTSTEEG GCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCAGGTT
SPAGSPTSTEEGTS CTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGCGCTAC
ESATPESGPGSPAG TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT
SPTSTEEGSPAGSP TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA
TSTEEGSTSSTAES CTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCCG
PGPGSTSESPSGTA GCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGG
PGTSPSGESSTAPG AAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAG
STSESPSGTAPGST GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTC
SESPSGTAPGTSPS TACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCC
GESSTAPGTSTEPS CCTAGCGGTGAATCTTCTACTGCACCAGGTTCTACCAGCG
EGSAPGTSESATPE AATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATC
SGPGTSESATPESG CCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAA
PGSEPATSGSETPG TCTTCTACCGCACCAGGTACTTCTACCGAACCTTCCGAGG
TSESATPESGPGTS GCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGT
ESATPESGPGTSTE CCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGG
PSEGSAPGTSESAT TCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCC
PESGPGTSTEPSEG AGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGG
SAPGTSPSGESSTA TACTTCTGAAAGCGCTACTCCGGAATCCGGTCCAGGTACC
PGTSPSGESSTAPG TCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTG
TSPSGESSTAPGTS AAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACTG
TEPSEGSAPGSPAG AACCGTCCGAAGGTAGCGCACCAGGTACCTCCCCTAGCG
SPTSTEEGTSTEPS GCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGA
EGSAPGSSPSASTG ATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCT
TGPGSSTPSGATGS TCTACCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTA
PGSSTPSGATGSPG GCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCG
SSTPSGATGSPGSS AGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCAC
TPSGATGSPGASPG CAGGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGG
TSSTGSPGASASGA TAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGC
PSTGGTSPSGESST TCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTA
APGSTSSTAESPGP CCCCGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCC
GTSPSGESSTAPGT GTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGTACT
SESATPESGPGTST AGCTCTACCGGTTCTCCAGGTGCAAGCGCAAGCGGCGCG
EPSEGSAPGTSTEP CCAAGCACGGGAGGTACTTCTCCGAGCGGTGAATCTTCT
SEGSAPGSSPSAST ACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGG
GTGPGSSTPSGAT GCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCC
GSPGASPGTSSTGS AGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGG
PGTSTPESGSASPG TACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACT
TSPSGESSTAPGTS TCTACTGAACCGTCCGAAGGTAGCGCACCAGGTTCTAGC
PSGESSTAPGTSES CCTTCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACTC
ATPESGPGSEPATS CTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCCGGGTAC
GSETPGTSTEPSEG TAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAAAGC
SAPGSTSESPSGTA GGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT
PGSTSESPSGTAPG CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTAC
TSTPESGSASPGSP TGCTCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT
AGSPTSTEEGTSES CCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA
ATPESGPGTSTEPS GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGT
EGSAPGSPAGSPTS TCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTA
TEEGTSESATPESG CCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTAC
PGSEPATSGSETPG CCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCCCGGCAGG
SSTPSGATGSPGAS CTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA
PGTSSTGSPGSSTP ACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCT
SGATGSPGSTSESP GAGGGCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACC
SGTAPGTSPSGESS TCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA
TAPGSTSSTAESPG TCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAA
PGSSTPSGATGSPG ACCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCC
ASPGTSSTGSPGTP CAGGTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG
GSGTASSSPGSPAG TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCT
SPTSTEEGSPAGSP ACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCCC
TSTEEGTSTEPSEG CTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCAGCTC SAP
TACCGCAGAATCTCCGGGTCCAGGTAGCTCTACCCCTTCT
GGTGCAACCGGCTCTCCAGGTGCATCCCCGGGTACCAGC
TCTACCGGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTT
CTTCCTCTCCAGGTAGCCCTGCTGGCTCTCCGACTTCTACT
GAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAG
GAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AE288 LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTACCTCTGA TSESATPESGPGSE
AAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGC PATSGSETPGTSES
TACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCA ATPESGPGSEPATS
ACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCT GSETPGTSESATPE
GGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTG SGPGTSTEPSEGSA
AATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCA PGSPAGSPTSTEEG
GCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA TSESATPESGPGSE
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC PATSGSETPGTSES
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG ATPESGPGSPAGSP
TACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGC TSTEEGSPAGSPTS
CCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCG TEEGTSTEPSEGSA
GCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCG PGTSESATPESGPG
AACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCG TSESATPESGPGTS
CTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTAC ESATPESGPGSEPA
TCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCCG TSGSETPGSEPATS
GAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTG GSETPGSPAGSPTS
AAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAA TEEGTSTEPSEGSA
CTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGG PGTSTEPSEGSAPG
AAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAG SEPATSGSETPGTS
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAG ESATPESGPGTSTE
CGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCT PSEGSAP
GAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACTG AACCGTCCGAGGGCAGCGCACCA
FIX- YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AE864
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTAGCCCGGC SPAGSPTSTEEGTS
TGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGC ESATPESGPGTSTE
GCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGT PSEGSAPGSPAGSP
CCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCTCCGA TSTEEGTSTEPSEG
CTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGG SAPGTSTEPSEGSA
CAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAG PGTSESATPESGPG
CGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAATCTGGC SEPATSGSETPGSE
CCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA PATSGSETPGSPAG
GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGT SPTSTEEGTSESAT
AGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT PESGPGTSTEPSEG
TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCT SAPGTSTEPSEGSA
ACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACC PGSPAGSPTSTEEG
GAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGT TSTEPSEGSAPGTS
TCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGT TEPSEGSAPGTSES
CCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTCTG ATPESGPGTSTEPS
AGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG EGSAPGTSESATPE
AGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTA SGPGSEPATSGSET
GCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG PGTSTEPSEGSAPG
GTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCC TSTEPSEGSAPGTS
AGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGG ESATPESGPGTSES
TACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACT ATPESGPGSPAGSP
TCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACCTCT TSTEEGTSESATPE
GAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCT SGPGSEPATSGSET
GGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGC PGTSESATPESGPG
GCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACC TSTEPSEGSAPGTS
TCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTC TEPSEGSAPGTSTE
CGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGG PSEGSAPGTSTEPS
GTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTA EGSAPGTSTEPSEG
GCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCG SAPGTSTEPSEGSA
CTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC PGSPAGSPTSTEEG
AGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGG TSTEPSEGSAPGTS
TACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAG ESATPESGPGSEPA
CCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCT TSGSETPGTSESAT
ACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAA PESGPGSEPATSGS
AGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCT ETPGTSESATPESG
ACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCA PGTSTEPSEGSAPG
ACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTCT TSESATPESGPGSP
GGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTG AGSPTSTEEGSPAG
AATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCA SPTSTEEGSPAGSP
GCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCCG TSTEEGTSESATPE
GCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGG SGPGTSTEPSEGSA
AAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAG PGTSESATPESGPG
GTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTA SEPATSGSETPGTS
CTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCT ESATPESGPGSEPA
CTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTG TSGSETPGTSESAT
AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG PESGPGTSTEPSEG
CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC SAPGSPAGSPTSTE
AACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC EGTSESATPESGPG
TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT SEPATSGSETPGTS
GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC ESATPESGPGSPAG
AGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG SPTSTEEGSPAGSP
AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC TSTEEGTSTEPSEG
CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG SAPGTSESATPESG
GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG PGTSESATPESGPG
CCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC TSESATPESGPGSE
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC PATSGSETPGSEPA
GAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGC TSGSETPGSPAGSP
GCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTA TSTEEGTSTEPSEG
CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC SAPGTSTEPSEGSA
GGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT PGSEPATSGSETPG
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA TSESATPESGPGTS
ACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAG TEPSEGSAP
GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCACCA
FIX- YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AF864
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA
QSTQSFNDFTRVV GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTTCTACCAG STSESPSGTAPGTS
CGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGC PSGESSTAPGSTSE
GGCGAATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTC SPSGTAPGSTSESP
CTTCTGGCACTGCACCAGGTTCTACTAGCGAATCCCCGTC SGTAPGTSTPESGS
TGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCC ASPGTSTPESGSAS
GCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCAT PGSTSESPSGTAPG
CTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCC STSESPSGTAPGTS
AGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGT PSGESSTAPGSTSE
ACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCTA SPSGTAPGTSPSGE
CTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTTCTCC SSTAPGTSPSGESS
TAGCGGTGAATCTTCTACCGCTCCAGGTACTTCCCCTAGC TAPGSTSSTAESPG
GGCGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACTG PGTSPSGESSTAPG
CAGAATCTCCGGGCCCAGGTACCTCTCCTAGCGGTGAATC TSPSGESSTAPGST
TTCTACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCT SSTAESPGPGTSTP
ACCGCTCCAGGTTCTACTAGCTCTACTGCAGAATCTCCTG ESGSASPGTSTPES
GCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCC GSASPGSTSESPSG
AGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGT TAPGSTSESPSGTA
TCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTA PGTSTPESGSASPG
CCAGCGAATCTCCGTCTGGCACTGCACCAGGTACCTCTAC STSSTAESPGPGTS
CCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCTCT TPESGSASPGSTSE
ACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAA SPSGTAPGTSPSGE
GCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTC SSTAPGSTSSTAES
TGGCACTGCACCAGGTACTTCTCCGAGCGGTGAATCTTCT PGPGTSPSGESSTA
ACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGG PGTSTPESGSASPG
GCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCC STSSTAESPGPGST
AGGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGT SSTAESPGPGSTSS
TCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTA TAESPGPGSTSSTA
CTAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTACTAG ESPGPGTSPSGESS
CTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCT TAPGSTSESPSGTA
ACTGCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTG PGSTSESPSGTAPG
AATCTTCTACTGCACCAGGTTCTACTAGCGAATCTCCTTC TSTPESGPXXXGA
TGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGC SASGAPSTXXXXS
ACTGCACCAGGTACCTCTACCCCTGAAAGCGGTCCXXXX ESPSGTAPGSTSES
XXXXXXXXTGCAAGCGCAAGCGGCGCGCCAAGCACGGG PSGTAPGSTSESPS
AXXXXXXXXTAGCGAATCTCCTTCTGGTACCGCTCCAGG GTAPGSTSESPSGT
TTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCT APGSTSESPSGTAP
ACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTA GSTSESPSGTAPGT
GCGAATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGA STPESGSASPGTSP
ATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCT SGESSTAPGTSPSG
CCTTCTGGTACTGCACCAGGTACTTCTACTCCGGAAAGCG ESSTAPGSTSSTAE
GTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTTC SPGPGTSPSGESST
TACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACT APGTSTPESGSASP
GCTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTC GSTSESPSGTAPGS
CAGGTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGG TSESPSGTAPGTSP
TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTTCT SGESSTAPGSTSES
ACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTA PSGTAPGTSTPESG
GCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG SASPGTSTPESGSA
CGGCGAATCTTCTACCGCACCAGGTTCTACTAGCGAATCC SPGSTSESPSGTAP
CCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGC GTSTPESGSASPGS
GGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCT TSSTAESPGPGSTS
CCGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTAC ESPSGTAPGSTSES
CGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT PSGTAPGTSPSGES
CCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAG STAPGSTSSTAESP
GTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTC GPGTSPSGESSTAP
TACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCT GTSTPESGSASPGT
CCTAGCGGCGAATCTTCTACCGCACCAGGTTCTACCAGCT SPSGESSTAPGTSP
CTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAGCGG SGESSTAPGTSPSG
TGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGC ESSTAPGSTSSTAE
GGTTCCGCTTCTCCAGGTACCTCCCCTAGCGGCGAATCTT SPGPGSTSSTAESP
CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTAC GPGTSPSGESSTAP
CGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA GSSPSASTGTGPGS
CCAGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG STPSGATGSPGSST
GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGTAC PSGATGSP
CTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTAGC
CCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTC
CGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTC TGGTGCAACCGGCTCCCCA FIX-
YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AG864
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTGCTTCCCC ASPGTSSTGSPGSS
GGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCT PSASTGTGPGSSPS
GCTTCTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTC ASTGTGPGTPGSG
CACTGGTACTGGTCCAGGTACCCCGGGTAGCGGTACCGC TASSSPGSSTPSGA
TTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCG TGSPGSNPSASTGT
GCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGG GPGASPGTSSTGSP
CCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA GTPGSGTASSSPGS
GGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTA STPSGATGSPGTPG
GCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTACTCC SGTASSSPGASPGT
TGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCT SSTGSPGASPGTSS
GGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCA TGSPGTPGSGTASS
CTAGCTCTACTGGTTCTCCAGGTACCCCGGGTAGCGGTAC SPGSSTPSGATGSP
TGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAA GASPGTSSTGSPGT
CCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGG PGSGTASSSPGSST
TTCTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCT PSGATGSPGSNPSA
CCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG STGTGPGSSPSAST
GTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTC GTGPGSSTPSGAT
TAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCT GSPGSSTPSGATGS
ACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTC PGASPGTSSTGSPG
CTTCTGGTGCAACTGGCTCTCCAGGTGCATCTCCGGGCAC ASPGTSSTGSPGAS
TAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGC PGTSSTGSPGTPGS
TCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTA GTASSSPGASPGTS
CTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTC STGSPGASPGTSST
TTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTC GSPGASPGTSSTGS
CAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGG PGSSPSASTGTGPG
TGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCT TPGSGTASSSPGAS
AGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGG PGTSSTGSPGASPG
GCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGG TSSTGSPGASPGTS
CACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACT STGSPGSSTPSGAT
AGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCT GSPGSSTPSGATGS
CTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAAC PGASPGTSSTGSPG
CGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGC TPGSGTASSSPGSS
TCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTC TPSGATGSPGSSTP
CAGGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGG SGATGSPGSSTPSG
TAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGC ATGSPGSSPSASTG
TCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTA TGPGASPGTSSTGS
CTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTC PGASPGTSSTGSPG
TGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACC TPGSGTASSSPGAS
AGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCT PGTSSTGSPGASPG
CTACCGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATC TSSTGSPGASPGTS
TTCCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGT STGSPGASPGTSST
TCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTC GSPGTPGSGTASSS
CAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG PGSSTPSGATGSPG
TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACC TPGSGTASSSPGSS
CCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTA TPSGATGSPGTPGS
CTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTAG GTASSSPGSSTPSG
CGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCT ATGSPGSSTPSGAT
GGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG GSPGSSPSASTGTG
CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACT PGSSPSASTGTGPG
GGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCT ASPGTSSTGSPGTP
CCCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCC GSGTASSSPGSSTP
AGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGT SGATGSPGSSPSAS
GCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTC TGTGPGSSPSASTG
CTGGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTAC TGPGASPGTSSTGS
TCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTG PGASPGTSSTGSPG
CATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTC SSTPSGATGSPGSS
TACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCT PSASTGTGPGASPG
ACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTG TSSTGSPGSSPSAS
GTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTC TGTGPGTPGSGTA
TCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA SSSPGSSTPSGATG
GGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTT SPGSSTPSGATGSP
CTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCC GASPGTSSTGSP
TGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACT
CCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTT
CTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAG CTCTACCGGTTCTCCA FIX-
YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AM875
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTACTTCTAC TSTEPSEGSAPGSE
TGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGC PATSGSETPGSPAG
TACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCT SPTSTEEGSTSSTA
CCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAG ESPGPGTSTPESGS
AATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTC ASPGSTSESPSGTA
TGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACT PGSTSESPSGTAPG
GCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTC TSTPESGSASPGTS
CAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG TPESGSASPGSEPA
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGC TSGSETPGTSESAT
GAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCT PESGPGSPAGSPTS
GAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCA TEEGTSTEPSEGSA
GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAAC PGTSESATPESGPG
CTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTAC TSTEPSEGSAPGTS
CCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAA TEPSEGSAPGSPAG
GGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGT SPTSTEEGTSTEPS
AGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACC EGSAPGTSTEPSEG
GAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCA SAPGTSESATPESG
CCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA PGTSESATPESGPG
GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGT TSTEPSEGSAPGTS
ACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCT TEPSEGSAPGTSES
CTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTAC ATPESGPGTSTEPS
CGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAG EGSAPGSEPATSGS
CGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCT ETPGSPAGSPTSTE
TCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTG EGSSTPSGATGSPG
GTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTC TPGSGTASSSPGSS
CACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGT TPSGATGSPGTSTE
TCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTC PSEGSAPGTSTEPS
CAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGG EGSAPGSEPATSGS
TACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC ETPGSPAGSPTSTE
CTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGA EGSPAGSPTSTEEG
ACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCT TSTEPSEGSAPGAS
GGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTT ASGAPSTGGTSES
CTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC ATPESGPGSPAGSP
CGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCC TSTEEGSPAGSPTS
AAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTC TEEGSTSSTAESPG
CGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGA
PGSTSESPSGTAPG GGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA
TSPSGESSTAPGTP AGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGT
GSGTASSSPGSSTP TCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTT
SGATGSPGSSPSAS CCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGG
TGTGPGSEPATSGS CAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCG
ETPGTSESATPESG TCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCAT
PGSEPATSGSETPG CTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCG
STSSTAESPGPGST GCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGA
SSTAESPGPGTSPS ATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGA
GESSTAPGSEPATS AACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGC
GSETPGSEPATSGS CCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG
ETPGTSTEPSEGSA GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAG
PGSTSSTAESPGPG CGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGA
TSTPESGSASPGST ACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT
SESPSGTAPGTSTE GAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCT
PSEGSAPGTSTEPS ACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAA
EGSAPGTSTEPSEG GCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTC
SAPGSSTPSGATGS TGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGG
PGSSPSASTGTGPG CAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAG
ASPGTSSTGSPGSE CGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCA
PATSGSETPGTSES CCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAG
ATPESGPGSPAGSP GTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGC
TSTEEGSSTPSGAT TTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAA
GSPGSSPSASTGTG CCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAA
PGASPGTSSTGSPG GCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTT
TSESATPESGPGTS CTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGG
TEPSEGSAPGTSTE TGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACT PSEGSAP
GGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTG
GTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGG
CCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AG864 LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTGCTTCCCC ASPGTSSTGSPGSS
GGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCT PSASTGTGPGSSPS
GCTTCTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTC ASTGTGPGTPGSG
CACTGGTACTGGTCCAGGTACCCCGGGTAGCGGTACCGC TASSSPGSSTPSGA
TTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCG TGSPGSNPSASTGT
GCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGG GPGASPGTSSTGSP
CCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA GTPGSGTASSSPGS
GGTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGGTA STPSGATGSPGTPG
GCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTACTCC SGTASSSPGASPGT
TGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCT SSTGSPGASPGTSS
GGTACTAGCTCTACTGGTTCTCCAGGTGCTTCTCCGGGCA TGSPGTPGSGTASS
CTAGCTCTACTGGTTCTCCAGGTACCCCGGGTAGCGGTAC SPGSSTPSGATGSP
TGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAA GASPGTSSTGSPGT
CCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGG PGSGTASSSPGSST
TTCTCCAGGTACCCCGGGTAGCGGTACCGCTTCTTCTTCT PSGATGSPGSNPSA
CCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG STGTGPGSSPSAST
GTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTC GTGPGSSTPSGAT
TAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCT GSPGSSTPSGATGS
ACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCTACTC PGASPGTSSTGSPG
CTTCTGGTGCAACTGGCTCTCCAGGTGCATCTCCGGGCAC ASPGTSSTGSPGAS
TAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGC PGTSSTGSPGTPGS
TCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCTCTA GTASSSPGASPGTS
CTGGTTCTCCAGGTACTCCTGGCAGCGGTACCGCTTCTTC STGSPGASPGTSST
TTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTC GSPGASPGTSSTGS
CAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGG PGSSPSASTGTGPG
TGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTCT TPGSGTASSSPGAS
AGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGG PGTSSTGSPGASPG
GCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGG TSSTGSPGASPGTS
CACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACT STGSPGSSTPSGAT
AGCTCTACTGGTTCTCCAGGTGCTTCTCCTGGTACCAGCT GSPGSSTPSGATGS
CTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAAC PGASPGTSSTGSPG
CGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGC TPGSGTASSSPGSS
TCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTTCTC TPSGATGSPGSSTP
CAGGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGG SGATGSPGSSTPSG
TAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGC ATGSPGSSPSASTG
TCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTA TGPGASPGTSSTGS
CTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTC PGASPGTSSTGSPG
TGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACC TPGSGTASSSPGAS
AGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCT PGTSSTGSPGASPG
CTACCGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATC TSSTGSPGASPGTS
TTCCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACTGGT STGSPGASPGTSST
TCTCCAGGTGCATCTCCGGGCACTAGCTCTACTGGTTCTC GSPGTPGSGTASSS
CAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG PGSSTPSGATGSPG
TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACC TPGSGTASSSPGSS
CCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTA TPSGATGSPGTPGS
CTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTAG GTASSSPGSSTPSG
CGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCT ATGSPGSSTPSGAT
GGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG GSPGSSPSASTGTG
CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACT PGSSPSASTGTGPG
GGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCT ASPGTSSTGSPGTP
CCCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCC GSGTASSSPGSSTP
AGGTTCTAGCCCGTCTGCATCTACTGGTACTGGTCCAGGT SGATGSPGSSPSAS
GCATCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTACTC TGTGPGSSPSASTG
CTGGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTAC TGPGASPGTSSTGS
TCCTTCTGGTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTG PGASPGTSSTGSPG
CATCCACCGGTACCGGCCCAGGTTCTAGCCCGTCTGCTTC SSTPSGATGSPGSS
TACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGCTCT PSASTGTGPGASPG
ACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTG TSSTGSPGSSPSAS
GTTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTC TGTGPGTPGSGTA
TCCAGGTTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA SSSPGSSTPSGATG
GGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTT SPGSSTPSGATGSP
CTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCC GASPGTSSTGSP
TGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGCTCTACT
CCGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTT
CTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAG CTCTACCGGTTCTCCA FIX-
YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AM875
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTTTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTACTTCTAC TSTEPSEGSAPGSE
TGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGC PATSGSETPGSPAG
TACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCT SPTSTEEGSTSSTA
CCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAG ESPGPGTSTPESGS
AATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTC ASPGSTSESPSGTA
TGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACT PGSTSESPSGTAPG
GCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTC TSTPESGSASPGTS
CAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG TPESGSASPGSEPA
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGC TSGSETPGTSESAT
GAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCT PESGPGSPAGSPTS
GAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCA TEEGTSTEPSEGSA
GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAAC PGTSESATPESGPG
CTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTAC TSTEPSEGSAPGTS
CCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAA TEPSEGSAPGSPAG
GGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGT SPTSTEEGTSTEPS
AGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACC EGSAPGTSTEPSEG
GAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCA SAPGTSESATPESG
CCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA PGTSESATPESGPG
GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGT TSTEPSEGSAPGTS
ACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCT TEPSEGSAPGTSES
CTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTAC ATPESGPGTSTEPS
CGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAG EGSAPGSEPATSGS
CGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCT ETPGSPAGSPTSTE
TCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTG EGSSTPSGATGSPG
GTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTC TPGSGTASSSPGSS
CACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGT TPSGATGSPGTSTE
TCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTC PSEGSAPGTSTEPS
CAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGG EGSAPGSEPATSGS
TACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC ETPGSPAGSPTSTE
CTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGA EGSPAGSPTSTEEG
ACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCT TSTEPSEGSAPGAS
GGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTT ASGAPSTGGTSES
CTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC ATPESGPGSPAGSP
CGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGCC TSTEEGSPAGSPTS
AAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTC TEEGSTSSTAESPG
CGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGA PGSTSESPSGTAPG
GGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA TSPSGESSTAPGTP
AGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGT GSGTASSSPGSSTP
TCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTT SGATGSPGSSPSAS
CCCCTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGG TGTGPGSEPATSGS
CAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTACCCCG ETPGTSESATPESG
TCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCAT PGSEPATSGSETPG
CTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCG STSSTAESPGPGST
GCTCTGAAACTCCAGGTACTTCTGAAAGCGCTACTCCGGA SSTAESPGPGTSPS
ATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGA GESSTAPGSEPATS
AACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGC GSETPGSEPATSGS
CCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG ETPGTSTEPSEGSA
GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTAG PGSTSSTAESPGPG
CGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGA TSTPESGSASPGST
ACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT SESPSGTAPGTSTE
GAACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCT PSEGSAPGTSTEPS
ACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAA EGSAPGTSTEPSEG
GCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTC SAPGSSTPSGATGS
TGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGG PGSSPSASTGTGPG
CAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAG ASPGTSSTGSPGSE
CGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCA PATSGSETPGTSES
CCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAG ATPESGPGSPAGSP
GTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGC TSTEEGSSTPSGAT
TTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTAGCGAA GSPGSSPSASTGTG
CCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAA PGASPGTSSTGSPG
GCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTT TSESATPESGPGTS
CTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGG TEPSEGSAPGTSTE
TGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACT PSEGSAP
GGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTG
GTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGG
CCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC AM1318 LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC
TPICIADKEYTNIFL ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG
KFGSGYVSGWGR GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGG
TTAAGGAAAAAACAAAGCTCACTGGGGTGGTACTTCTAC TSTEPSEGSAPGSE
TGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGC PATSGSETPGSPAG
TACTTCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCT SPTSTEEGSTSSTA
CCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCAG ESPGPGTSTPESGS
AATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTC ASPGSTSESPSGTA
TGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACT PGSTSESPSGTAPG
GCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTC TSTPESGSASPGTS
CAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG TPESGSASPGSEPA
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGC TSGSETPGTSESAT
GAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCT PESGPGSPAGSPTS
GAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGCA TEEGTSTEPSEGSA
GGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAAC PGTSESATPESGPG
CTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTAC TSTEPSEGSAPGTS
CCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAA TEPSEGSAPGSPAG
GGTAGCGCACCAGGTACTTCTACCGAACCGTCCGAGGGT SPTSTEEGTSTEPS
AGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACC EGSAPGTSTEPSEG
GAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCA SAPGTSESATPESG
CCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA PGTSESATPESGPG
GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGT TSTEPSEGSAPGTS
ACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCT TEPSEGSAPGTSES
CTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTAC ATPESGPGTSTEPS
CGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAG EGSAPGSEPATSGS
CGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCT ETPGSPAGSPTSTE
TCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTG EGSSTPSGATGSPG
GTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTC TPGSGTASSSPGSS
CACCGAGGAAGGTAGCTCTACCCCGTCTGGTGCTACTGGT TPSGATGSPGTSTE
TCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTC PSEGSAPGTSTEPS
CAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGG EGSAPGSEPATSGS
TACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAC ETPGSPAGSPTSTE
CTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGA EGSPAGSPTSTEEG
ACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCT TSTEPSEGSAPGPE
GGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTT PTGPAPSGGSEPAT
CTCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTC SGSETPGTSESATP
CGAAGGTAGCGCTCCAGGTCCAGAACCAACGGGGCCGGC ESGPGSPAGSPTST
CCCAAGCGGAGGTAGCGAACCGGCAACCTCCGGCTCTGA EEGTSESATPESGP
AACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGG GSPAGSPTSTEEGS
CCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGA PAGSPTSTEEGTSE
AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGT SATPESGPGSPAGS
AGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC PTSTEEGSPAGSPT
CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTG STEEGSTSSTAESP
AAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTG GPGSTSESPSGTAP
GCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCT GTSPSGESSTAPGS
CTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGC TSESPSGTAPGSTS
TGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCT ESPSGTAPGTSPSG
GGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTA ESSTAPGTSTEPSE
CTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGC GSAPGTSESATPES
TCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA GPGTSESATPESGP
GGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTA GSEPATSGSETPGT
CTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTC SESATPESGPGTSE
TGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGA SATPESGPGTSTEP
AAGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACCGGC SEGSAPGTSESATP
AACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCT ESGPGTSTEPSEGS
ACTCCGGAATCTGGTCCAGGTACTTCTGAAAGCGCTACTC APGTSPSGESSTAP
CGGAATCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGG GTSPSGESSTAPGT
CAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTC SPSGESSTAPGTST
CGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGC EPSEGSAPGSPAGS
ACCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCA PTSTEEGTSTEPSE
GGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTA GSAPGSSPSASTGT
CCTCCCCTAGCGGTGAATCTTCTACCGCACCAGGTACTTC GPGSSTPSGATGSP
TACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGC GSSTPSGATGSPGS
AGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAA STPSGATGSPGSST
CCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTT PSGATGSPGASPGT
CCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTGGTGC SSTGSPGASASGAP
AACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACC STGGTSPSGESSTA
GGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCT PGSTSSTAESPGPG
CTCCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCC TSPSGESSTAPGTS
AGGTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCAGGT ESATPESGPGTSTE
GCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCT PSEGSAPGTSTEPS
CCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCT EGSAPGSSPSASTG
CTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGG TGPGSSTPSGATGS
TGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACT PGASPGTSSTGSPG
CCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAG TSTPESGSASPGTS
GGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGT PSGESSTAPGTSPS
AGCGCACCAGGTTCTAGCCCTTCTGCATCTACTGGTACTG GESSTAPGTSESAT
GCCCAGGTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCC PESGPGSEPATSGS
AGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT ETPGTSTEPSEGSA
ACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTT PGSTSESPSGTAPG
CTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCC STSESPSGTAPGTS
TAGCGGCGAATCTTCTACTGCTCCAGGTACTTCTGAAAGC TPESGSASPGSPAG
GCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT SPTSTEEGTSESAT
TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCG PESGPGTSTEPSEG
AAGGTAGCGCACCAGGTTCTACCAGCGAATCCCCTTCTG SAPGSPAGSPTSTE
GTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGGCAC EGTSESATPESGPG
CGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT SEPATSGSETPGSS
CCAGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA TPSGATGSPGASPG
GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGT TSSTGSPGSSTPSG
ACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGC ATGSPGSTSESPSG
CCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTG TAPGTSPSGESSTA
AAAGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGG PGSTSSTAESPGPG
CAACCTCCGGTTCTGAAACCCCAGGTAGCTCTACCCCGTC SSTPSGATGSPGAS
TGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGC PGTSSTGSPGTPGS
TCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTA GTASSSPGSPAGSP
CTGGCTCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTAC TSTEEGSPAGSPTS
TGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCT TEEGTSTEPSEGSAP
CCAGGTTCTACCAGCTCTACCGCAGAATCTCCGGGTCCAG
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGC
ATCCCCGGGTACCAGCTCTACCGGTTCTCCAGGTACTCCG
GGTAGCGGTACCGCTTCTTCCTCTCCAGGTAGCCCTGCTG
GCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTC
TCCGACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCC GAAGGTAGCGCTCCA *Sequence
name reflects N- to C-terminus configuration of the coagulation
factor and XTEN components
TABLE-US-00048 TABLE 42 Exemplary CFXTEN comprising CF, cleavage
sequences and XTEN sequences CFXTEN Name* Amino Acid Sequence DNA
Nucleotide Sequence FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FXIa- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE288 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT KLTRAETGGTSES
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT ATPESGPGSEPATS
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT GSETPGTSESATPE
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA SGPGSEPATSGSET
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT PGTSESATPESGPG
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT TSTEPSEGSAPGSP
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG AGSPTSTEEGTSES
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA ATPESGPGSEPATS
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA GSETPGTSESATPE
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC SGPGSPAGSPTSTE
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EGSPAGSPTSTEEG
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA TSTEPSEGSAPGTS
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG ESATPESGPGTSES
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC ATPESGPGTSESAT
AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT PESGPGSEPATSGS
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC ETPGSEPATSGSET
GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA PGSPAGSPTSTEEG
CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA TSTEPSEGSAPGTS
GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC TEPSEGSAPGSEPA
CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC TSGSETPGTSESAT
TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG PESGPGTSTEPSEG
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA SAP
TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FXIa- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE864 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT KLTRAETGGSPAG
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT SPTSTEEGTSESAT
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT PESGPGTSTEPSEG
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA SAPGSPAGSPTSTE
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT EGTSTEPSEGSAPG
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT TSTEPSEGSAPGTS
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA ESATPESGPGSEPA
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC TSGSETPGSEPATS
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC GSETPGSPAGSPTS
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA TEEGTSESATPESG
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG PGTSTEPSEGSAPG
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA TSTEPSEGSAPGSP
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG AGSPTSTEEGTSTE
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG PSEGSAPGTSTEPS
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG EGSAPGTSESATPE
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC SGPGTSTEPSEGSA
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT PGTSESATPESGPG
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA SEPATSGSETPGTS
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT TEPSEGSAPGTSTE
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT PSEGSAPGTSESAT
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG PESGPGTSESATPE
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG SGPGSPAGSPTSTE
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT EGTSESATPESGPG
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA SEPATSGSETPGTS
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC ESATPESGPGTSTE
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG PSEGSAPGTSTEPS
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC EGSAPGTSTEPSEG
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA SAPGTSTEPSEGSA
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG PGTSTEPSEGSAPG
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC TSTEPSEGSAPGSP
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC AGSPTSTEEGTSTE
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA PSEGSAPGTSESAT
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC PESGPGSEPATSGS
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG ETPGTSESATPESG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG PGSEPATSGSETPG
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA TSESATPESGPGTS
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT TEPSEGSAPGTSES
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC ATPESGPGSPAGSP
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA TSTEEGSPAGSPTS
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA TEEGSPAGSPTSTE
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC EGTSESATPESGPG
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC TSTEPSEGSAPGTS
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC ESATPESGPGSEPA
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA TSGSETPGTSESAT
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG PESGPGSEPATSGS
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG ETPGTSESATPESG
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA PGTSTEPSEGSAPG
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT SPAGSPTSTEEGTS
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC ESATPESGPGSEPA
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA TSGSETPGTSESAT
CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG PESGPGSPAGSPTS
GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG TEEGSPAGSPTSTE
CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT EGTSTEPSEGSAPG
CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC TSESATPESGPGTS
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC ESATPESGPGTSES
ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT ATPESGPGSEPATS
GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA GSETPGSEPATSGS
CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA ETPGSPAGSPTSTE
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA EGTSTEPSEGSAPG
CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG TSTEPSEGSAPGSE
AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC PATSGSETPGTSES
CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG ATPESGPGTSTEPS
GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC EGSAP
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FXIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE288 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGK
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTACCT LTRAETGGTSESA
CTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAAC TPESGPGSEPATSG
CTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAG SETPGTSESATPES
CGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC GPGSEPATSGSETP
CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT GTSESATPESGPGT
CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGG STEPSEGSAPGSPA
GCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCAC GSPTSTEEGTSESA
CGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGG TPESGPGSEPATSG
CCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC SETPGTSESATPES
AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGT GPGSPAGSPTSTEE
AGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC GSPAGSPTSTEEGT
CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTA STEPSEGSAPGTSE
CCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA SATPESGPGTSESA
GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCG TPESGPGTSESATP
CTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC ESGPGSEPATSGSE
CCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGG TPGSEPATSGSETP
TTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCT GSPAGSPTSTEEGT
GAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT STEPSEGSAPGTST
GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA EPSEGSAPGSEPAT
CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG SGSETPGTSESATP
GTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA ESGPGTSTEPSEGS
CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC AP
TACTGAACCGTCCGAGGGCAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FXIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE864 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGK
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTAGCC LTRAETGGSPAGS
CGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA PTSTEEGTSESATP
AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA ESGPGTSTEPSEGS
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCT
APGSPAGSPTSTEE CCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG
GTSTEPSEGSAPGT AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG
STEPSEGSAPGTSE GCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAAT
SATPESGPGSEPAT CTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAAC
SGSETPGSEPATSG CCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC
SETPGSPAGSPTST AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGG
EEGTSESATPESGP TACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC
GTSTEPSEGSAPGT CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCT
STEPSEGSAPGSPA ACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCA
GSPTSTEEGTSTEP GGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAAC
SEGSAPGTSTEPSE CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTC
GSAPGTSESATPES TGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCC
GPGTSTEPSEGSAP GGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGG
GTSESATPESGPGS TAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATC
EPATSGSETPGTST CGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACT
EPSEGSAPGTSTEP CCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA
SEGSAPGTSESATP GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGT
ESGPGTSESATPES ACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACC
GPGSPAGSPTSTEE TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCT
GTSESATPESGPGS GCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAA
EPATSGSETPGTSE AGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA
SATPESGPGTSTEP ACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTA
SEGSAPGTSTEPSE CTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGA
GSAPGTSTEPSEGS GGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG
APGTSTEPSEGSAP TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAG
GTSTEPSEGSAPGT CGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCT
STEPSEGSAPGSPA CCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA
GSPTSTEEGTSTEP GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT
SEGSAPGTSESATP AGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTT
ESGPGSEPATSGSE CTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTG
TPGTSESATPESGP AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG
GSEPATSGSETPGT CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC
SESATPESGPGTST AACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC
EPSEGSAPGTSESA TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT
TPESGPGSPAGSPT GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC
STEEGSPAGSPTST AGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC
EEGSPAGSPTSTEE GGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAG
GTSESATPESGPGT GAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA
STEPSEGSAPGTSE GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT
SATPESGPGSEPAT ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC
SGSETPGTSESATP TCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCT
ESGPGSEPATSGSE GAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT
TPGTSESATPESGP GCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG
GTSTEPSEGSAPGS CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCT
PAGSPTSTEEGTSE CTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCC
SATPESGPGSEPAT TGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC
SGSETPGTSESATP AGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG
ESGPGSPAGSPTST AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC
EEGSPAGSPTSTEE CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG
GTSTEPSEGSAPGT GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG
SESATPESGPGTSE CCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
SATPESGPGTSESA GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC
TPESGPGSEPATSG GAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGC
SETPGSEPATSGSE GCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTA
TPGSPAGSPTSTEE CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC
GTSTEPSEGSAPGT GGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT
STEPSEGSAPGSEP GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA
ATSGSETPGTSESA ACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAG
TPESGPGTSTEPSE GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCACCA
FVII- ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FXIIa-
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE288
REIFKDAERTKLF GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT TMTRIVGGGGTSE
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT SATPESGPGSEPAT
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT SGSETPGTSESATP
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA ESGPGSEPATSGSE
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT TPGTSESATPESGP
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT GTSTEPSEGSAPGS
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG PAGSPTSTEEGTSE
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA SATPESGPGSEPAT
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA SGSETPGTSESATP
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC ESGPGSPAGSPTST
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EEGSPAGSPTSTEE
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA GTSTEPSEGSAPGT
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG SESATPESGPGTSE
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC SATPESGPGTSESA
AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT TPESGPGSEPATSG
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC SETPGSEPATSGSE
GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA TPGSPAGSPTSTEE
CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA GTSTEPSEGSAPGT
GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC STEPSEGSAPGSEP
CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC ATSGSETPGTSESA
TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG TPESGPGTSTEPSE
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA GSAP
TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FXIIa- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE864 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT TMTRIVGGGGSPA
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT GSPTSTEEGTSESA
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT TPESGPGTSTEPSE
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA GSAPGSPAGSPTST
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT EEGTSTEPSEGSAP
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT GTSTEPSEGSAPGT
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA SESATPESGPGSEP
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC ATSGSETPGSEPAT
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC SGSETPGSPAGSPT
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA STEEGTSESATPES
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG GPGTSTEPSEGSAP
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA GTSTEPSEGSAPGS
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG PAGSPTSTEEGTST
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG EPSEGSAPGTSTEP
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG SEGSAPGTSESATP
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC ESGPGTSTEPSEGS
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT APGTSESATPESGP
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA GSEPATSGSETPGT
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT STEPSEGSAPGTST
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT EPSEGSAPGTSESA
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG TPESGPGTSESATP
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG ESGPGSPAGSPTST
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT EEGTSESATPESGP
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA GSEPATSGSETPGT
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC SESATPESGPGTST
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG EPSEGSAPGTSTEP
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC SEGSAPGTSTEPSE
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA GSAPGTSTEPSEGS
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG APGTSTEPSEGSAP
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC GTSTEPSEGSAPGS
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC PAGSPTSTEEGTST
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA EPSEGSAPGTSESA
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC TPESGPGSEPATSG
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG SETPGTSESATPES
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG GPGSEPATSGSETP
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GTSESATPESGPGT
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT STEPSEGSAPGTSE
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SATPESGPGSPAGS
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA PTSTEEGSPAGSPT
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA STEEGSPAGSPTST
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC EEGTSESATPESGP
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC GTSTEPSEGSAPGT
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC SESATPESGPGSEP
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA ATSGSETPGTSESA
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG TPESGPGSEPATSG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG SETPGTSESATPES
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GPGTSTEPSEGSAP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT GSPAGSPTSTEEGT
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SESATPESGPGSEP
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA ATSGSETPGTSESA
CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG TPESGPGSPAGSPT
GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG STEEGSPAGSPTST
CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT EEGTSTEPSEGSAP
CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC GTSESATPESGPGT
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC SESATPESGPGTSE
ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT SATPESGPGSEPAT
GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA SGSETPGSEPATSG
CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA SETPGSPAGSPTST
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA EEGTSTEPSEGSAP
CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG GTSTEPSEGSAPGS
AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC EPATSGSETPGTSE
CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG SATPESGPGTSTEP
GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC SEGSAP
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FXIIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE288 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGT
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTACCT MTRIVGGGGTSES
CTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAAC
ATPESGPGSEPATS CTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAG
GSETPGTSESATPE CGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC
SGPGSEPATSGSET CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT
PGTSESATPESGPG CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGG
TSTEPSEGSAPGSP GCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCAC
AGSPTSTEEGTSES CGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGG
ATPESGPGSEPATS CCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC
GSETPGTSESATPE AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGT
SGPGSPAGSPTSTE AGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC
EGSPAGSPTSTEEG CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTA
TSTEPSEGSAPGTS CCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA
ESATPESGPGTSES GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCG
ATPESGPGTSESAT CTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC
PESGPGSEPATSGS CCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGG
ETPGSEPATSGSET TTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCT
PGSPAGSPTSTEEG GAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT
TSTEPSEGSAPGTS GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA
TEPSEGSAPGSEPA CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG
TSGSETPGTSESAT GTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA
PESGPGTSTEPSEG CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC SAP
TACTGAACCGTCCGAGGGCAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FXIIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE864 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGT
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTAGCC MTRIVGGGGSPAG
CGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA SPTSTEEGTSESAT
AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA PESGPGTSTEPSEG
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCT SAPGSPAGSPTSTE
CCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG EGTSTEPSEGSAPG
AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG TSTEPSEGSAPGTS
GCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAAT ESATPESGPGSEPA
CTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAAC TSGSETPGSEPATS
CCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC GSETPGSPAGSPTS
AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGG TEEGTSESATPESG
TACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC PGTSTEPSEGSAPG
CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCT TSTEPSEGSAPGSP
ACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCA AGSPTSTEEGTSTE
GGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAAC PSEGSAPGTSTEPS
CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSESATPE
TGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCC SGPGTSTEPSEGSA
GGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGG PGTSESATPESGPG
TAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATC SEPATSGSETPGTS
CGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACT TEPSEGSAPGTSTE
CCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA PSEGSAPGTSESAT
GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGT PESGPGTSESATPE
ACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACC SGPGSPAGSPTSTE
TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCT EGTSESATPESGPG
GCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAA SEPATSGSETPGTS
AGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA ESATPESGPGTSTE
ACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTA PSEGSAPGTSTEPS
CTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGA EGSAPGTSTEPSEG
GGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG SAPGTSTEPSEGSA
TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAG PGTSTEPSEGSAPG
CGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCT TSTEPSEGSAPGSP
CCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA AGSPTSTEEGTSTE
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT PSEGSAPGTSESAT
AGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTT PESGPGSEPATSGS
CTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTG ETPGTSESATPESG
AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG PGSEPATSGSETPG
CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC TSESATPESGPGTS
AACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC TEPSEGSAPGTSES
TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT ATPESGPGSPAGSP
GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC TSTEEGSPAGSPTS
AGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC TEEGSPAGSPTSTE
GGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAG EGTSESATPESGPG
GAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA TSTEPSEGSAPGTS
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT ESATPESGPGSEPA
ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC TSGSETPGTSESAT
TCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCT PESGPGSEPATSGS
GAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT ETPGTSESATPESG
GCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG PGTSTEPSEGSAPG
CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCT SPAGSPTSTEEGTS
CTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCC ESATPESGPGSEPA
TGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC TSGSETPGTSESAT
AGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG PESGPGSPAGSPTS
AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC TEEGSPAGSPTSTE
CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG EGTSTEPSEGSAPG
GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG TSESATPESGPGTS
CCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC ESATPESGPGTSES
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC ATPESGPGSEPATS
GAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGC GSETPGSEPATSGS
GCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTA ETPGSPAGSPTSTE
CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC EGTSTEPSEGSAPG
GGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT TSTEPSEGSAPGSE
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA PATSGSETPGTSES
ACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAG ATPESGPGTSTEPS
GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA EGSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCACCA
FVII- ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG
Kallikrein- RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA
AE288 REIFKDAERTKLF GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT
WISYSDGDQCASS GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA
PCQNGGSCKDQLQ AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC
SYICFCLPAFEGRN CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC
CETHKDDQLICVN GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT SPFRSTGGGGTSES
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT ATPESGPGSEPATS
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT GSETPGTSESATPE
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA SGPGSEPATSGSET
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT PGTSESATPESGPG
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT TSTEPSEGSAPGSP
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG AGSPTSTEEGTSES
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA ATPESGPGSEPATS
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA GSETPGTSESATPE
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC SGPGSPAGSPTSTE
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EGSPAGSPTSTEEG
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA TSTEPSEGSAPGTS
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG ESATPESGPGTSES
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC ATPESGPGTSESAT
AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT PESGPGSEPATSGS
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC ETPGSEPATSGSET
GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA PGSPAGSPTSTEEG
CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA TSTEPSEGSAPGTS
GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC TEPSEGSAPGSEPA
CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC TSGSETPGTSESAT
TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG PESGPGTSTEPSEG
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA SAP
TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG Kallikrein- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE864 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT SPFRSTGGGGSPA
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT GSPTSTEEGTSESA
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT TPESGPGTSTEPSE
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA GSAPGSPAGSPTST
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT EEGTSTEPSEGSAP
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT GTSTEPSEGSAPGT
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA SESATPESGPGSEP
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC ATSGSETPGSEPAT
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC SGSETPGSPAGSPT
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA STEEGTSESATPES
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG GPGTSTEPSEGSAP
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA GTSTEPSEGSAPGS
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG PAGSPTSTEEGTST
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG EPSEGSAPGTSTEP
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG SEGSAPGTSESATP
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC ESGPGTSTEPSEGS
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT APGTSESATPESGP
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA GSEPATSGSETPGT
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT STEPSEGSAPGTST
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT EPSEGSAPGTSESA
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG TPESGPGTSESATP
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG ESGPGSPAGSPTST
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT EEGTSESATPESGP
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA GSEPATSGSETPGT
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC SESATPESGPGTST
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG EPSEGSAPGTSTEP
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC SEGSAPGTSTEPSE
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA GSAPGTSTEPSEGS
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG APGTSTEPSEGSAP
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC GTSTEPSEGSAPGS
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC PAGSPTSTEEGTST
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA EPSEGSAPGTSESA
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC TPESGPGSEPATSG
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG SETPGTSESATPES
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG GPGSEPATSGSETP
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GTSESATPESGPGT
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT STEPSEGSAPGTSE
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SATPESGPGSPAGS
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA PTSTEEGSPAGSPT
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA STEEGSPAGSPTST
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC EEGTSESATPESGP
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC GTSTEPSEGSAPGT
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC SESATPESGPGSEP
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA ATSGSETPGTSESA
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG
TPESGPGSEPATSG CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG
SETPGTSESATPES CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA
GPGTSTEPSEGSAP GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT
GSPAGSPTSTEEGT AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC
SESATPESGPGSEP TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA
ATSGSETPGTSESA CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG
TPESGPGSPAGSPT GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG
STEEGSPAGSPTST CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT
EEGTSTEPSEGSAP CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC
GTSESATPESGPGT TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC
SESATPESGPGTSE ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT
SATPESGPGSEPAT GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA
SGSETPGSEPATSG CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA
SETPGSPAGSPTST GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA
EEGTSTEPSEGSAP CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG
GTSTEPSEGSAPGS AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC
EPATSGSETPGTSE CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG
SATPESGPGTSTEP GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC SEGSAP
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC Kallikrein- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE288 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGS
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTACCT PFRSTGGGGTSESA
CTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAAC TPESGPGSEPATSG
CTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAG SETPGTSESATPES
CGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC GPGSEPATSGSETP
CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT GTSESATPESGPGT
CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGG STEPSEGSAPGSPA
GCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCAC GSPTSTEEGTSESA
CGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGG TPESGPGSEPATSG
CCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC SETPGTSESATPES
AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGT GPGSPAGSPTSTEE
AGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC GSPAGSPTSTEEGT
CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTA STEPSEGSAPGTSE
CCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA SATPESGPGTSESA
GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCG TPESGPGTSESATP
CTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC ESGPGSEPATSGSE
CCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGG TPGSEPATSGSETP
TTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCT GSPAGSPTSTEEGT
GAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT STEPSEGSAPGTST
GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA EPSEGSAPGSEPAT
CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG SGSETPGTSESATP
GTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA ESGPGTSTEPSEGS
CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC AP
TACTGAACCGTCCGAGGGCAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC Kallikrein- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE864 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGS
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTAGCC PFRSTGGGGSPAG
CGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA SPTSTEEGTSESAT
AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA PESGPGTSTEPSEG
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCT SAPGSPAGSPTSTE
CCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG EGTSTEPSEGSAPG
AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG TSTEPSEGSAPGTS
GCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAAT ESATPESGPGSEPA
CTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAAC TSGSETPGSEPATS
CCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC GSETPGSPAGSPTS
AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGG TEEGTSESATPESG
TACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC PGTSTEPSEGSAPG
CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCT TSTEPSEGSAPGSP
ACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCA AGSPTSTEEGTSTE
GGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAAC PSEGSAPGTSTEPS
CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTC EGSAPGTSESATPE
TGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCC SGPGTSTEPSEGSA
GGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGG PGTSESATPESGPG
TAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATC SEPATSGSETPGTS
CGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACT TEPSEGSAPGTSTE
CCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA PSEGSAPGTSESAT
GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGT PESGPGTSESATPE
ACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACC SGPGSPAGSPTSTE
TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCT EGTSESATPESGPG
GCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAA SEPATSGSETPGTS
AGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA ESATPESGPGTSTE
ACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTA PSEGSAPGTSTEPS
CTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGA EGSAPGTSTEPSEG
GGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG SAPGTSTEPSEGSA
TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAG PGTSTEPSEGSAPG
CGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCT TSTEPSEGSAPGSP
CCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA AGSPTSTEEGTSTE
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT PSEGSAPGTSESAT
AGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTT PESGPGSEPATSGS
CTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTG ETPGTSESATPESG
AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG PGSEPATSGSETPG
CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC TSESATPESGPGTS
AACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC TEPSEGSAPGTSES
TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT ATPESGPGSPAGSP
GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC TSTEEGSPAGSPTS
AGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC TEEGSPAGSPTSTE
GGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAG EGTSESATPESGPG
GAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA TSTEPSEGSAPGTS
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT ESATPESGPGSEPA
ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC TSGSETPGTSESAT
TCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCT PESGPGSEPATSGS
GAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT ETPGTSESATPESG
GCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG PGTSTEPSEGSAPG
CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCT SPAGSPTSTEEGTS
CTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCC ESATPESGPGSEPA
TGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC TSGSETPGTSESAT
AGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG PESGPGSPAGSPTS
AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC TEEGSPAGSPTSTE
CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG EGTSTEPSEGSAPG
GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG TSESATPESGPGTS
CCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC ESATPESGPGTSES
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC ATPESGPGSEPATS
GAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGC GSETPGSEPATSGS
GCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTA ETPGSPAGSPTSTE
CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC EGTSTEPSEGSAPG
GGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT TSTEPSEGSAPGSE
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA PATSGSETPGTSES
ACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAG ATPESGPGTSTEPS
GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA EGSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCACCA
FVII- ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FIIa-
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE288
REIFKDAERTKLF GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT LTPRSLLVGGTSES
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT ATPESGPGSEPATS
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT GSETPGTSESATPE
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA SGPGSEPATSGSET
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT PGTSESATPESGPG
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT TSTEPSEGSAPGSP
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG AGSPTSTEEGTSES
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA ATPESGPGSEPATS
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA GSETPGTSESATPE
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC SGPGSPAGSPTSTE
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EGSPAGSPTSTEEG
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA TSTEPSEGSAPGTS
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG ESATPESGPGTSES
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC ATPESGPGTSESAT
AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT PESGPGSEPATSGS
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC ETPGSEPATSGSET
GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA PGSPAGSPTSTEEG
CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA TSTEPSEGSAPGTS
GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC TEPSEGSAPGSEPA
CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC TSGSETPGTSESAT
TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG PESGPGTSTEPSEG
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA SAP
TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG FIIa- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE864 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT
NHDIALLRLHQPV CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT LTPRSLLVGGSPA
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT GSPTSTEEGTSESA
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT TPESGPGTSTEPSE
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA GSAPGSPAGSPTST
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT EEGTSTEPSEGSAP
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT GTSTEPSEGSAPGT
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA SESATPESGPGSEP
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC ATSGSETPGSEPAT
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC SGSETPGSPAGSPT
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA STEEGTSESATPES
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG GPGTSTEPSEGSAP
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA GTSTEPSEGSAPGS
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG PAGSPTSTEEGTST
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG EPSEGSAPGTSTEP
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG SEGSAPGTSESATP
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC ESGPGTSTEPSEGS
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT APGTSESATPESGP
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA GSEPATSGSETPGT
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT STEPSEGSAPGTST
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT EPSEGSAPGTSESA
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG TPESGPGTSESATP
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG ESGPGSPAGSPTST
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT EEGTSESATPESGP
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA GSEPATSGSETPGT
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC SESATPESGPGTST
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG EPSEGSAPGTSTEP
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC SEGSAPGTSTEPSE
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA GSAPGTSTEPSEGS
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG APGTSTEPSEGSAP
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC GTSTEPSEGSAPGS
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC PAGSPTSTEEGTST
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA EPSEGSAPGTSESA
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC TPESGPGSEPATSG
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG SETPGTSESATPES
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG GPGSEPATSGSETP
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GTSESATPESGPGT
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT STEPSEGSAPGTSE
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SATPESGPGSPAGS
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA PTSTEEGSPAGSPT
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA STEEGSPAGSPTST
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC EEGTSESATPESGP
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC GTSTEPSEGSAPGT
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC SESATPESGPGSEP
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA ATSGSETPGTSESA
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG TPESGPGSEPATSG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG SETPGTSESATPES
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GPGTSTEPSEGSAP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT GSPAGSPTSTEEGT
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SESATPESGPGSEP
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA ATSGSETPGTSESA
CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG TPESGPGSPAGSPT
GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG STEEGSPAGSPTST
CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT EEGTSTEPSEGSAP
CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC GTSESATPESGPGT
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC SESATPESGPGTSE
ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT SATPESGPGSEPAT
GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA SGSETPGSEPATSG
CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA SETPGSPAGSPTST
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA EEGTSTEPSEGSAP
CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG GTSTEPSEGSAPGS
AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC EPATSGSETPGTSE
CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG SATPESGPGTSTEP
GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC SEGSAP
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FIIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE288 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGL
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTACCT TPRSLLVGGTSESA
CTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAAC TPESGPGSEPATSG
CTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAG SETPGTSESATPES
CGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC GPGSEPATSGSETP
CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT GTSESATPESGPGT
CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGG STEPSEGSAPGSPA
GCAGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCAC GSPTSTEEGTSESA
CGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGG TPESGPGSEPATSG
CCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC SETPGTSESATPES
AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGT GPGSPAGSPTSTEE
AGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGC GSPAGSPTSTEEGT
CCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTA STEPSEGSAPGTSE
CCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAA SATPESGPGTSESA
GCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCG TPESGPGTSESATP
CTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTAC ESGPGSEPATSGSE
CCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGG TPGSEPATSGSETP
TTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCT GSPAGSPTSTEEGT
GAAACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACT STEPSEGSAPGTST
GAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCA EPSEGSAPGSEPAT
CCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAG SGSETPGTSESATP
GTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTA ESGPGTSTEPSEGS
CCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTC AP
TACTGAACCGTCCGAGGGCAGCGCACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC FIIa- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA AE864 EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGL
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGTGGTAGCC TPRSLLVGGSPAGS
CGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGA PTSTEEGTSESATP
AAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTGAA ESGPGTSTEPSEGS
CCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTCT APGSPAGSPTSTEE
CCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCG GTSTEPSEGSAPGT
AAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGG STEPSEGSAPGTSE
GCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAAT SATPESGPGSEPAT
CTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAAC SGSETPGSEPATSG
CCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC SETPGSPAGSPTST
AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGG EEGTSESATPESGP
TACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAC GTSTEPSEGSAPGT
CTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCT STEPSEGSAPGSPA
ACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCA GSPTSTEEGTSTEP
GGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCGAAC SEGSAPGTSTEPSE
CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCTTC GSAPGTSESATPES
TGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCC GPGTSTEPSEGSAP
GGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGG GTSESATPESGPGS
TAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATC EPATSGSETPGTST
CGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACT EPSEGSAPGTSTEP
CCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA SEGSAPGTSESATP
GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGT ESGPGTSESATPES
ACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAGGTACC GPGSPAGSPTSTEE
TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCT GTSESATPESGPGS
GCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAA EPATSGSETPGTSE
AGCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCA SATPESGPGTSTEP
ACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGCTA SEGSAPGTSTEPSE
CTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGA GSAPGTSTEPSEGS
GGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGG APGTSTEPSEGSAP
TAGCGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAG GTSTEPSEGSAPGT
CGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCT STEPSEGSAPGSPA
CCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA GSPTSTEEGTSTEP
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT SEGSAPGTSESATP
AGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTT ESGPGSEPATSGSE
CTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTG TPGTSESATPESGP
AAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTG GSEPATSGSETPGT
CTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGC SESATPESGPGTST
AACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCTC EPSEGSAPGTSESA
TGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT TPESGPGSPAGSPT
GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC STEEGSPAGSPTST
AGCGCACCAGGTACTTCTGAAAGCGCTACTCCTGAGTCC EEGSPAGSPTSTEE
GGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAG GTSESATPESGPGT
GAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA STEPSEGSAPGTSE
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGT SATPESGPGSEPAT
ACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACC SGSETPGTSESATP
TCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCT ESGPGSEPATSGSE
GAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCT TPGTSESATPESGP
GCTACCTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCG GTSTEPSEGSAPGS
CAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAACCT PAGSPTSTEEGTSE
CTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCC SATPESGPGSEPAT
TGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC SGSETPGTSESATP
AGCGCACCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCG ESGPGSPAGSPTST
AAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCC EEGSPAGSPTSTEE
CAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAG GTSTEPSEGSAPGT
GTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAG SESATPESGPGTSE
CCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC SATPESGPGTSESA
GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACC TPESGPGSEPATSG
GAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGC SETPGSEPATSGSE
GCTACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTA TPGSPAGSPTSTEE
CTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGCTACCCC GTSTEPSEGSAPGT
GGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT STEPSEGSAPGSEP
GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAA ATSGSETPGTSESA
ACTCCAGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAG TPESGPGTSTEPSE
GAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTA
GCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTC
TGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTACT GAACCGTCCGAGGGCAGCGCACCA
FVII- ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG MMP-
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA 17-
REIFKDAERTKLF GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT AE288
WISYSDGDQCASS GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA
PCQNGGSCKDQLQ AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC
SYICFCLPAFEGRN CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC
CETHKDDQLICVN GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC
LSEHDGDEQSRRV GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT APLGLRLRGGTSE
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT SATPESGPGSEPAT
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT SGSETPGTSESATP
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA ESGPGSEPATSGSE
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT TPGTSESATPESGP
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT GTSTEPSEGSAPGS
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG PAGSPTSTEEGTSE
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA SATPESGPGSEPAT
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA SGSETPGTSESATP
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC ESGPGSPAGSPTST
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EEGSPAGSPTSTEE
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA GTSTEPSEGSAPGT
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG SESATPESGPGTSE
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC SATPESGPGTSESA
AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT TPESGPGSEPATSG
ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC SETPGSEPATSGSE
GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA TPGSPAGSPTSTEE
CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA GTSTEPSEGSAPGT
GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC STEPSEGSAPGSEP
CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC ATSGSETPGTSESA
TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG TPESGPGTSTEPSE
CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA GSAP
TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GCACCA FVII- ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG MMP- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA 17- REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT AE864 WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT APLGLRLRGGSPA
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT GSPTSTEEGTSESA
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT TPESGPGTSTEPSE
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA GSAPGSPAGSPTST
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT EEGTSTEPSEGSAP
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT GTSTEPSEGSAPGT
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA SESATPESGPGSEP
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC ATSGSETPGSEPAT
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC SGSETPGSPAGSPT
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA STEEGTSESATPES
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG GPGTSTEPSEGSAP
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA GTSTEPSEGSAPGS
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG PAGSPTSTEEGTST
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG EPSEGSAPGTSTEP
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG SEGSAPGTSESATP
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC ESGPGTSTEPSEGS
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT APGTSESATPESGP
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA GSEPATSGSETPGT
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT STEPSEGSAPGTST
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT EPSEGSAPGTSESA
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG TPESGPGTSESATP
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG ESGPGSPAGSPTST
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT EEGTSESATPESGP
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA GSEPATSGSETPGT
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC SESATPESGPGTST
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG EPSEGSAPGTSTEP
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC SEGSAPGTSTEPSE
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA GSAPGTSTEPSEGS
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG APGTSTEPSEGSAP
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC GTSTEPSEGSAPGS
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC PAGSPTSTEEGTST
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA EPSEGSAPGTSESA
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC TPESGPGSEPATSG
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG SETPGTSESATPES
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG GPGSEPATSGSETP
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GTSESATPESGPGT
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT STEPSEGSAPGTSE
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SATPESGPGSPAGS
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA PTSTEEGSPAGSPT
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA STEEGSPAGSPTST
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC EEGTSESATPESGP
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC GTSTEPSEGSAPGT
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC SESATPESGPGSEP
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA ATSGSETPGTSESA
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG TPESGPGSEPATSG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG SETPGTSESATPES
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA GPGTSTEPSEGSAP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT GSPAGSPTSTEEGT
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC SESATPESGPGSEP
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA ATSGSETPGTSESA
CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG TPESGPGSPAGSPT
GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG STEEGSPAGSPTST
CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT EEGTSTEPSEGSAP
CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC GTSESATPESGPGT
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC SESATPESGPGTSE
ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT SATPESGPGSEPAT
GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA SGSETPGSEPATSG
CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA SETPGSPAGSPTST
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA EEGTSTEPSEGSAP
CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG GTSTEPSEGSAPGS
AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC EPATSGSETPGTSE
CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG SATPESGPGTSTEP
GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC SEGSAP
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA FIX- YNSGKLEEFVQGN
TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC MMP- LERECMEEKCSFE
CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA 17-FXIa EAREVFENTERTT
GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT EFWKQYVDGDQC
GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGA
TTAAGGAAAAAACAAAGCTCACTGGGGT0GGT0 PLGLRLRGKLTRA ET FIX-
YNSGKLEEFVQGN TATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC MMP-
LERECMEEKCSFE CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAA 17-
EAREVFENTERTT GAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACT Elastase
EFWKQYVDGDQC GAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAG ESNPCLNGGSCKD
TCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGAC DINSYECWCPFGF
ATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG EGKNCELDVTCNI
GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA KNGRCEQFCKNSA
ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA DNKVVCSCTEGYR
ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC LAENQKSCEPAVP
AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCC FPCGRVSVSQTSK
ATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACC LTRAETVFPDVDY
CGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATT VNSTEAETILDNIT
CTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAA QSTQSFNDFTRVV
GCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGG GGEDAKPGQFPW
AGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGT QVVLNGKVDAFC
TTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATC GGSIVNEKWIVTA
GTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG AHCVETGVKITVV
AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATA AGEHNIEETEHTE
ATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT QKRNVIRIIPHHNY
GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTA NAAINKYNHDIAL
TTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA LELDEPLVLNSYV
CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGC TPICIADKEYTNIFL
ATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG KFGSGYVSGWGR
GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACA VFHKGRSALVLQY
AAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCC LRVPLVDRATCLR
ACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTC STKFTIYNNMFCA
ACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAG GFHEGGRDSCQGD
GAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCC SGGPHVTEVEGTS
ATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAA FLTGIISWGEECAM
TTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT KGKYGIYTKVSRY
ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA VNWIKEKTKLTGA
TTAAGGAAAAAACAAAGCTCACTGGGGT0 PLGLRLR FVII-FIX ANAFLEELRPGSLE
GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AP- RECKEEQCSFEEA
GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE288 REIFKDAERTKLF
GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT KLTRAETVFPDVD
CCCGGT0GGTGGTACCTCTGAAAGCGCAACTCCTGAGTCT YVNSTEAETILDNI
GGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACT TQSTQSFNDFTRV
CCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA VGGE
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT GGTSESATPESGPG
ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTT SEPATSGSETPGTS
CTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTG ESATPESGPGSEPA
CTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAA TSGSETPGTSESAT
GCGCAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAA PESGPGTSTEPSEG
CCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTAC SAPGSPAGSPTSTE
TCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACT EGTSESATPESGPG
TCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTA SEPATSGSETPGTS
CTGAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCG ESATPESGPGSPAG
CACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC
SPTSTEEGSPAGSP AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT
TSTEEGTSTEPSEG ACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGC
SAPGTSESATPESG GAACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAA
PGTSESATPESGPG CCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCA
TSESATPESGPGSE GGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAAC
PATSGSETPGSEPA CTTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTC
TSGSETPGSPAGSP TGAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGG
TSTEEGTSTEPSEG CTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAA
SAPGTSTEPSEGSA TCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
PGSEPATSGSETPG GCACCA TSESATPESGPGTS TEPSEGSAP FVII-FIX
ANAFLEELRPGSLE GCCAACGCGTTCCTGGAGGAGCTACGGCCGGGCTCCCTG AP-
RECKEEQCSFEEA GAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGA AE864
REIFKDAERTKLF GGCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCT WISYSDGDQCASS
GTTCTGGATTTCTTACAGTGATGGGGACCAGTGTGCCTCA PCQNGGSCKDQLQ
AGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTC SYICFCLPAFEGRN
CAGTCCTATATCTGCTTCTGCCTCCCTGCCTTCGAGGGCC CETHKDDQLICVN
GGAACTGTGAGACGCACAAGGATGACCAGCTGATCTGTG ENGGCEQYCSDHT
TGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC GTKRSCRCHEGYS
ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGT LLADGVSCTPTVE
ACTCTCTGCTGGCAGACGGGGTGTCCTGCACACCCACAGT YPCGKIPILEKRNA
TGAATATCCATGTGGAAAAATACCTATTCTAGAAAAAAG SKPQGRIVGGKVC
AAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAA PKGECPWQVLLLV
GGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTT NGAQLCGGTLINTI
GTTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGAT WVVSAAHCFDKIK
CAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTCGAC NWRNLIAVLGEHD
AAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGC LSEHDGDEQSRRV
GAGCACGACCTCAGCGAGCACGACGGGGATGAGCAGAG AQVIIPSTYVPGTT
CCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGT NHDIALLRLHQPV
CCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCT VLTDHVVPLCLPE
GCACCAGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTC RTFSERTLAFVRFS
TGCCTGCCCGAACGGACGTTCTCTGAGAGGACGCTGGCC LVSGWGQLLDRG
TTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAGCTGC ATALELMVLNVPR
TGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCA LMTQDCLQQSRK
ACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGT VGDSPNITEYMFC
CACGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACA AGYSDGSKDSCKG
TGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTG DSGGPHATHYRGT
CAAGGGGGACAGTGGAGGCCCACATGCCACCCACTACCG WYLTGIVSWGQG
GGGCACGTGGTACCTGACGGGCATCGTCAGCTGGGGCCA CATVGHFGVYTRV
GGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACCAG SQYIEWLQKLMRS
GGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCG EPRPGVLLRAPFPG
CTCAGAGCCACGCCCAGGAGTCCTCCTGCGAGCCCCATTT KLTRAETVFPDVD
CCCGGT0GGTGGTAGCCCGGCTGGCTCTCCTACCTCTACT YVNSTEAETILDNI
GAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGT TQSTQSFNDFTRV
CCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA VGGE
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGT GGSPAGSPTSTEEG
ACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCT TSESATPESGPGTS
CTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGA TEPSEGSAPGSPAG
AAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGC SPTSTEEGTSTEPS
TACTTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACC EGSAPGTSTEPSEG
TCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTCTCCGA SAPGTSESATPESG
CCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGG PGSEPATSGSETPG
AGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCA SEPATSGSETPGSP
GCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG AGSPTSTEEGTSES
CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGG ATPESGPGTSTEPS
AAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAG EGSAPGTSTEPSEG
GTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAC SAPGSPAGSPTSTE
TTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT EGTSTEPSEGSAPG
ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAA TSTEPSEGSAPGTS
AGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCT ESATPESGPGTSTE
ACTTCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGT PSEGSAPGTSESAT
CCGAAGGTAGCGCACCAGGTACTTCTACTGAACCGTCTG PESGPGSEPATSGS
AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG ETPGTSTEPSEGSA
AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT PGTSTEPSEGSAPG
CCGGCCCAGGTAGCCCTGCTGGCTCTCCAACCTCCACCGA TSESATPESGPGTS
AGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC ESATPESGPGSPAG
AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGG SPTSTEEGTSESAT
TACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC PESGPGSEPATSGS
TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTA ETPGTSESATPESG
CTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCG PGTSTEPSEGSAPG
AACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAAC TSTEPSEGSAPGTS
CTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTC TEPSEGSAPGTSTE
TGAAGGTAGCGCACCAGGTACTTCTACCGAACCGTCCGA PSEGSAPGTSTEPS
GGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTC EGSAPGTSTEPSEG
CACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAG SAPGSPAGSPTSTE
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG EGTSTEPSEGSAPG
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA TSESATPESGPGSE
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT PATSGSETPGTSES
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC ATPESGPGSEPATS
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA GSETPGTSESATPE
CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAA SGPGTSTEPSEGSA
GCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC PGTSESATPESGPG
TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCC SPAGSPTSTEEGSP
AACTTCTACTGAAGAAGGTAGCCCGGCAGGCTCTCCGAC AGSPTSTEEGSPAG
CTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGA SPTSTEEGTSESAT
GTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAG PESGPGTSTEPSEG
CGCACCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGG SAPGTSESATPESG
CCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA PGSEPATSGSETPG
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGT TSESATPESGPGSE
AGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACC PATSGSETPGTSES
TCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA ATPESGPGTSTEPS
CTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTG EGSAPGSPAGSPTS
GCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCG TEEGTSESATPESG
CAACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCT PGSEPATSGSETPG
CCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGCTACTCC TSESATPESGPGSP
TGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCC AGSPTSTEEGSPAG
ACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACT SPTSTEEGTSTEPS
GAAGAAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCA EGSAPGTSESATPE
CCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA SGPGTSESATPESG
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTA PGTSESATPESGPG
CTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCG SEPATSGSETPGSE
AACCGGCTACTTCTGGTTCTGAAACCCCAGGTAGCGAAC PATSGSETPGSPAG
CGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAG SPTSTEEGTSTEPS
GCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC EGSAPGTSTEPSEG
TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCT SAPGSEPATSGSET
GAGGGCAGCGCTCCAGGTAGCGAACCTGCAACCTCTGGC PGTSESATPESGPG
TCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAAT TSTEPSEGSAP
CTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCG CACCA *Sequence name
reflects N- to C-terminus configuration of the CF, cleavage
sequence and XTEN components
TABLE-US-00049 TABLE 43 Exemplary FVII variants incorporating FIX
AP sequence, cleavage sequences and XTEN** FVII Construct Name*
Amino Acid Sequence FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP EGF2)-(AP)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILD
NITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTF
SERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDS
PNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP EGF2)-(AP)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE AE288
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILD
NITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTF
SERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDS
PNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP EGF2)-(AP)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE AE864
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGSKLTRAETVFPDVDYVNSTEAETILD
NITQSTQSFNDFTRVVGGEGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTF
SERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDS
PNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES
GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTE
EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT
STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT
STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS AP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-V149)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQP
VVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR
LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP EGF2)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG (K142-V149)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE FVII(Pro)-
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL AE288
SEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQP
VVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR
LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPG
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)- CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG
(K142-V149)- TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE
FVII(Pro)- CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
AE864 SEHDGDEQSRRVAQVIIPSTYVPGTTNGKLTRAETVGSKPQGRHDIALLRLHQP
VVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR
LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPG
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP
ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-G184)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE
CPWQVILLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQ
PVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVP
RLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-G184)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE AE288
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQ
PVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVP
RLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPG
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP
GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-G184)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQIVGGKVCPKGE AE864
CPWQVLLLVNGAQLCGGGRTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNGDFTRVVGGGSKPQGRHDIALLRLHQ
PVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVP
RLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPG
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP
ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSE
SATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP
ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE
GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP
ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP
GTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRIVGGKVCPK
GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL
SEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERT
FSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRIVGGKVCPK KLTRAET-
GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL AE288
SEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERT
FSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRIVGGKVCPK KLTRAET-
GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDL AE864
SEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERT
FSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATV
GHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSP
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKLTRAETVFPDVDYVNS
TEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLIN
TIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPG
TTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKG
DSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRS EPRPGVLLRAPFP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKLTRAETVFPDVDYVNS KLTRAET-
TEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLIN AE288
TIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPG
TTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKG
DSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRS
EPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT
SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS
GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE
PATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKLTRAETVFPDVDYVNS KLTRAET-
TEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLIN AE864
TIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPG
TTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKG
DSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRS
EPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE
SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP
GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN
STEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLI
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR SEPRPGVLLRAPFP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN KLTRAET-
STEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE288
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN KLTRAET-
STEAETILDNITQSTQSFNDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE864
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCP
KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHD
LSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPER
TFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVG
DSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCAT
VGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCP KLTRAET-
KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHD AE288
LSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPER
TFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVG
DSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCAT
VGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(D177-T179)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCP KLTRAET-
KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHD AE864
LSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPER
TFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVG
DSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCAT
VGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPG
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT
PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG
PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS
ESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKLTRAETVFPDVDYVNS
TEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR SEPRPGVLLRAPFP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKLTRAETVFPDVDYVNS KLTRAET-
TEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE288
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(K142-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKLTRAETVFPDVDYVNS KLTRAET-
TEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE864
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN
STEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR SEPRPGVLLRAPFP
FVII(Gla- ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN KLTRAET-
STEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE288
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST
EEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS
EPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVII(Gla-
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSP
EGF2)-(R134-V181)-
CQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTG FVII(Pro)-
TKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSKLTRAETVFPDVDYVN KLTRAET-
STEAETILDNITQSTQSFNDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLI AE864
NTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVP
GTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD
RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMR
SEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGT
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP
SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT
SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA
TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA
GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS *Sequence
name reflects N- to C-terminus configuration of the FVII variant
(Gla-EGF2 domains), FIX AP cleavage sequence, FVII protease domain,
XTEN cleavage sequence and XTEN components (the latter when
included) **Not all sequences incorporate XTEN
* * * * *