U.S. patent application number 14/248661 was filed with the patent office on 2014-09-18 for modified coagulation factors with prolonged in vivo half-life.
This patent application is currently assigned to CSL Behring GmbH. The applicant listed for this patent is CSL Behring GmbH. Invention is credited to Hubert METZNER, Stefan SCHULTE, Thomas WEIMER.
Application Number | 20140273096 14/248661 |
Document ID | / |
Family ID | 39155545 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140273096 |
Kind Code |
A1 |
SCHULTE; Stefan ; et
al. |
September 18, 2014 |
MODIFIED COAGULATION FACTORS WITH PROLONGED IN VIVO HALF-LIFE
Abstract
The present invention relates to nucleic acid sequences coding
for modified coagulation factors, preferably coagulation factor
VIII, and their derivatives; recombinant expression vectors
containing such nucleic acid sequences; host cells transformed with
such recombinant expression vectors; and recombinant polypeptides
and derivatives coded for by said nucleic acid sequences, whereby
said recombinant polypeptides and derivatives have biological
activities and prolonged in vivo half-lives compared to the
unmodified wild-type proteins. The invention also relates to
corresponding sequences that result in improved in vitro stability.
The present invention further relates to processes for the
manufacture of such recombinant proteins and their derivatives. The
invention also relates to a transfer vector for use in human gene
therapy, which comprises such nucleic acid sequences.
Inventors: |
SCHULTE; Stefan; (Marburg,
DE) ; WEIMER; Thomas; (Gladenbach, DE) ;
METZNER; Hubert; (Marburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSL Behring GmbH |
Marburg |
|
DE |
|
|
Assignee: |
CSL Behring GmbH
Marburg
DE
|
Family ID: |
39155545 |
Appl. No.: |
14/248661 |
Filed: |
April 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12520840 |
Jul 23, 2009 |
8754194 |
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PCT/EP2007/011356 |
Dec 21, 2007 |
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14248661 |
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60879334 |
Jan 9, 2007 |
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Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/328; 435/352; 435/354; 435/356; 435/358; 435/365;
435/369; 536/23.4 |
Current CPC
Class: |
C07K 14/745 20130101;
C07K 2319/31 20130101; C07K 14/755 20130101; C07K 14/765 20130101;
C07K 16/18 20130101; A61P 7/04 20180101 |
Class at
Publication: |
435/69.6 ;
536/23.4; 435/320.1; 435/328; 435/365; 435/356; 435/354; 435/352;
435/369; 435/358 |
International
Class: |
C07K 14/755 20060101
C07K014/755; C07K 14/765 20060101 C07K014/765; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2006 |
EP |
06026747.3 |
Claims
1-32. (canceled)
33. A polynucleotide or a group of polynucleotides encoding a
modified factor VIII (FVIII) polypeptide, comprising a FVIII
polypeptide having an N-terminal amino acid and a C-terminal amino
acid, and a half-life enhancing polypeptide (HLEP) inserted within
the B-domain between the N-terminal amino acid and the C-terminal
amino acid of the FVIII polypeptide, wherein the FVIII polypeptide
is capable of being cleaved from the HLEP moiety during activation
in vivo, wherein the modified FVIII polypeptide exhibits a
prolonged half-life prior to activation during a bleeding event and
a half-life substantially the same as that of an unmodified FVIII
peptide following activation, and wherein the HLEP comprises
albumin or an immunoglobulin constant region polypeptide.
34. The polynucleotide or group of polynucleotides according to
claim 33, wherein the modified FVIII polypeptide has a prolonged
functional or antigenic half-life as compared to a FVIII
polypeptide lacking an inserted HLEP.
35. The polynucleotide or group of polynucleotides according to
claim 33, wherein the modified FVIII polypeptide has an improved in
vivo recovery as compared to the FVIII polypeptide lacking an
inserted HLEP.
36. The polynucleotide or group of polynucleotides according to
claim 33, wherein the modified FVIII polypeptide has increased
stability in serum-free culture media and/or in animal protein-free
culture media as compared to the FVIII polypeptide lacking an
inserted HLEP.
37. The polynucleotide or group of polynucleotides according to
claim 33, wherein the B-domain of FVIII or a part thereof is
replaced with the HLEP.
38. The polynucleotide or group of polynucleotides according to
claim 37, wherein more than 75% of the B-domain is deleted, or more
than 75% of the B-domain is replaced by linker sequences.
39. The polynucleotide or group of polynucleotides according to
claim 33, wherein the modified FVIII polypeptide has at least 10%
of the biological activity of the FVIII polypeptide lacking an
inserted HLEP.
40. The polynucleotide or group of polynucleotides according to
claim 33, wherein the half-life enhancing polypeptide is
albumin.
41. The polynucleotide or group of polynucleotides according to
claim 33, wherein the B-domain of FVIII has been replaced partly or
completely with human albumin.
42. The polynucleotide or group of polynucleotides according to
claim 33, wherein the immunoglobulin constant region polypeptide is
an immunoglobulin G Fc domain.
43. A plasmid or vector comprising the polynucleotide according to
claim 33, or a group of plasmids or vectors comprising the group of
polynucleotides according to claim 33.
44. The plasmid or vector, or the group of plasmids or vectors,
according to claim 43, wherein the plasmid(s) or vector(s) are
expression vector(s).
45. The vector, or the group of vectors, according to claim 43,
wherein the vector(s) are transfer vector(s) for use in human gene
therapy.
46. A host cell comprising the polynucleotide or group of
polynucleotides according to claim 33.
47. A method of producing a modified FVIII polypeptide, comprising
culturing the host cell according to claim 46 under conditions such
that the modified FVIII polypeptide is expressed.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to modified nucleic acid
sequences coding for coagulation factors preferably coagulation
factor VIII and their derivatives, recombinant expression vectors
containing such nucleic acid sequences, host cells transformed with
such recombinant expression vectors, recombinant polypeptides and
derivatives coded for by said nucleic acid sequences which
recombinant polypeptides and derivatives do have biological
activities together with prolonged in vivo half-life and/or
improved in vivo recovery compared to the unmodified wild-type
protein. The invention also relates to corresponding sequences that
result in improved in vitro stability. The present invention
further relates to processes for the manufacture of such
recombinant proteins and their derivatives. The invention also
relates to a transfer vector for use in human gene therapy, which
comprises such modified nucleic acid sequences.
BACKGROUND OF THE INVENTION
[0002] There are various bleeding disorders caused by deficiencies
of blood coagulation factors. The most common disorders are
hemophilia A and B, resulting from deficiencies of blood
coagulation factor VIII and IX, respectively. Another known
bleeding disorder is von Willebrand's disease.
[0003] Classic hemophilia or hemophilia A is an inherited bleeding
disorder. It results from a chromosome X-linked deficiency of blood
coagulation Factor VIII, and affects almost exclusively males with
an incidence of between one and two individuals per 10.000. The
X-chromosome defect is transmitted by female carriers who are not
themselves hemophiliacs. The clinical manifestation of hemophilia A
is an increased bleeding tendency. Before treatment with Factor
VIII concentrates was introduced the mean life span for a person
with severe hemophilia was less than 20 years. The use of
concentrates of Factor VIII from plasma has considerably improved
the situation for the hemophilia A patients increasing the mean
life span extensively, giving most of them the possibility to live
a more or less normal life. However, there have been certain
problems with the plasma derived concentrates and their use, the
most serious of which have been the transmission of viruses. So
far, viruses causing hepatitis B, non-A non-B hepatitis and AIDS
have hit the population seriously. Since then different virus
inactivation methods and new highly purified Factor VIII
concentrates have recently been developed which established a very
high safety standard also for plasma derived Factor VIII.
[0004] The cloning of the cDNA for Factor VIII (Wood et al. 1984.
Nature 312:330-336; Vehar et al. 1984. Nature 312:337-342) made it
possible to express Factor VIII recombinantly leading to the
development of several recombinant Factor VIII products, which were
approved by the regulatory authorities between 1992 and 2003. The
fact that the central B domain of the Factor VIII polypeptide chain
residing between amino acids Arg-740 and Glu-1649 does not seem to
be necessary for full biological activity has also led to the
development of a B domain deleted Factor VIII.
[0005] The mature Factor VIII molecule consists of 2332 amino acids
which can be grouped into three homologous A domains, two
homologous C domains and a B Domain which are arranged in the
order: A1-A2-B-A3-C1-C2. The complete amino acid sequence of mature
human Factor VIII is shown in SEQ ID NO:2. During its secretion
into plasma Factor VIII is processed intracellularly into a series
of metal-ion linked heterodimers as single chain Factor VIII is
cleaved at the B-A3 boundary and at different sites within the
B-domain. This processing leads to heterogenoeous heavy chain
molecules consisting of the A1, the A2 and various parts of the
B-domain which have a molecular size ranging from 90 kDa to 200
kDa. The heavy chains are bound via a metal ion to the light
chains, which consist of the A3, the C1 and the C2 domain (Saenko
et al. 2002. Vox Sang. 83:89-96). In plasma this heterodimeric
Factor VIII binds with high affinity to von Willebrand Factor
(vWF), which protects it from premature catabolism. The half-life
of non-activated Factor VIII bound to vWF is about 12 hours in
plasma.
[0006] Coagulation Factor VIII is activated via proteolytic
cleavage by FXa and thrombin at amino acids Arg372 and Arg740
within the heavy chain and at Arg1689 in the light chain resulting
in the release of von Willebrand Factor and generating the
activated Factor VIII heterotrimer which will form the tenase
complex on phospholipid surfaces with FIXa and FX provided that
Ca.sup.2+ is present. The heterotrimer consists of the A1 domain, a
50 kDa fragment, the A2 domain, a 43 kDa fragment and the light
chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of Factor
VIII (Factor VIIIa) consists of an A1-subunit associated through
the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light
chain and a free A2 subunit relatively loosely associated with the
A1 and the A3 domain.
[0007] To avoid excessive coagulation, Factor VIIIa must be
inactivated soon after activation. The inactivation of Factor VIIIa
via activated Protein C (APC) by cleavage at Arg336 and Arg562 is
not considered to be the major rate-limiting step. It is rather the
dissociation of the non covalently attached A2 subunit from the
heterotrimer which is thought to be the rate limiting step in
Factor VIIIa inactivation after thrombin activation (Fay et al.
1991. J. Biol. Chem. 266 8957, Fay & Smudzin 1992. J. Biol.
Chem. 267:13246-50). This is a rapid process, which explains the
short half-life of Factor VIIIa in plasma, which is only 2.1
minutes (Saenko et al. 2002. Vox Sang. 83:89-96).
[0008] In severe hemophilia A patients undergoing prophylactic
treatment Factor VIII has to be administered intravenously (i.v.)
about 3 times per week due to the short plasma half-life of Factor
VIII of about 12 hours. Each i.v. administration is cumbersome,
associated with pain and entails the risk of an infection
especially as this is mostly done at home by the patients
themselves or by the parents of children being diagnosed for
hemophilia A.
[0009] It would thus be highly desirable to create a Factor VIII
with increased functional half-life allowing the manufacturing of
pharmaceutical compositions containing Factor VIII, which have to
be administered less frequently.
[0010] Several attempts have been made to prolong the half-life of
non-activated Factor VIII either by reducing its interaction with
cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently
attaching polymers to Factor VIII (WO 94/15625, WO 97/11957 and
U.S. Pat. No. 4,970,300) or by encapsulation of Factor VIII (WO
99/55306).
[0011] In WO 97/03193 it was speculated that the introduction of
novel metal binding sites could stabilize Factor VIII and in
particular mutants in which His or Met is substituted for any of
Phe652, Tyr1786, Lys1818, Asp1840 and/or Asn1864. However no
rationale was provided how to determine the success meaning the
stabilization resulting from such modifications nor a rationale why
the proposed amino acids were chosen. This approach remains
speculative, as no further evidence was published since.
[0012] Another approach has been made in creating a Factor VIIIa,
which is inactivation resistant by first covalently attaching the
A2 domain to the A3 domain and secondly by mutating the APC
cleavage sites (Pipe & Kaufman. 1997. PNAS 94:11851-11856, WO
97/40145 and WO 03/087355.). The underlying genetic construct was
also used to produce transgenic animals as described in WO
02/072023A2. The instant variant showed still 38% of its peak
activity 4 h after thrombin activation but lacks the vWF binding
domain since by fusing the A2 to the A3 domain this particular
domain was deleted. For the reason that vWF binding significantly
prolongs half-life of FVIII in vivo, it is to be expected that
half-life of the non-activated form of the instant FVIII variant is
compromised. The inventors themselves recognized this and tried to
overcome the problem by adding an antibody which stablizes the
light chain in a conformation which retains some affinity for
vWF.
[0013] Gale et al. 2002 (Protein Science 11:2091-2101) published
the stabilization of FVa by covalently attaching the A3 domain to
the A2 domain. They identified two neighbouring amino acids
according to structural predictions, one on the A2 domain and the
other being located on the A3 domain, and replaced these two amino
acids with cysteine residues, which formed a disulfide bridge
during export into the endoplasmatic reticulum. The same approach
was used to covalently attach via disulfide bridges the A2 to the
A3 domain of Factor VIII (WO 02/103024A2). Such Factor VIII mutants
with covalently attached A3 and A2 domains, thus stabilizing
FVIIIa, retained about 90% of their initial highest activity for 40
minutes after activation whereas the activity of wild type Factor
VIII quickly diminished to 10% of its initial highest activity. The
Factor VIII mutants retained their 90% activity for additional 3 h
without any further loss of activity (Gale et al. 2003. J. Thromb.
Haemost. 1:1966-1971).
[0014] WO2006/108590 discloses several stabilized FVIII mutants
characterized by the insertion of different peptidic linkers
substituting the thrombin activation site at Arg372 also
stabilizing the activated form of FVIII. The level of FVIII
activity increased concomitantly with the length of the linker
reaching a maximum when 99 amino acids (L99) were inserted. Using a
chromogenic assay method, the FVIII activity detected with FVIII
L99 was similar to FVIII WT. Activated FVIII L99 was almost stable
during more than 1 hour.
[0015] As none of the above described approaches has yet resulted
in an improved FVIII molecule applicable in patients there is an
ongoing need to develop modified coagulation factor VIII molecules
which exhibit prolonged half-life.
[0016] In view of a potential thrombogenic risk it is more
desirable to prolong the half-life of the non-activated form of
FVIII than that of FVIIIa.
[0017] Another problem generally encountered with rec FVIII
production is poor yield. Various methods known to the man of the
art have been tried, but have not resolved such problem of poor
yield.
DESCRIPTION OF THE INVENTION
[0018] It is an objective of this invention to provide blood
coagulation molecules with enhanced in vivo half-life.
[0019] It is another objective of this invention to provide blood
coagulation molecules with improved in vivo recovery.
[0020] Another objective of the invention is that these modified
blood coagulation molecules can be expressed by mammalian cells and
retain their biological activity in the expressed modified
proteins.
[0021] Another objective of the invention is to provide an improved
yield by increased expression and/or increased stability of the
coagulation molecules in mammalian cell culture.
[0022] Yet another objective of the invention is to provide FVIII
molecules with increased stability in mammalian cell culture in
serum- and/or animal protein-free culture media, especially in the
absence of vWF.
[0023] It was now surprisingly found that inserting heterologous
polypeptides such as albumin into the FVIII molecule, preferably
such that they replace the FVIII B domain almost completely or in
part, not only permits expression and secretion of FVIII chimeric
proteins from mammalian cells but also results in modified FVIII
molecules that retain significant FVIII activity. In addition, such
modified FVIII molecules exhibit prolonged in vivo half-life and/or
improved in vivo recovery.
[0024] An additional potential benefit of those embodiments of the
present invention in which FVIII is the coagulation factor and the
A2 domain remains not covalently attached to the A3 domain after
activation is that only the half-life of the non-activated form of
FVIII is increased, whereas the half-life of the activated form of
FVIII remains essentially the same, which might result in a
decreased risk of thrombogenicity.
[0025] It was furthermore found that the FVIII molecules of the
invention are more stable than wild-type FVIII in mammalian cell
culture, especially in the absence of stabilizing von Willebrand
factor (vWF) in serum- and/or animal protein-free culture
media.
[0026] Such molecules can be generated by inserting a half-life
enhancing protein (HLEP) moiety into the amino acid sequence of the
blood coagulation factor, e.g. into the FVIII molecule. If FVIII is
the blood coagulation factor the HLEP is preferably inserted into
or replaces the B domain of FVIII or part of it.
[0027] HLEPs in the sense of the present invention are selected
from a group consisting of members of the albumin family, which
includes albumin, afamin, alpha-fetoprotein and the vitamin D
binding protein, as well as portions of an immunoglobulin constant
region and polypeptides capable of binding under physiological
conditions to members of the albumin family as well as to portions
of an immunoglobulin constant region. The most preferred HLEP is
human albumin.
[0028] Also encompassed by the invention are other proteins in
which HLEPs are inserted into other coagulation factors such as von
Willebrand factor, factor V and prothrombin factors including
factor VII, factor IX, factor X, protein C, protein S, protein Z
and prothrombin. Similar to FVIII described above the particular
HLEP, preferably albumin, is inserted in preferred embodiments at
or in the vicinity of junction sites of domains or subunits of the
coagulation factors above.
[0029] In the prior art fusions of coagulation factors to albumin
(WO 01/79271), alpha-fetoprotein (WO 2005/024044) and
immunoglobulin (WO 2004/101740) as half-life enhancing polypeptides
have been described. These were taught to be attached to the
carboxy- or the amino-terminus or to both termini of the respective
therapeutic protein moiety, occasionally linked by peptidic
linkers, preferably by linkers consisting of glycine and
serine.
[0030] Ballance et al. (WO 01/79271) described N- or C-terminal
fusion polypeptides of a multitude of different therapeutic
polypeptides fused to human serum albumin. Long lists of potential
fusion partners are described without disclosing experimental data
for almost any of these polypeptides whether or not the respective
albumin fusion proteins actually retain biological activity and
have improved properties. Among said list of therapeutic
polypeptides also Factor VIII is mentioned.
[0031] Contrary to prior art fusion proteins, the heterologous
amino acid sequence in the modified coagulation factor of this
invention is not fused to the very N-terminus or C-terminus of the
coagulation factor, but inserted within an internal region of the
amino acid sequence of the coagulation factor. Surprisingly, the
insertion of even large polypeptides did not result in a complete
loss of biological activity of the coagulation factor. Rather, the
thus modified coagulation factor had biological activity, increased
in vivo functional half-life, in vivo recovery and increased
stability.
[0032] The present invention therefore relates to a modified
coagulation factor having at an internal region of the coagulation
factor an insertion of a half-life enhancing polypeptide (HLEP),
characterized in that the modified coagulation factor has prolonged
functional half-life compared to the functional half-life of the
coagulation factor lacking said insertion, and/or compared to the
functional half-life of the wild type coagulation factor.
[0033] The present invention also relates to the insertion of more
than one HLEP wherein the HLEP, which is inserted several times,
may be the same HLEP or may be a combination of different HLEPs.
Also combinations of insertions of one or more HLEPs at an internal
region of the coagulation factor with additional N- and/or
C-terminal fusions of one or more HLEPs, which could be the same
HLEP or a combination of different HLEPs are encompassed by the
invention.
[0034] The present invention also relates to a modified coagulation
factor having at an internal region of the coagulation factor an
insertion of a half-life enhancing polypeptide (HLEP),
characterized in that the modified coagulation factor has improved
in vivo recovery compared to the in vivo recovery of the
coagulation factor lacking said insertion, and/or compared to the
in vivo recovery of the wild type coagulation factor.
[0035] In another aspect of the invention the modified coagulation
factor has increased stability in serum-free culture media,
compared to that of the coagulation factor lacking said insertion,
and/or compared to the stability of the wild type coagulation
factor. In another aspect of the invention the modified coagulation
factor has increased stability in animal protein-free culture
media, compared to that of the coagulation factor lacking said
insertion, and/or compared to the stability of the wild type
coagulation factor. The increased stability in serum-free and/or
animal-free culture media is especially pronounced if stabilizing
amounts of vWF are missing.
[0036] Animal protein-free media in the sense of the invention are
media free from proteins or protein fragments derived from
animals.
[0037] Another aspect of the invention are polynucleotides or sets
of polynucleotides encoding the modified coagulation factor of the
invention.
[0038] The invention further relates to plasmids or vectors
comprising a polynucleotide described herein, to host cells
comprising a polynucleotide or a plasmid or vector described
herein.
[0039] Another aspect of the invention is a method of producing a
modified coagulation factor, comprising: [0040] (a) culturing host
cells of the invention under conditions such that the modified
coagulation factor is expressed; and [0041] (b) optionally
recovering the modified coagulation factor from the host cells or
from the culture medium.
[0042] The invention further pertains to pharmaceutical
compositions comprising a modified coagulation factor, a
polynucleotide, or a plasmid or vector described herein.
[0043] Yet another aspect of the invention is the use of a modified
coagulation factor, a polynucleotide, or a plasmid or vector, or of
a host cell according to this invention for the manufacture of a
medicament for the treatment or prevention of a blood coagulation
disorder.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The invention pertains to a modified coagulation factor
comprising at an internal region between the N-terminal amino acid
and the C-terminal amino acid of the primary translation
polypeptide of the coagulation factor an insertion of a half-life
enhancing polypeptide (HLEP), characterized in that the modified
coagulation factor has prolonged functional half-life compared to
the functional half-life of the coagulation factor lacking said
insertion, and/or compared to the functional half-life of the wild
type coagulation factor.
[0045] The "functional half-life" according to the present
invention is the half-life of the biological function of the
coagulation factor once it has been administered to a mammal and
can be measured in vitro in blood samples taken at different time
intervals from said mammal after the coagulation factors has been
administered.
[0046] The phrases "insertion", "inserting" and "inserted" refer to
the addition of amino acids at an internal position of the
coagulation factor amino acid sequence. Other than in the case of
N-terminal or C-terminal fusion proteins, the amino acids are
according to this invention not added to the very N-terminus or
C-terminus of the coagulation factor amino acid sequence, but
inserted at an internal position within the amino acid sequence of
the coagulation factor. "Insertion" encompasses not only the
addition of amino acids (without deleting amino acids from the
coagulation factor amino acid sequence), but also the replacement
of one or more amino acids of the coagulation factor amino acid
sequence with the amino acids to be "inserted". For example, a
complete internal domain or a substantial part thereof may be
replaced with the HLEP.
[0047] In one embodiment, the modified coagulation factor has the
following structure:
N-L1-H-L2-C, [formula 1]
[0048] wherein
[0049] N is an N-terminal portion of a coagulation factor,
[0050] L1 and L2 independently are chemical bonds or linker
sequences, which linker sequences can be different linker sequences
or the same linker sequences,
[0051] H is a HLEP, and
[0052] C is a C-terminal portion of the coagulation factor.
[0053] Preferably, N comprises one or two or three or four or five
protein domains that are present at the N-terminus of the wild type
coagulation factor. C preferably comprises one or two or three or
four or five protein domains that are present at the C-terminus of
the wild type coagulation factor. In one embodiment, the wild type
coagulation factor has substantially the structure N--C. In another
embodiment, the wild type coagulation factor has substantially the
structure N-D-C, wherein D represents a domain or a part thereof
that is replaced with the HLEP in the modified coagulation factor
or in other words D represents a deletion of a part of the wild
type coagulation factor (i.e. a complete domain or part thereof)
which is replaced with the HLEP in the modified coagulation factor.
Preferred coagulation factor sequences are described infra.
Usually, the length of N+C does not exceed that of the wild type
coagulation factor.
[0054] L1 and L2 may independently be chemical bonds or linker
sequences consisting of one or more amino acids, e.g. of 1 to 20, 1
to 15, 1 to 10, 1 to 5 or 1 to 3 (e.g. 1, 2 or 3) amino acids and
which may be equal or different from each other. Usually, the
linker sequences are not present at the corresponding position in
the wild type coagulation factor. Examples of suitable amino acids
present in L1 and L2 include Gly and Ser.
[0055] Preferred HLEP sequences are described infra. The modified
coagulation factor of the invention may comprise more than one HLEP
sequence, e.g. two or three HLEP sequences. These multiple HLEP
sequences may be inserted in tandem, e.g. as successive repeats, or
they may be present at different positions of the coagulation
factor sequence including also fusions of HLEP sequences at the
very N-terminus or at the very C-terminus or at both termini of the
coagulation factor sequence, wherein at least one HLEP sequence
must be inserted at an internal position within the coagulation
factor sequence. In these embodiments, the modified coagulation
factor may have one of the following structures:
N-L1-H-L2-I-L3-H-L4-C [formula 2]
N-L1-H-L2-C-L3-H [formula 3]
H-L1-N-L2-H-L3-C [formula 4]
H-L1-N-L2-H-L3-C-L4-H [formula 5]
[0056] wherein
[0057] N is an N-terminal portion of a coagulation factor,
[0058] L1, L2, L3 and L4 independently are chemical bonds or linker
sequences, which linker sequences can be different linker sequences
or the same linker sequences,
[0059] H is a HLEP,
[0060] I is an internal sequence of the coagulation factor and
[0061] C is a C-terminal portion of the coagulation factor.
[0062] Coagulation factors may be processed proteolytically at
various stages. For example, as mentioned supra, during its
secretion into plasma single chain Factor VIII is cleaved
intracellularly at the B-A3 boundary and at different sites within
the B-domain. The heavy chain is bound via a metal ion to the light
chain having the domain structure A3-C1-C2. Factor VIII is
activated via proteolytic cleavage at amino acids Arg372 and Arg740
within the heavy chain and at Arg1689 in the light chain generating
the activated Factor VIII heterotrimer consisting of the A1 domain,
the A2 domain, and the light chain (A3-C1-C2), a 73 kDa fragment.
Thus the active form of Factor VIII (Factor VIIIa) consists of an
A1-subunit associated through the divalent metal ion linkage to a
thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit
relatively loosely associated with the A1 and the A3 domain.
[0063] Accordingly, the present invention encompasses also modified
coagulation factors that are not present as single chain
polypeptides but consist of several polypeptides (e.g. one or two
or three) that are associated with each other via non-covalent
linkages. By way of example, the structure of the modified
coagulation factor may be as follows:
N-L1-H-L2 . . . C, [formula 6]
N-L1-H . . . L2-C, [formula 7]
N-L1 . . . H-L2-C, [formula 8]
N . . . L1-H-L2-C, [formula 9]
wherein " . . . " signifies a non-covalent linkage, and the meaning
of N, L1, L2, H and C is as defined above. Cleaved forms analogous
to those of formula 6 to formula 9 of polypeptides according to
formula 2 to formula 5 are also encompassed by the invention.
[0064] Usually, the site of insertion is chosen such that the
biological activity of the coagulation factor is retained in full
or at least in part. Preferably, the biological activity of the
modified coagulation factor of the invention is at least 25%, more
preferably at least 50%, most preferably at least 75% of biological
activity of the coagulation factor lacking the insertion or of the
wild type form of the coagulation factor.
[0065] Generally, insertion between two domains of the coagulation
factor or within the vicinity of the boundary between two domains
is preferred. The two domains may be adjacent domains in the wild
type coagulation factor or not.
[0066] When referring herein to an insertion between two domains
(e.g. an "insertion between domain X and domain Y"), this
preferably means an insertion exactly between the C-terminal amino
acid of domain X and the N-terminal amino acid of domain Y.
However, an "insertion between domain X and domain Y" in the sense
of this invention may also include an insertion at an amino acid
position up to n amino acids upstream to the C-terminal amino acid
of domain X, or at an amino acid position up to n amino acids
downstream to the N-terminal amino acid of domain Y. The figure n
is an integer that should not be greater than 10%, preferably not
greater than 5% of the total number of amino acids of the domain
referred to. Usually, n is 20, preferably 15, more preferably 10,
still more preferably 5 or less (e.g. 1, 2, 3, 4 or 5).
[0067] It is also preferred that the stability of the modified
coagulation factor in serum-free medium is greater than that of the
coagulation factor lacking the insertion and/or that of the wild
type form of the coagulation factor. It is also preferred that the
stability of the modified coagulation factor in animal protein-free
medium is greater than that of the coagulation factor lacking the
insertion and/or that of the wild type form of the coagulation
factor. Preferably the increase in stability compared to the
coagulation factor lacking the insertion and/or to the wild type
form of the coagulation factor is at least 10%, more preferably at
least 25%, most preferably at least 50%. The stability of the
coagulation factor in those media can be determined as described in
example 7.
[0068] The functional half-life according to the present invention
is the half-life of the biological function of the coagulation
factor once it has been administered to a mammal and is measured in
vitro. The functional half-life of the modified coagulation factor
according to the invention is greater than that of the coagulation
factor lacking the modification as tested in the same species. The
functional half-life is preferably increased by at least 25%, more
preferably by at least 50%, and even more preferably by at least
100% compared to the coagulation factor lacking the modification
and/or to the wild type form of the coagulation factor.
[0069] The functional half-life of a modified coagulation factor
comprising a HLEP modification, can be determined by administering
the respective modified coagulation factor (and in comparison that
of the non-modified coagulation factor) to rats, rabbits or other
experimental animal species intravenously or subcutaneously and
following the elimination of the biological activity of said
modified or respectively non-modified coagulation factor in blood
samples drawn at appropriate intervals after application. Suitable
test methods are the activity tests described herein.
[0070] As a surrogate marker for the half-life of biological
activity also the levels of antigen of the modified or respectively
non-modified coagulation factor can be measured. Thus also
encompassed by the invention are modified coagulation factors
having at an internal region between the N-terminal amino acid and
the C-terminal amino acid of the primary translation polypeptide of
the coagulation factor an insertion of a half-life enhancing
polypeptide (HLEP), characterized in that the modified coagulation
factor has a prolonged half-life of the coagulation factor antigen
compared to the half-life of the coagulation factor antigen lacking
said insertion. The "half-life of the coagulation factor antigen"
according to the present invention is the half-life of the antigen
of the coagulation factor once it has been administered to a mammal
and is measured in vitro. Antigen test methods based on specific
antibodies in an enzyme immunoassay format as known to the man of
the art and commercially available (e.g. Dade Behring,
Instrumentation Laboratory, Abbott Laboratories, Diagnostica
Stago). Functional and antigen half-lives can be calculated using
the time points of the beta phase of elimination according to the
formula t.sub.1/2=ln 2/k, whereas k is the slope of the regression
line.
[0071] Once a coagulation factor is activated in vivo during
coagulation, it may be no longer desirable to maintain the
increased half-life of the now activated coagulation factor as this
might lead to thrombotic complications what is already the case for
a wild type activated coagulation factor FVIIa (Aledort 2004. J
Thromb Haemost 2:1700-1708) and what should be much more possibly
threatening if the activated factor would have an increased
half-life. It is therefore another objective of the present
invention to provide long-lived coagulation factor molecules, which
after endogenous activation in vivo or after availability of a
cofactor in vivo do have a functional half-life comparable to that
of an unmodified coagulation factor. This can be achieved by
maintaining certain cleavage sites in the modified coagulation
factor (see infra) leading to a proteolytic cleavage during
activation which separates the coagulation factor from the HLEP.
Accordingly, in one embodiment, the functional half-life of the
endogenously activated modified coagulation factor is substantially
the same as that of the activated non-modified coagulation factor
lacking the modification, and/or it is substantially the same as
that of the activated wild type coagulation factor (e.g. .+-.15%,
preferably .+-.10%).
[0072] In another embodiment, the functional half-life of the
endogenously activated modified coagulation factor is prolonged
compared to that of the activated non-modified coagulation factor
lacking the insertion, or compared to that of the activated wild
type coagulation factor. The increase may be more than 15%, for
example at least 20% or at least 50%. Again, such functional
half-life values can be measured and calculated as described for
functional half-lives supra. Increased half-lives of the
endogenously activated modified coagulation factors may be
beneficial in situations were only very low levels of the
coagulation factors are available that therefore are not
thrombogenic. Such situations may occur e.g. upon gene therapy
treatment where often only low expression rates can be achieved.
Therefore, such stabilized coagulation factors might be beneficial
in e.g. gene therapy despite a thrombogenic risk connected to such
coagulation factors if administered as proteins in high or
physiologic doses.
[0073] Half-Life Enhancing Polypeptides (HLEPs)
[0074] A "half-life enhancing polypeptide" as used herein is
selected from the group consisting of albumin, a member of the
albumin-family, the constant region of immunoglobulin G and
fragments thereof and polypeptides capable of binding under
physiological conditions to albumin, to members of the albumin
family as well as to portions of an immunoglobulin constant region.
It may be a full-length half-life-enhancing protein described
herein (e.g. albumin, a member of the albumin-family or the
constant region of immunoglobulin G) or one or more fragments
thereof that are capable of stabilizing or prolonging the
therapeutic activity or the biological activity of the coagulation
factor. Such fragments may be of 10 or more amino acids in length
or may include at least about 15, at least about 20, at least about
25, at least about 30, at least about 50, at least about 100, or
more contiguous amino acids from the HLEP sequence or may include
part or all of specific domains of the respective HLEP, as long as
the HLEP fragment provides a functional half-life extension of at
least 25% compared to a wild type coagulation factor.
[0075] The HLEP portion of the proposed coagulation factor
insertion constructs of the invention may be a variant of a normal
HLEP. The term "variants" includes insertions, deletions and
substitutions, either conservative or non-conservative, where such
changes do not substantially alter the active site, or active
domain which confers the biological activities of the modified
coagulation factors.
[0076] In particular, the proposed FVIII HLEP insertion or B domain
replacement constructs of the invention may include naturally
occurring polymorphic variants of HLEPs and fragments of HLEPs. The
HLEP may be derived from any vertebrate, especially any mammal, for
example human, monkey, cow, sheep, or pig. Non-mammalian HLEPs
include, but are not limited to, hen and salmon.
[0077] Albumin as HLEP
[0078] The terms, "human serum albumin" (HSA) and "human albumin"
(HA) and "albumin" (ALB) are used interchangeably in this
application. The terms "albumin" and "serum albumin" are broader,
and encompass human serum albumin (and fragments and variants
thereof) as well as albumin from other species (and fragments and
variants thereof).
[0079] As used herein, "albumin" refers collectively to albumin
polypeptide or amino acid sequence, or an albumin fragment or
variant, having one or more functional activities (e.g., biological
activities) of albumin. In particular, "albumin" refers to human
albumin or fragments thereof, especially the mature form of human
albumin as shown in SEQ ID NO:3 herein or albumin from other
vertebrates or fragments thereof, or analogs or variants of these
molecules or fragments thereof.
[0080] In particular, the proposed coagulation factor insertion
constructs of the invention may include naturally occurring
polymorphic variants of human albumin and fragments of human
albumin. Generally speaking, an albumin fragment or variant will be
at least 10, preferably at least 40, most preferably more than 70
amino acids long. The albumin variant may preferentially consist of
or alternatively comprise at least one whole domain of albumin or
fragments of said domains, for example domains 1 (amino acids 1-194
of SEQ ID NO:3), 2 (amino acids 195-387 of SEQ ID NO: 3), 3 (amino
acids 388-585 of SEQ ID NO: 3), 1+2 (1-387 of SEQ ID NO: 3), 2+3
(195-585 of SEQ ID NO: 3) or 1+3 (amino acids 1-194 of SEQ ID NO:
3+amino acids 388-585 of SEQ ID NO: 3). Each domain is itself made
up of two homologous subdomains namely 1-105, 120-194, 195-291,
316-387, 388-491 and 512-585, with flexible inter-subdomain linker
regions comprising residues Lys106 to Glu119, Glu292 to Val315 and
Glu492 to Ala511.
[0081] The albumin portion of the proposed coagulation factor
insertion constructs of the invention may comprise at least one
subdomain or domain of HA or conservative modifications
thereof.
[0082] Afamin, Alpha-Fetoprotein and Vitamin D Binding Protein as
HLEPs
[0083] Besides albumin, alpha-fetoprotein, another member of the
albumin family, has been claimed to enhance the half-life of an
attached therapeutic polypeptide in vivo (WO 2005/024044). The
albumin family of proteins, evolutionarily related serum transport
proteins, consists of albumin, alpha-fetoprotein (AFP; Beattie
& Dugaiczyk 1982. Gene 20:415-422), afamin (AFM; Lichenstein et
al. 1994. J. Biol. Chem. 269:18149-18154) and vitamin D binding
protein (DBP; Cooke & David 1985. J. Clin. Invest.
76:2420-2424). Their genes represent a multigene cluster with
structural and functional similarities mapping to the same
chromosomal region in humans, mice and rat. The structural
similarity of the albumin family members suggest their usability as
HLEPs. It is therefore another object of the invention to use such
albumin family members, fragments and variants thereof as HLEPs.
The term "variants" includes insertions, deletions and
substitutions, either conservative or non-conservative as long as
the desired function is still present.
[0084] Albumin family members may comprise the full length of the
respective protein AFP, AFM and DBP, or may include one or more
fragments thereof that are capable of stabilizing or prolonging the
therapeutic activity. Such fragments may be of 10 or more amino
acids in length or may include about 15, 20, 25, 30, 50, or more
contiguous amino acids of the respective protein sequence or may
include part or all of specific domains of the respective protein,
as long as the HLEP fragments provide a half-life extension of at
least 25%. Albumin family members of the insertion proteins of the
invention may include naturally occurring polymorphic variants of
AFP, AFM and DBP.
[0085] Immunoglobulins as HLEPs
[0086] Immunoglobulin G (IgG) constant regions (Fc) are known in
the art to increase the half-life of therapeutic proteins (Dumont J
A et al. 2006. BioDrugs 20:151-160). The IgG constant region of the
heavy chain consists of 3 domains (CH1-CH3) and a hinge region. The
immunoglobulin sequence may be derived from any mammal, or from
subclasses IgG1, IgG2, IgG3 or IgG4, respectively. IgG and IgG
fragments without an antigen-binding domain may also be used as
HLEPs. The therapeutic polypeptide portion is connected to the IgG
or the IgG fragments preferably via the hinge region of the
antibody or a peptidic linker, which may even be cleavable. Several
patents and patent applications describe the fusion of therapeutic
proteins to immunoglobulin constant regions to enhance the
therapeutic protein's in vivo half-lifes. US 2004/0087778 and WO
2005/001025 describe fusion proteins of Fc domains or at least
portions of immunoglobulin constant regions with biologically
active peptides that increase the half-life of the peptide, which
otherwise would be quickly eliminated in vivo. Fc-IFN-.beta. fusion
proteins were described that achieved enhanced biological activity,
prolonged circulating half-life and greater solubility (WO
2006/000448). Fc-EPO proteins with a prolonged serum half-life and
increased in vivo potency were disclosed (WO 2005/063808) as well
as Fc fusions with G-CSF (WO 2003/076567), glucagon-like peptide-1
(WO 2005/000892), clotting factors (WO 2004/101740) and
interleukin-10 (U.S. Pat. No. 6,403,077), all with half-life
enhancing properties.
[0087] Coagulation Factors
[0088] The term "coagulation factor" as used herein denotes a blood
coagulation factor or blood clotting factor. Coagulation factors
include factor VIII, von Willebrand factor, prothrombin factors
(comprising factor VII, Factor IX, factor X, protein C, protein S,
protein Z and prothrombin) and coagulation factor V.
[0089] Coagulation factors of the present invention may also be
variants of wild-type coagulation factors. The term "variants"
includes insertions, deletions and substitutions, either
conservative or non-conservative, where such changes do not
substantially alter the active site, or active domain, which
confers the biological activities of the respective coagulation
factor.
[0090] FVIII
[0091] The terms "blood coagulation Factor VIII", "Factor VIII" and
FVIII'' are used interchangeably herein. "Blood coagulation Factor
VIII" includes wild type blood coagulation Factor VIII as well as
derivatives of wild type blood coagulation Factor VIII having the
procoagulant activity of wild type blood coagulation Factor VIII.
Derivatives may have deletions, insertions and/or additions
compared with the amino acid sequence of wild type Factor VIII. The
term FVIII includes proteolytically processed forms of Factor VIII,
e.g. the form before activation, comprising heavy chain and light
chain.
[0092] The term "Factor VIII" includes any Factor VIII variants or
mutants having at least 10%, preferably at least 25%, more
preferably at least 50%, most preferably at least 75% of the
biological activity of wild type factor VIII.
[0093] As non-limiting examples, Factor VIII molecules include
Factor VIII mutants preventing or reducing APC cleavage (Amano
1998. Thromb. Haemost. 79:557-563), Factor VIII mutants further
stabilizing the A2 domain (WO 97/40145), FVIII mutants resulting in
increased expression (Swaroop et al. 1997. JBC 272:24121-24124),
Factor VIII mutants reducing its immunogenicity (Lollar 1999.
Thromb. Haemost. 82:505-508), FVIII reconstituted from differently
expressed heavy and light chains (Oh et al. 1999. Exp. Mol. Med.
31:95-100), FVIII mutants reducing binding to receptors leading to
catabolism of FVIII like HSPG (heparan sulfate proteoglycans)
and/or LRP (low density lipoprotein receptor related protein)
(Ananyeva et al. 2001. TCM, 11:251-257), disulfide bond-stabilized
FVIII variants (Gale et al., 2006. J. Thromb. Hemost. 4:1315-1322),
FVIII mutants with improved secretion properties (Miao et al.,
2004. Blood 103:3412-3419), FVIII mutants with increased cofactor
specific activity (Wakabayashi et al., 2005. Biochemistry
44:10298-304), FVIII mutants with improved biosynthesis and
secretion, reduced ER chaperone interaction, improved ER-Golgi
transport, increased activation or resistance to inactivation and
improved half-life (summarized by Pipe 2004. Sem. Thromb. Hemost.
30:227-237). All of these factor VIII mutants and variants are
incorporated herein by reference in their entirety.
[0094] A suitable test to determine the biological activity of
Factor VIII is the one stage or the two stage coagulation assay
(Rizza et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom
ed. The Hemophilias. NY Churchchill Livingston 1992) or the
chromogenic substrate FVIII:C assay (S. Rosen, 1984. Scand J
Haematol 33: 139-145, suppl.). The content of these references is
incorporated herein by reference.
[0095] The cDNA sequence and the amino acid sequence of the mature
wild type form of human blood coagulation Factor VIII are shown in
SEQ ID NO:1 and SEQ ID NO:2, respectively. The reference to an
amino acid position of a specific sequence means the position of
said amino acid in the FVIII wild-type protein and does not exclude
the presence of mutations, e.g. deletions, insertions and/or
substitutions at other positions in the sequence referred to. For
example, a mutation in "Glu2004" referring to SEQ ID NO:2 does not
exclude that in the modified homologue one or more amino acids at
positions 1 through 2332 of SEQ ID NO:2 are missing.
[0096] FVIII Proteins with a HLEP Insertion
[0097] Modified FVIII proteins of the invention in the most general
sense are characterized in that they comprise FVIII molecules with
a HLEP integrated into the FVIII molecules such that the HLEP does
not reduce the molar specific FVIII activity of the chimeric
protein below about 10% of the molar specific FVIII activity of
wild type FVIII. The insertion of the HLEP can take place in any
place between the N-terminal and the C-terminal amino acid of the
FVIII sequence. Preferentially the HLEP is integrated between
domains of the wild-type FVIII protein.
[0098] The domains of FVIII comprise the following amino acid
positions (amino acid numbers refer to SEQ ID NO:2):
[0099] A1: . . . 1-336
[0100] a1: . . . 337-372
[0101] A2: . . . 373-710
[0102] a2: . . . 711-740
[0103] B: . . . 741-1648
[0104] a3: . . . 1649-1689
[0105] A3: . . . 1690-2019
[0106] C1: . . . 2020-2172
[0107] C2: . . . 2173-2332
[0108] Preferred integration sites for a HLEP within the FVIII
molecule are defined as such sites where the insertion of a HLEP
moiety has the least negative effect on FVIII functional activity.
Potential integration sites include, but are not limited to, the
region between the C-terminus of acidic region 1 (a1) and the
N-terminus of the A2 domain, the region between the C-terminus of
the A3 domain and the N-terminus of the C1 domain, the region
between the C-terminus of the C1 domain and the N-terminus of the
C2 domain and preferably the region of the B domain, where the B
domain may be replaced partially or in its entirety (FIG. 2).
[0109] In a preferred embodiment of the invention chimeric FVIII
proteins of the invention are characterized in that they comprise
FVIII molecules with partial or full deletion of the B domain and a
HLEP integrated into the FVIII molecules such that the HLEP is
inserted between a functional A1/A2 domain at its amino terminus
and a functional A3/C1/C2 domain at its carboxy terminus.
[0110] It was found that it is possible to insert HLEPs or HLEP
derivatives within the B domain (the FVIII sequence between the A2
and A3 domains [amino acids 741 to 1648] which seems dispensable
for the biological function of FVIII (Pittman et al. 1992. Blood
81:2925-2935) to provide FVIII molecules with new and improved
properties while retaining FVIII biological activity. The B domain
has a length of about 900 amino acids and the HLEP may either be
inserted at any place within the B domain without any deletion of
the B domain or the B domain may be replaced by a HLEP partially or
in its entirety. Partial deletion refers to deletions of at least 1
amino acid, preferably to deletions of 100 to 600 amino acids and
most preferred to deletions of more than 600 amino acids of the B
domain (FIG. 2e-h).
[0111] In a preferred embodiment of the invention most of the B
domain is replaced by a HLEP, while a few amino acids of the amino
and carboxy terminal sequence of the B domain containing processing
sites important for cleavage and activation of the FVIII molecules
of the invention are conserved (FIGS. 1a,b,d and 2h-i). Preferably
about 1 to 20 amino acids, more preferably 3 to 10 amino acids, at
the C- and at the N-terminus of the B domain, which are required to
conserve the processing sites for thrombin at amino acid position
740 of the FVIII sequence (SEQ ID NO 2) and the protease cleaving
between the B domain and the A3 domain during the secretion
process, are maintained within the FVIII molecule of the invention
(FIG. 1 and FIG. 2h). Alternatively, the amino acids retained from
the B domain might be replaced by artificial cleavage sites. A
PACE/Furin cleavage site (Nakayama 1997. Biochem. J. 327:625-635)
may be used to guide the processing during secretion, and
artificial thrombin cleavage sites as described in WO 2004/005347
(FIG. 1c) or other protease cleavage sites may be introduced for
activation processing (FIG. 1e).
[0112] Another aspect of the invention is the insertion of more
than one HLEP wherein the HLEP, which is inserted several times,
may be the same HLEP or may be a combination of different HLEPs.
Also combinations of insertions of one or more HLEPs into FVIII
with additional N- and/or C-terminal fusions of one or more HLEPs,
which could be the same HLEP or a combination of different HLEPs
are encompassed by the invention.
[0113] Once a coagulation factor is endogenously activated during
coagulation in vivo, it may be no longer desirable to maintain the
increased functional half-life of the now activated coagulation
factor as this might lead to thrombotic complications what is
already the case for a wild type activated coagulation factor as
FVIIa (Aledort 2004. J Thromb Haemost 2:1700-1708) and what should
be much more relevant if the activated factor would have an
increased functional half-life. It is therefore another objective
of the present invention to provide long-lived coagulation factor
VIII molecules, which after endogenous activation in vivo or after
availability of a cofactor do have a functional half-life
comparable to that of unmodified FVIII. This can by way of
non-limiting example be achieved by maintaining the cleavage sites
for thrombin at amino acid position 740 of the FVIII sequence (SEQ
ID NO 2) and for the protease cleaving between the B domain and the
A3 domain during the secretion process. With such FVIII-HLEP
connecting sequences the activation of the FVIII chimeric protein
of the invention will lead to a concomitant complete separation of
FVIIIa from the HLEP moiety.
[0114] In yet another embodiment of the invention, however, one or
more of the proteolytical cleavage sites, preferably the thrombin
cleavage sites at Arg740 (e.g. FIG. 2i) and/or Arg372, are mutated
or deleted in order to prevent cleavage and result in an insertion
protein which displays improved properties like enhanced functional
half-life even as an activated molecule.
[0115] In another embodiment of the invention the deletion of the B
domain may be extended into the flanking acidic regions a2 and a3
(FIG. 2 k and l). With regard to a2 this region may be deleted in
part (FIG. 2k) or completely. Therefore the HLEP moiety will not be
released upon FVIII activation but instead remain attached to the
A2 domain. Such an activated insertion protein will have an
enhanced functional half-life. Acidic region a3 may be deleted in
part (FIG. 2l) as long as the vWF binding properties of a3 remain
unaffected.
[0116] In one embodiment of the invention another potential
integration site within the FVIII molecule is represented by the
region between the C-terminus of acidic region 1 (a1)) and the
N-terminus of the A2 domain (FIG. 2a-d). FIG. 2a describes an
integration scheme where an additional thrombin cleavage site has
been introduced at the albumin C-terminus. In such an insertion
protein the HLEP moiety will be cleaved off during endogenous FVIII
activation in vivo and the activated FVIII molecule will have a
functional half-life comparable to wild-type FVIII. In the case of
an insertion protein as depicted in FIG. 2b the additional thrombin
cleavage site at the HLEP C-terminus is lacking. Therefore the HLEP
will not be released upon FVIII activation but instead remain
attached to the A2 domain. Such an activated insertion protein will
have an enhanced functional half-life. In the case of an insertion
protein as depicted in FIG. 2c the thrombin cleavage site at Arg372
is lacking. Therefore the HLEP will not be released upon FVIII
activation but instead remain attached to the A1 domain. Such an
activated insertion protein will have an enhanced half-life. An
insertion protein as depicted in FIG. 2d will keep A1 and A2
domains covalently linked and generate an insertion protein with
functional half-life extension also of the activated form.
[0117] In another embodiment of the invention another potential
integration site within the FVIII molecule is represented by the
region between the C-terminus of the A3 domain and the N-terminus
of the C1 domain (FIG. 2m). In such an insertion protein the HLEP
moiety will be an integral component of the FVIII light chain and
both the non-activated and the activated insertion protein will
have enhanced functional half-lives.
[0118] In another embodiment of the invention another potential
integration site within the FVIII molecule is represented by the
region between the C-terminus of the C1 domain and the N-terminus
of the C2 domain (FIG. 2n). In such an insertion protein the HLEP
moiety will be an integral component of the FVIII light chain and
both the non-activated and the activated insertion protein will
have enhanced functional half-lives.
[0119] In another embodiment of the invention the FVIII proteins of
the invention may be expressed as two separate chains (see
infra).
[0120] The modified coagulation factor VIII according to this
invention may be a single chain polypeptide, or it may be composed
of two or three polypeptide chains that are associated via
non-covalent linkages, due to proteolytic processing.
[0121] In another embodiment of the invention, the amino acids at
or near the PACE/Furin cleavage site (Arg1648, e.g. FIG. 1a) are
mutated or deleted in order to prevent cleavage by PACE/Furin. This
is thought to result in a one-chain Factor VIII/HLEP fusion
molecule with improved half-life.
[0122] In one embodiment of the invention, the modified FVIII of
the invention exhibits an increased functional half-life compared
to the corresponding FVIII form containing no integrated HLEP
and/or to the wild type form FVIII. The functional half-life e.g.
can be determined in vivo in animal models of hemophilia A, like
FVIII knockout mice, in which one would expect a longer lasting
hemostatic effect as compared to wild type FVIII. The hemostatic
effect could be tested for example by determining time to arrest of
bleeding after a tail clip.
[0123] The functional half-life is preferably increased by at least
25%, more preferably by at least 50%, and even more preferably by
at least 100% compared to the form without inclusion of a HLEP
and/or to the wild type form of FVIII.
[0124] In another embodiment of the invention, the modified FVIII
of the invention exhibits an improved in vivo recovery compared to
the corresponding FVIII form containing no integrated HLEP and/or
to the wild type form FVIII. The in vivo recovery can be determined
in vivo in normal animals or in animal models of hemophilia A, like
FVIII knockout mice, in which one would expect an increased
percentage of the modified FVIII of the invention be found by
antigen or activity assays in the circulation shortly (5 to 10
min.) after i.v. administration compared to the corresponding FVIII
form containing no integrated HLEP and/or to the wild type form
FVIII.
[0125] The in vivo recovery is preferably increased by at least
10%, more preferably by at least 20%, and even more preferably by
at least 40% compared to the form without inclusion of a HLEP
and/or to the wild type form of FVIII.
[0126] In yet another embodiment of the invention immunoglobulin
constant regions or portions thereof are used as HLEPs. Preferably
the Fc region comprised of a CH2 and CH3 domain and a hinge region
of an IgG, more preferably of an IgG1 or fragments or variants
thereof are used, variants including mutations which enhance
binding to the neonatal Fc receptor (FcRn). The Fc region is not
used to generate monomeric or dimeric Fc insertions as described in
the art, but rather is inserted into the FVIII molecule such that
part of the FVIII molecule is fused to its N-terminus and another
part is fused to its C-terminus (FIG. 2a-n). In a preferred
embodiment of the invention an unfused Fc region is coexpressed
from another expression vector or even from the same expression
vector which through disulfide bridge linking forms a Fc
heterodimer with the Fc region within the chimeric FVIII
molecule.
[0127] In addition to the extension of functional half-life of
FVIII, HLEP moieties as described in this invention may also be
used for insertion into other multi-domain proteins for the same
purpose of half-life extension.
[0128] Therefore the invention also encompasses other modified
proteins, preferably modified coagulation factors, with insertions
of HLEP moieties within their amino acid sequence.
[0129] Von Willebrand Factor
[0130] Von Willebrand factor (vWF) is a multimeric plasma
glycoprotein with a prominent role in primary hemostasis. The
mature protein consists of 2050 amino acids and is composed of
homologous domains arranged in the order
D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK. The amino acid sequence and
the cDNA sequence of wild type vWF are disclosed in Collins et al.
1987. Proc Natl. Acad. Sci. USA 84:4393-4397. The term "von
Willebrand factor" includes any mutants and variants of wild type
vWF having at least 10%, preferably at least 25%, more preferably
at least 50%, most preferably at least 75% of the biological
activity of wild type vWF. The biological activity of wild type vWF
can be determined by the man of the art using methods for
ristocetin co-factor activity (Federici A B et al. 2004.
Haematologica 89:77-85), binding of vWF to GP lba of the platelet
glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb
Hemost. 12:305-310), or a collagen binding assay (Kailas &
Talpsep. 2001. Annals of Hematology 80:466-471).
[0131] One or more HLEPs may be inserted into the vWF molecule.
HLEP insertion is chosen as not to interfere with the binding
capabilities of vWF to e.g. FVIII, platelets, Heparin or collagen.
Suitable insertion sites include, but are not limited to, the D3-A1
junction, the D4-B1 junction, the C2-CK junction as well as A2,
into which a HLEP moiety may be inserted upon partial or complete
removal of the A2 domain. VWF functional activities may be assessed
as described supra.
[0132] Prothrombin Factors
[0133] Prothrombin factors, including factor VII (FVII), factor IX
(FIX), factor X (FX), protein C (PC), protein S, protein Z and
prothrombin (PT) are a family of proteins characterized by a gla
domain containing y-carboxylated glutamic acid residues and EGF- or
Kringle domains on the light chain, which is separated from the
heavy chain containing the trypsin protease domain (two laminin-G
domains for protein S) by a short intervening sequence which is
cleaved upon activation of the protein.
[0134] The amino acid sequences and the cDNA sequences of these
coagulation factors are known in the art and are disclosed for
example in the PubMed protein sequence library
(http://www.ncbi.nlm.nih.gov/entrez/querv.fcgi?db=Protein) with
accession numbers NP.sub.--000122 (FVII), NP.sub.--000124 (FIX),
NP.sub.--000495 (FX), NP.sub.--000303 (PC), NP.sub.--000304
(Protein S), NP.sub.--003882 (Protein Z) and NP.sub.--000497
(Prothrombin).
[0135] Also prothrombin factors may be stabilized by the insertion
of a HLEP moiety as described in this invention. Prothrombin
factors include factor VII (FVII), factor IX (FIX), factor X (FX),
protein C(PC), protein S, protein Z and prothrombin (PT). As
described supra, prothrom bin factors are characterized by a gla
domain containing y-carboxylated glutamic acid residues and EGF- or
Kringle domains on the light chain, which is separated from the
heavy chain containing the trypsin protease domain (two laminin-G
domains for protein S) by a short intervening sequence which is
cleaved upon activation of the protein. This peptide sequence is
the preferred integration site for a HLEP moiety. Preferably, the
HLEP is inserted such that the activation cleavage is not hampered
by maintaining the natural activation sequence or by inserting
artificial cleavage sites like a PACE/Furin cleavage site (Nakayama
1997. Biochem. J. 327:625-635), an artificial thrombin cleavage
site (as described in WO 2004/005347) or another suitable protease
cleavage site. The conservation of the activity of the respective
prothrombin factor after HLEP insertion may be assessed by assays
known to the man of the art. FVII activity may be determined using
a commercially available chromogenic test kit (Chromogenix Coaset
FVII) based on the method described by Seligsohn et al. (1978.
Blood 52:978-988) and FVIIa activity can be determined using the
STACLOT.RTM. FVIIa-rTF kit (Diagnostica Stago) based on the method
described by Morissey et al. (1993. Blood 81:734-744). FIX activity
may be assessed by a clotting assay as described by Chavin &
Weidner (1984. J. Biol. Chem. 259:3387-3390). FX activity may be
measured using a chromogenic assay as described by Van Wijk et al.
(1981. Thromb. Res. 22:681-686). Protein C activity may be assessed
by a chromogenic assay as supplied by Instrumentation Laboratory
(HaemoslL Protein C) based on the method described by Comb et al.
(1984. Blood 63:15-21) and protein S activity by a method described
by Heeb et al. (2006. J. Thromb. Haemost. 4:385-391). Petrovan et
al. (1999. Am. J. Clin. Pathol. 112:705-711 describe an activity
assay for prothrombin and Tabatabai et al. (2001. Thromb. Haemost.
85:655-660) published a protein Z activity assay.
[0136] Coagulation factor V
[0137] Coagulation factor V (FV) is a high molecular weight plasma
glycoprotein that participates as a cofactor in the activation of
Prothrombin by factor Xa. It is homologous to factor VIII and
Ceruloplasmin and has a similar domain structure of
A1-A2-B-A3-C1-C2. The amino acid sequence and the cDNA sequence of
wild type FV are disclosed for example in PubMed with accession
numbers NP.sub.--000121 and NM.sub.--000130, respectively.
[0138] As described above for Factor VIII, HLEP moieties could be
inserted into the FV molecule for half-life extension at comparable
inter-domain sites, preferably into the B domain or replacing part
or all of the B domain. The FV activity can be assessed as
described by Bick et al. (1973. Beitr. Pathol. 150:311-315).
[0139] Polynucleotides
[0140] The invention further relates to a polynucleotide encoding a
modified coagulation factor, preferably a modified FVIII variant as
described in this application. The term "polynucleotide(s)"
generally refers to any polyribonucleotide or
polydeoxyribonucleotide that may be unmodified RNA or DNA or
modified RNA or DNA. The polynucleotide may be single- or
double-stranded DNA, single or double-stranded RNA. As used herein,
the term "polynucleotide(s)" also includes DNAs or RNAs that
comprise one or more modified bases and/or unusual bases, such as
inosine. It will be appreciated that a variety of modifications may
be made to DNA and RNA that serve many useful purposes known to
those of skill in the art. The term "polynucleotide(s)" as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including, for example, simple and complex cells.
[0141] The skilled person will understand that, due to the
degeneracy of the genetic code, a given polypeptide can be encoded
by different polynucleotides. These "variants" are encompassed by
this invention.
[0142] Preferably, the polynucleotide of the invention is an
isolated polynucleotide. The term "isolated" polynucleotide refers
to a polynucleotide that is substantially free from other nucleic
acid sequences, such as and not limited to other chromosomal and
extrachromosomal DNA and RNA. Isolated polynucleotides may be
purified from a host cell. Conventional nucleic acid purification
methods known to skilled artisans may be used to obtain isolated
polynucleotides. The term also includes recombinant polynucleotides
and chemically synthesized polynucleotides.
[0143] The invention further relates to a group of polynucleotides
which together encode the modified coagulation factor of the
invention. A first polynucleotide in the group may encode the
N-terminal part of the modified coagulation factor, and a second
polynucleotide may encode the C-terminal part of the modified
coagulation factor.
[0144] Yet another aspect of the invention is a plasmid or vector
comprising a polynucleotide according to the invention. Preferably,
the plasmid or vector is an expression vector. In a particular
embodiment, the vector is a transfer vector for use in human gene
therapy.
[0145] The invention also relates to a group of plasmids or vectors
that comprise the above group of polynucleotides. A first plasmid
or vector may contain said first polynucleotide, and a second
plasmid or vector may contain said second polynucleotide. By way of
example, and with reference to coagulation factor VIII, the coding
sequences of the signal peptide, the A1 and A2 domains, the B
domain sequence remainder and the HLEP may be cloned into the first
expression vector and the coding sequences of A3, C1 and C2 with an
appropriate signal peptide sequence may be cloned into the second
expression vector (FIG. 20). Both expression vectors are
cotransfected into a suitable host cell, which will lead to the
expression of the light and heavy chains of the FVIII molecule of
the invention and the formation of a functional protein.
[0146] Alternatively, the coding sequence of the FVIII signal
peptide, the A1 and A2 domains are cloned into the first expression
vector and the coding sequences of the HLEP, FVIII A3, C1 and C2
with an appropriate signal peptide sequence are cloned into the
second expression vector (FIG. 2p). Both expression vectors are
cotransfected into a suitable host cell, which will lead to the
expression of the light and heavy chains of the FVIII molecule of
the invention and the formation of a functional protein.
[0147] Alternatively, both coding sequences are cloned into one
expression vector either using two separate promoter sequences or
one promoter and an internal ribosome entry site (IRES) element to
direct the expression of both FVIII chains.
[0148] Still another aspect of the invention is a host cell
comprising a polynucleotide, a plasmid or vector of the invention,
or a group of polynucleotides or a group of plasmids or vectors as
described herein.
[0149] The host cells of the invention may be employed in a method
of producing a modified coagulation factor, preferably a modified
FVIII molecule, which is part of this invention. The method
comprises: [0150] (a) culturing host cells of the invention under
conditions such that the desired insertion protein is expressed;
and [0151] (b) optionally recovering the desired insertion protein
from the host cells or from the culture medium.
[0152] It is preferred to purify the modified coagulation factors
of the present invention to .gtoreq.80% purity, more preferably
.gtoreq.95% purity, and particularly preferred is a
pharmaceutically pure state that is greater than 99.9% pure with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, an isolated or purified modified coagulation
factor of the invention is substantially free of other, non-related
polypeptides.
[0153] The various products of the invention are useful as
medicaments. Accordingly, the invention relates to a pharmaceutical
composition comprising a modified coagulation factor, preferably
the modified FVIII molecule as described herein, a polynucleotide
of the invention, or a plasmid or vector of the invention.
[0154] The invention also concerns a method of treating an
individual suffering from a blood coagulation disorder such as
hemophilia A or B. The method comprises administering to said
individual an efficient amount of the modified coagulation factor,
preferably modified FVIII or FIX as described herein. In another
embodiment, the method comprises administering to the individual an
efficient amount of a polynucleotide of the invention or of a
plasmid or vector of the invention. Alternatively, the method may
comprise administering to the individual an efficient amount of the
host cells of the invention described herein.
[0155] The invention also relates to polynucleotides and their use
encoding the modified VWF and Prothrombin factor variants as
described above.
[0156] Expression of the Proposed Mutants
[0157] The production of recombinant mutant proteins at high levels
in suitable host cells requires the assembly of the above-mentioned
modified cDNAs into efficient transcriptional units together with
suitable regulatory elements in a recombinant expression vector
that can be propagated in various expression systems according to
methods known to those skilled in the art. Efficient
transcriptional regulatory elements could be derived from viruses
having animal cells as their natural hosts or from the chromosomal
DNA of animal cells. Preferably, promoter-enhancer combinations
derived from the Simian Virus 40, adenovirus, BK polyoma virus,
human cytomegalovirus, or the long terminal repeat of Rous sarcoma
virus, or promoter-enhancer combinations including strongly
constitutively transcribed genes in animal cells like beta-actin or
GRP78 can be used. In order to achieve stable high levels of mRNA
transcribed from the cDNAs, the transcriptional unit should contain
in its 3'-proximal part a DNA region encoding a transcriptional
termination-polyadenylation sequence. Preferably, this sequence is
derived from the Simian Virus 40 early transcriptional region, the
rabbit beta-globin gene, or the human tissue plasminogen activator
gene.
[0158] The cDNAs are then integrated into the genome of a suitable
host cell line for expression of the Factor VIII proteins.
Preferably this cell line should be an animal cell-line of
vertebrate origin in order to ensure correct folding, disulfide
bond formation, asparagine-linked glycosylation and other
post-translational modifications as well as secretion into the
cultivation medium. Examples on other post-translational
modifications are tyrosine O-sulfation and proteolytic processing
of the nascent polypeptide chain. Examples of cell lines that can
be use are monkey COS-cells, mouse L-cells, mouse C127-cells,
hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster
CHO-cells.
[0159] The recombinant expression vector encoding the corresponding
cDNAs can be introduced into an animal cell line in several
different ways. For instance, recombinant expression vectors can be
created from vectors based on different animal viruses. Examples of
these are vectors based on baculovirus, vaccinia virus, adenovirus,
and preferably bovine papilloma virus.
[0160] The transcription units encoding the corresponding DNA's can
also be introduced into animal cells together with another
recombinant gene which may function as a dominant selectable marker
in these cells in order to facilitate the isolation of specific
cell clones which have integrated the recombinant DNA into their
genome. Examples of this type of dominant selectable marker genes
are Tn5 amino glycoside phosphotransferase, conferring resistance
to geneticin (G418), hygromycin phosphotransferase, conferring
resistance to hygromycin, and puromycin acetyl transferase,
conferring resistance to puromycin. The recombinant expression
vector encoding such a selectable marker can reside either on the
same vector as the one encoding the cDNA of the desired protein, or
it can be encoded on a separate vector which is simultaneously
introduced and integrated to the genome of the host cell,
frequently resulting in a tight physical linkage between the
different transcription units.
[0161] Other types of selectable marker genes which can be used
together with the cDNA of the desired protein are based on various
transcription units encoding dihydrofolate reductase (dhfr). After
introduction of this type of gene into cells lacking endogenous
dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44), it will
enable these to grow in media lacking nucleosides. An example of
such a medium is Ham's F12 without hypoxanthine, thymidin, and
glycine. These dhfr-genes can be introduced together with the
Factor VIII cDNA transcriptional units into CHO-cells of the above
type, either linked on the same vector or on different vectors,
thus creating dhfr-positive cell lines producing recombinant
protein.
[0162] If the above cell lines are grown in the presence of the
cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to
methotrexate will emerge. These cell lines may produce recombinant
protein at an increased rate due to the amplified number of linked
dhfr and the desired protein's transcriptional units. When
propagating these cell lines in increasing concentrations of
methotrexate (1-10000 nM), new cell lines can be obtained which
produce the desired protein at very high rate.
[0163] The above cell lines producing the desired protein can be
grown on a large scale, either in suspension culture or on various
solid supports. Examples of these supports are micro carriers based
on dextran or collagen matrices, or solid supports in the form of
hollow fibres or various ceramic materials. When grown in cell
suspension culture or on micro carriers the culture of the above
cell lines can be performed either as a bath culture or as a
perfusion culture with continuous production of conditioned medium
over extended periods of time. Thus, according to the present
invention, the above cell lines are well suited for the development
of an industrial process for the production of the desired
recombinant mutant proteins
[0164] Purification and Formulation
[0165] The recombinant mutant protein, which accumulates in the
medium of secreting cells of the above types, can be concentrated
and purified by a variety of biochemical and chromatographic
methods, including methods utilizing differences in size, charge,
hydrophobicity, solubility, specific affinity, etc. between the
desired protein and other substances in the cell cultivation
medium.
[0166] An example of such purification is the adsorption of the
recombinant mutant protein to a monoclonal antibody, directed to
e.g. a HLEP, preferably human albumin, or directed to the
respective coagulation factor, which is immobilised on a solid
support. After adsorption of the FVIII mutant to the support,
washing and desorption, the protein can be further purified by a
variety of chromatographic techniques based on the above
properties. The order of the purification steps is chosen e.g.
according to capacity and selectivity of the steps, stability of
the support or other aspects. Preferred purification steps e.g. are
but are not limited to ion exchange chromatography steps, immune
affinity chromatography steps, affinity chromatography steps,
hydrophobic interaction chromatography steps, dye chromatography
steps, and size exclusion chromatography steps.
[0167] In order to minimize the theoretical risk of virus
contaminations, additional steps may be included in the process
that allow effective inactivation or elimination of viruses. Such
steps e.g. are heat treatment in the liquid or solid state,
treatment with solvents and/or detergents, radiation in the visible
or UV spectrum, gamma-radiation or nanofiltration.
[0168] The modified polynucleotides (e.g. DNA) of this invention
may also be integrated into a transfer vector for use in the human
gene therapy.
[0169] The various embodiments described herein may be combined
with each other. The present invention will be further described in
more detail in the following examples thereof. This description of
specific embodiments of the invention will be made in conjunction
with the appended figures.
[0170] The insertion proteins as described in this invention can be
formulated into pharmaceutical preparations for therapeutic use.
The purified protein may be dissolved in conventional
physiologically compatible aqueous buffer solutions to which there
may be added, optionally, pharmaceutical excipients to provide
pharmaceutical preparations.
[0171] Such pharmaceutical carriers and excipients as well as
suitable pharmaceutical formulations are well known in the art (see
for example "Pharmaceutical Formulation Development of Peptides and
Proteins", Frokjaer et al., Taylor & Francis (2000) or
"Handbook of Pharmaceutical Excipients", 3.sup.rd edition, Kibbe et
al., Pharmaceutical Press (2000)). In particular, the
pharmaceutical composition comprising the polypeptide variant of
the invention may be formulated in lyophilized or stable liquid
form. The polypeptide variant may be lyophilized by a variety of
procedures known in the art. Lyophilized formulations are
reconstituted prior to use by the addition of one or more
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0172] Formulations of the composition are delivered to the
individual by any pharmaceutically suitable means of
administration. Various delivery systems are known and can be used
to administer the composition by any convenient route.
Preferentially, the compositions of the invention are administered
systemically. For systemic use, insertion proteins of the invention
are formulated for parenteral (e.g. intravenous, subcutaneous,
intramuscular, intraperitoneal, intracerebral, intrapulmonar,
intranasal or transdermal) or enteral (e.g., oral, vaginal or
rectal) delivery according to conventional methods. The most
preferential routes of administration are intravenous and
subcutaneous administration. The formulations can be administered
continuously by infusion or by bolus injection. Some formulations
encompass slow release systems.
[0173] The insertion proteins of the present invention are
administered to patients in a therapeutically effective dose,
meaning a dose that is sufficient to produce the desired effects,
preventing or lessening the severity or spread of the condition or
indication being treated without reaching a dose which produces
intolerable adverse side effects. The exact dose depends on many
factors as e.g. the indication, formulation, mode of administration
and has to be determined in preclinical and clinical trials for
each respective indication.
[0174] The pharmaceutical composition of the invention may be
administered alone or in conjunction with other therapeutic agents.
These agents may be incorporated as part of the same
pharmaceutical. One example of such an agent is von Willebrand
factor.
[0175] FIG. 1 shows the replacement of FVIII B domain by albumin.
cDNA organisation of FVIII wild-type (FVIII wt) and FVIII with the
B domain replacement by albumin (FVIII-HA) are outlined. Transition
sequences and the remaining amino acids of the B domain in the
FVIII-HA constructs are shown. Amino acid numbering refers to the
FVIII wild-type sequence as outlined in SEQ ID NO:2. The C1636S
amino acid exchange in DNA pF8-1211 and the R740 deletion in
pF8-1413 are indicated.
[0176] FIG. 2 schematically shows various embodiments of the cDNA
encoding the modified Factor VIII polypeptides of the present
invention. The HLEP may be inserted at various positions within the
FVIII sequence, as described supra.
[0177] FIG. 3 shows the pharmacokinetic profile of two modified
FVIII molecules with albumin integrated and partial deletion of the
B-domain (DNA pF8-1211 and pF8-1413, see FIG. 1) in comparison to
wild type FVIII (see example 5).
EXAMPLES
Example 1
Generation of Expression Vectors for FVIII Molecules with Albumin
Replacing the FVIII B Domain
[0178] An expression plasmid based on pIRESpuro3 (BD Biosciences)
containing the full length FVIII cDNA sequence in its multiple
cloning site (pF8-FL) was first used to delete the majority of the
B domain sequence and create a restriction site for insertion of
foreign sequences. For that oligonucleotides We1356 and We1357 (SEQ
ID NO. 5 and 6) were used in a PCR reaction using pF8-FL as a
template to amplify a part of the A2 domain and the N-terminus of
the B domain (fragment 1) and oligonucleotides We1358 and We1359
(SEQ ID NO. 7 and 8) were used in another PCR reaction using pF8-FL
as a template to amplify the C-terminus of the B domain, the A3
domain and part of the C1 domain (fragment 2). Both fragments were
gel purified. Fragment 1 was subsequently digested with restricion
endonucleases PinAl and BamH1, fragment 2 was digested with
restriction endonucleases PinAl and BspEl; both fragments were then
purified and ligated into pF8-FL, where the BamH1/BspEl fragment
encompassing part of the A2 domain, the B and A3 domains and part
of the C1 domain had been removed. The resulting plasmid, pF8-DB,
now basically contained a major B domain deletion with a remainder
of N- and C-terminal B domain sequences joined by a PinAl site.
Into this site a human albumin fragment was inserted, which had
been generated by PCR amplification on albumin cDNA using primers
We2502 and We2503 (SEQ ID NO. 9 and 10), PinAl digestion and
purification. To remove the PinAl sites the resulting plasmid was
subjected to two rounds of site-directed mutagenesis according to
standard protocols (QuickChange XL Site Directed Mutagenesis Kit,
Stratagene). For this oligonucleotides We2504 and We2505 (SEQ ID
NO. 11 and 12) were used as mutagenic primers in the first round,
and oligonucleotides We2506 and We2507 (SEQ ID NO. 13 and 14) were
used in the second round of mutagenesis. The final expression
plasmid was designated pF8-1210. In order to remove a free cysteine
residue (amino acid 1636, SEQ ID NO. 2 and FIG. 1) site-directed
mutagenesis was applied using oligonucleotides We2508 and We2509
(SEQ ID NO. 15 and 16) giving rise to plasmid pF8-1211.
[0179] Site directed mutagenesis was applied according to standard
protocols (QuickChange XL Site Directed Mutagenesis Kit,
Stratagene) to delete the arginine in position 740 in plasmid
pF8-1211. For this oligonucleotides We2768 and We2769 (SEQ ID NO.
17 and 18) were used as mutagenic primers. The resulting expression
plasmid was designated pF8-1413. A FVIII molecule where the B
domain had been replaced by amino acid sequence RRGR was used as
the wild-type FVIII control, the encoding plasmid was called
pF8-457.
[0180] Using the protocols and plasmids described above and by
applying molecular biology techniques known to those skilled in the
art (and as described e.g. in Current Protocols in Molecular
Biology, Ausubel F M et al. (eds.) including supplement 80, October
2007, John Wiley & Sons, Inc.;
http://www.currentprotocols.com/WileyCDA/) other constructs can be
made by the artisan with insertions of a HLEP molecule in positions
described in FIG. 2 and linker sequences as shown exemplarily in
FIGS. 1b-e.
Example 2
Generation of Expression Vectors for FVIII Molecules with an
Immunoglobulin Constant Region Replacing the FVIII B Domain
[0181] The insertion of an IgG Fc domain into the FVIII molecule
replacing the majority of the B domain was performed in analogy to
the protocols and reference described above. The resulting plasmid
was called pF8-1518 and the mature protein translated from this is
shown in SEQ ID NO. 19.
[0182] As recycling of IgG by the neonatal Fc receptor only works
with the Fc being dimeric pF8-1518 was cotransfected into HEK-293
cells with a plasmid encoding a human immunoglobulin G heavy chain
region (p1335, SEQ ID No. 20). The coexpression of plasmids
pF8-1518 and p1335 led to the expression of a functional FVIII
molecule (table 1).
[0183] In another set of constructs FVIII heavy and light chains
were expressed separately. For that pF8-1518 was mutated in that a
stop codon was introduced at the very 3''-end of the IgG heavy
chain sequence. The expression of such construct (pF8-1515) led to
a FVIII heavy chain (A1 and A2 domain) with a few amino acids of
the B domain followed by the IgG heavy chain (SEQ ID NO. 21). The
FVIII light chain construct was also based on plasmid pF8-1518 in
that the A1 and A2 domain coding sequences were replaced by a
signal peptide. The expression of such construct (pF8-1517) led to
a FVIII light chain with an IgG heavy chain attached to its
N-terminus (SEQ ID NO. 22). The coexpression of plasmids pF8-1515
and pF8-1517 led to the expression of a functional FVIII molecule
(table 1).
Example 3
Transfection and Expression of FVIII Mutants
[0184] Expression plasmids were grown up. in E. coli TOP10
(Invitrogen) and purified using standard protocols (Qiagen).
HEK-293 cells were transfected using the Lipofectamine 2000 reagent
(Invitrogen) and grown up in serum-free medium (Invitrogen 293
Express) in the presence of 4 .mu.g/ml Puromycin and optionally 0.5
IU/ml vWF. Transfected cell populations were spread through
T-flasks into roller bottles or small scale fermenters from which
supernatants were harvested for purification.
[0185] Table 1 lists expression data of a number of constructs
outlined in FIGS. 1 and 2 and described in examples 1 and 2. Unless
otherwise indicated, the HLEP used is albumin.
TABLE-US-00001 TABLE 1 Activity Antigen Ratio activity/ Construct
[U/mL] [U/mL] antigen FIG. 2c 1.0 7.3 0.14 FIG. 2d 0.4 4.7 0.09
FIG. 2f 0.44 1.09 0.40 FIG. 2h 1.04 0.94 1.11 FIG. 2i 0.33 0.47
0.70 FIG. 2i (HLEP = 0.31 1.01 0.31 Afamin) FIG. 2i (HLEP = 0.53
1.16 0.46 Alpha-fetoprotein) FIG. 2o 0.22 0.75 0.30 pF8-1518 +
p1335 1.19 1.78 0.67 (HLEP = Fc) pF8-1515 + pF8-1517 1.75 6.68 0.26
(HLEP = Fc)
Example 4
Purification of Factor VIII Mutants
[0186] To the expression supernatant containing the chimeric Factor
VIII molecule a sufficient amount of an immune affinity resin was
added to bind the FVIII activity almost completely. The immune
affinity resin had been prepared by binding an appropriate
anti-FVIII MAb covalently to Sephacryl S1000 resin used as a
support. After washing of the resin it was filled into a
chromatography column and washed again. Elution was done using a
buffer containing 250 mM CaCl2 and 50% ethylene glycol.
[0187] The immune affinity chromatography (IAC) fractions
containing FVIII:C activity were pooled, dialyzed against
formulation buffer (excipients: sodium chloride, sucrose,
histidine, calcium chloride, and Tween 80), and concentrated.
Samples are either stored frozen or are freeze-dried using an
appropriate freeze-drying cycle. Table 2 shows the results of a
purification run using a FVIII mutant (pF8-1211 from HEK-293) and
IAC as main purification step.
TABLE-US-00002 TABLE 2 Volume FVIII:C FVIII:Ag Total protein*
Specific activity FVIII:C/FVIII:Ag Sample (mL) (IU/mL) (IU/mL)
(mg/mL) (IU/mg) (IU/IU) Supernatant 890 3.3 1.92 1.72 1.9 1.72 IAC
Eluate 26 52.2 30.6 0.036 1450 1.71 *determined by measurement of
Optical density (OD) at 280 nm (OD.sub.280, 1% = 10.0)
[0188] Alternatively, the FVIII containing cell culture supernatant
is concentrated/purified by a first ion exchange chromatography
followed by further purification using immune affinity
chromatography (IAC). In this case the eluate of the ion exchange
chromatography is loaded onto an IAC column using the above
mentioned resin.
Example 5
Analysis of Chimeric Factor VIII Activity and Antigen
[0189] For activity determination of FVIII:C in vitro either a
clotting assay (e.g. Pathromtin SL reagent and FVIII deficient
plasma delivered by Dade Behring, Germany) or a chromogenic assay
(e.g. Coamatic FVIII:C assay delivered by Haemochrom) were used.
The assays were performed according to the manufacturers
instructions.
[0190] FVIII antigen (FVIII:Ag) was determined by an ELISA whose
performance is known to those skilled in the art. Briefly,
microplates were incubated with 100 .mu.L per well of the capture
antibody (sheep anti-human FVIII IgG, Cedarlane CL20035K-C, diluted
1:200 in Buffer A [Sigma C3041]) for 2 hours at ambient
temperature. After washing plates three times with buffer B (Sigma
P3563), serial dilutions of the test sample in sample diluent
buffer (Cedarlane) as well as serial dilutions of a FVIII
preparation (ZLB Behring; 200-2 mU/mL) in sample diluent buffer
(volumes per well: 100 .mu.L) were incubated for two hours at
ambient temperature. After three wash steps with buffer B, 100
.mu.L of a 1:2 dilution in buffer B of the detection antibody
(sheep anti-human FVIII IgG, Cedarlane CL20035K-D, peroxidase
labelled) were added to each well and incubated for another hour at
ambient temperature. After three wash steps with buffer B, 100
.mu.L of substrate solution (1:10 (v/v) TMB OUVF:TMB Buffer OUVG,
Dade Behring) were added per well and incubated for 30 minutes at
ambient temperature in the dark. Addition of 100 .mu.L stop
solution (Dade Behring, OSFA) prepared the samples for reading in a
suitable microplate reader at 450 nm wavelength. Concentrations of
test samples were then calculated using the standard curve with the
FVIII preparation as reference.
Example 6
Pharmacokinetics of Factor VIII Mutants in Rats
[0191] The FVIII mutants were administered intravenously to
narcotized CD/Lewis rats (6 rats per substance) with a dose of 100
IU/kg body weight. Blood samples were drawn at appropriate
intervals starting at 5 minutes after application of the test
substances. FVIII antigen content was subsequently quantified by an
ELISA assay specific for human Factor VIII or by a mixed ELISA
specific for albumin and FVIII, respectively (see above). The mean
values of the treatment groups were used to calculate in vivo
recovery after 5 min. Half-lives for each protein were calculated
using the time points of the beta phase of elimination according to
the formula t.sub.1/2=ln 2/k, whereas k is the slope of the
regression line. The result is depicted in FIG. 3.
[0192] The terminal half-life calculated for the chimeric FVIII-HA
constructs between 2 and 24 h was 4.97 h for 1413 and 6.86 h for
1211, the terminal half-life calculated for wild type FVIII between
2 and 8 h was 2.17 h. Therefore, a clear increase of the terminal
half-life is shown for the chimeric FVIII-HA molecules extending
FVIII half-life 2-3-fold.
[0193] Bioavailabilities of the chimeric FVIII-HA constructs and
wild-type FVIII are shown in table 3 displaying superior
bioavailabilities of the FVIII-HA proteins of the invention.
TABLE-US-00003 TABLE 3 Increased in vivo recovery of FVIII-HA
proteins compared with FVIII wild-type (Helixate) in vivo recovery
increase in in vivo recovery [% of injected protein 5 compared to
Helixate .RTM. min. after i.v. application] (wild-type FVIII) [%]
1211 73.5 123.5 1413 87.7 147.8 Helixate 59.4
Example 7
Functional Half-Life of a Factor VIII Mutant in Rats
[0194] The FVIII mutant pF8-1211 (expressed in HEK-293 cells and
purified by IAC) as well as a control preparation (wild type FVIII
Helixate NexGen) were administered intravenously to narcotized
CD/Lewis rats (6 rats per substance) with a dose of 100 IU/kg body
weight. Blood samples were drawn at appropriate intervals starting
at 5 minutes after application of the test substances. FVIII
antigen content was subsequently quantified for the control group
using an ELISA assay specific for human Factor VIII (see example
4). In order to measure the FVIII:C activity of the FVIII mutant in
rat plasma an assay was established determining specifically the
FVIII mutant activity. In principle, the FVIII mutant was bound
from the rat plasma sample to a microtiter plate via an antibody
directed against human albumin and FVIII activity was then
determined by a chromogenic FVIII:C assay (Coatest VIII:C/4).
Briefly, 96-well microtiter plates were coated with the capture
antibody (mouse anti-human albumin Mab 3E8, diluted to 5 .mu.g/mL
in carbonate/bicarbonate buffer.) over night at ambient
temperature. After washing the plates with wash buffer (PBST,
=phosphate buffered saline containing 0.05% Tween 20, Sigma P3563),
the plates were blocked using non-fat milk in PBS (Phosphate
buffered saline) and washed again with wash buffer followed by
dilution buffer (50 mM Tris.times.HCl, 100 mM NaCl, 0.05% Tween 20
pH 7.2). Samples were applied in 40 .mu.L volume per well and
incubated for 1 h at 37.degree. C. Washing was done using dilution
buffer containing 300 mM CaCl2 followed by dilution buffer. The
FVIII:C activity determination was performed using Coatest VIII:C/4
reagents. 10 .mu.L dilution buffer and 50 .mu.L Coatest FIXa and FX
reagent were applied into the wells and incubated for 5 min at
37.degree. C. Then, 25 .mu.L of CaCl2 solution were added and again
incubated for 10 min at 37.degree. C. 50 .mu.L of substrate
solution was added and furthermore incubated for 10 min at
37.degree. C. This step was followed by addition of 25 .mu.L of
stopping solution (20% acetic acid). A microtiter plate reader was
used to read the absorbance at 405 nm. FVIII:C concentrations of
the samples were calculated using a standard curve prepared with
the FVIII mutant pF8-1211 as reference.
[0195] The FVIII:C respectively FVIII antigen results of the
treatment groups were used to calculate the terminal half-lives for
the corresponding proteins. The terminal functional half-life
calculated for the chimeric FVIII-HSA construct pF8-1211 between 2
and 24 h was 4.44 h, the terminal half-life of FVIII antigen
calculated for wild type FVIII between 2 and 8 h was 2.75 h.
Therefore, a clear increase of the functional half-life of FVIII:C
activity was shown for the chimeric FVIII-HSA molecule (increase by
61% compared to terminal FVIII:Ag half-life of wild type
FVIII).
Example 8
In Vitro Stability of FVIII Albumin Insertion Protein
[0196] Table 4 summarizes the results of an expression study of a
FVIII albumin insertion protein in serum-free cell culture. HEK-293
cells were transfected in triplicate with pF8-1439 (FVIII albumin
insertion) and pF8-457 (FVIII wild-type), respectively, seeded into
T80 flasks with equal cell numbers and grown in the absence of
stabilizing vWF. Culture supernatant was then harvested after 96,
120 and 144 hours and tested for FVIII activity and antigen
content.
TABLE-US-00004 TABLE 4 Culture FVIII FVIII activity/ time antigen*
SD** activity* SD** antigen [hrs] [mU/mL] [mU/mL] ratio pF8-457 96
679.0 48.9 1056.7 135.8 1.6 (FVIII wild type) pF8-1439 96 386.7
44.2 1060.0 115.3 2.7 (FVIII albumin) pF8-457 120 819.3 23.2 1720.0
65.6 2.1 (FVIII wild type) pF8-1439 120 389.3 74.9 1420.0 196.7 3.6
(FVIII albumin) pF8-457 144 595.7 59.9 1236.7 388.0 2.1 (FVIII wild
type) pF8-1439 144 381.3 50.1 1583.3 226.8 4.2 (FVIII albumin)
*mean value from triplicate experiment; **SD, standard
deviation
[0197] The results demonstrate a stabilizing effect of albumin when
present as an integral part of the FVIII molecule in cell culture.
The productivity is not necessarily higher in the case of the
insertion protein but the specific activity of the FVIII protein
(expressed in the ratio activity/antigen) is significantly higher
when the albumin is an integral part of the FVIII molecule (FIG. 3)
compared to wild-type FVIII.
Sequence CWU 1
1
4917056DNAHomo Sapiens 1atgcaaatag agctctccac ctgcttcttt ctgtgccttt
tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg
actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct
cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa
gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa
ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat
300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct
tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg
atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt
ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc
ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg
taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa
600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact
ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact
ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg
cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg
ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg
aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat
900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac
actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc
accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag
gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga
tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact
ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact
1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt
agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg
gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac
acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat
cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat
ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact
1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt
gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag
tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc
tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat
tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc
agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag
1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc
agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc
acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg
catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt
cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg
aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg
2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg
gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca
ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg
agtaaaaaca atgccattga accaagaagc 2280ttctcccaga attcaagaca
ccctagcact aggcaaaagc aatttaatgc caccacaatt 2340ccagaaaatg
acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa
2400atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc
tactccacat 2460gggctatcct tatctgatct ccaagaagcc aaatatgaga
ctttttctga tgatccatca 2520cctggagcaa tagacagtaa taacagcctg
tctgaaatga cacacttcag gccacagctc 2580catcacagtg gggacatggt
atttacccct gagtcaggcc tccaattaag attaaatgag 2640aaactgggga
caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca
2700tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac
tgataataca 2760agttccttag gacccccaag tatgccagtt cattatgata
gtcaattaga taccactcta 2820tttggcaaaa agtcatctcc ccttactgag
tctggtggac ctctgagctt gagtgaagaa 2880aataatgatt caaagttgtt
agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940aaaaatgtat
cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct
3000gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt
aaagacaaac 3060aaaacttcca ataattcagc aactaataga aagactcaca
ttgatggccc atcattatta 3120attgagaata gtccatcagt ctggcaaaat
atattagaaa gtgacactga gtttaaaaaa 3180gtgacacctt tgattcatga
cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240aatcatatgt
caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa
3300gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa
gatgctattc 3360ttgccagaat cagcaaggtg gatacaaagg actcatggaa
agaactctct gaactctggg 3420caaggcccca gtccaaagca attagtatcc
ttaggaccag aaaaatctgt ggaaggtcag 3480aatttcttgt ctgagaaaaa
caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540ggactcaaag
agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat
3600ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat
agaaaagaag 3660gaaacattaa tccaagagaa tgtagttttg cctcagatac
atacagtgac tggcactaag 3720aatttcatga agaacctttt cttactgagc
actaggcaaa atgtagaagg ttcatatgac 3780ggggcatatg ctccagtact
tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840aagaaacaca
cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga
3900aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc
tcctaataca 3960agccagcaga attttgtcac gcaacgtagt aagagagctt
tgaaacaatt cagactccca 4020ctagaagaaa cagaacttga aaaaaggata
attgtggatg acacctcaac ccagtggtcc 4080aaaaacatga aacatttgac
cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140aaaggggcca
ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct
4200caagcaaata gatctccatt acccattgca aaggtatcat catttccatc
tattagacct 4260atatatctga ccagggtcct attccaagac aactcttctc
atcttccagc agcatcttat 4320agaaagaaag attctggggt ccaagaaagc
agtcatttct tacaaggagc caaaaaaaat 4380aacctttctt tagccattct
aaccttggag atgactggtg atcaaagaga ggttggctcc 4440ctggggacaa
gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg
4500aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt
tcacatttat 4560cagaaggacc tattccctac ggaaactagc aatgggtctc
ctggccatct ggatctcgtg 4620gaagggagcc ttcttcaggg aacagaggga
gcgattaagt ggaatgaagc aaacagacct 4680ggaaaagttc cctttctgag
agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740ttggatcctc
ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa
4800tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat
tttgtccctg 4860aacgcttgtg aaagcaatca tgcaatagca gcaataaatg
agggacaaaa taagcccgaa 4920atagaagtca cctgggcaaa gcaaggtagg
actgaaaggc tgtgctctca aaacccacca 4980gtcttgaaac gccatcaacg
ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040attgactatg
atgataccat atcagttgaa atgaagaagg aagattttga catttatgat
5100gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta
ttttattgct 5160gcagtggaga ggctctggga ttatgggatg agtagctccc
cacatgttct aagaaacagg 5220gctcagagtg gcagtgtccc tcagttcaag
aaagttgttt tccaggaatt tactgatggc 5280tcctttactc agcccttata
ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340tatataagag
cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt
5400ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca
aggagcagaa 5460cctagaaaaa actttgtcaa gcctaatgaa accaaaactt
acttttggaa agtgcaacat 5520catatggcac ccactaaaga tgagtttgac
tgcaaagcct gggcttattt ctctgatgtt 5580gacctggaaa aagatgtgca
ctcaggcctg attggacccc ttctggtctg ccacactaac 5640acactgaacc
ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc
5700atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa
ctgcagggct 5760ccctgcaata tccagatgga agatcccact tttaaagaga
attatcgctt ccatgcaatc 5820aatggctaca taatggatac actacctggc
ttagtaatgg ctcaggatca aaggattcga 5880tggtatctgc tcagcatggg
cagcaatgaa aacatccatt ctattcattt cagtggacat 5940gtgttcactg
tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt
6000gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt
ggaatgcctt 6060attggcgagc atctacatgc tgggatgagc acactttttc
tggtgtacag caataagtgt 6120cagactcccc tgggaatggc ttctggacac
attagagatt ttcagattac agcttcagga 6180caatatggac agtgggcccc
aaagctggcc agacttcatt attccggatc aatcaatgcc 6240tggagcacca
aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt
6300cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat
ctctcagttt 6360atcatcatgt atagtcttga tgggaagaag tggcagactt
atcgaggaaa ttccactgga 6420accttaatgg tcttctttgg caatgtggat
tcatctggga taaaacacaa tatttttaac 6480cctccaatta ttgctcgata
catccgtttg cacccaactc attatagcat tcgcagcact 6540cttcgcatgg
agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag
6600agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa
tatgtttgcc 6660acctggtctc cttcaaaagc tcgacttcac ctccaaggga
ggagtaatgc ctggagacct 6720caggtgaata atccaaaaga gtggctgcaa
gtggacttcc agaagacaat gaaagtcaca 6780ggagtaacta ctcagggagt
aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840atctccagca
gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag
6900gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga
cccaccgtta 6960ctgactcgct accttcgaat tcacccccag agttgggtgc
accagattgc cctgaggatg 7020gaggttctgg gctgcgaggc acaggacctc tactga
705622332PRTHomo Sapiens 2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val
Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu
Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe
Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val
Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro
Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85
90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly
Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln
Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys
Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys
Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg
Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys
Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210
215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn
Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser
Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val
His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn
His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu
Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu
Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu
Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330
335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro
Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys
Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp
Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr
Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg
Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr
Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu
Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455
460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu
Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro
Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser
Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly
Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg
Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580
585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu
Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala
Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe
Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met
Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu
Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu
Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705
710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn
Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His
Arg Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro
Glu Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg
Thr Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu
Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu
Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser
Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825
830 Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val
835 840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys
Leu Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe
Lys Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile
Pro Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser
Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu
Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu
Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser
Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950
955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly
Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala
Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr
Ser Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp
Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp
Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val
Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn
Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065
Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070
1075 1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe
Lys 1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln
Arg Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly
Pro Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys
Ser Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys
Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly
Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe
Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185
Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190
1195 1200 Leu Ile Gln Glu Asn
Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215 Gly Thr Lys
Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230 Gln
Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240
1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys
1250 1255 1260 His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn
Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu
Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg Ile Ser Pro Asn Thr Ser
Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln Arg Ser Lys Arg Ala Leu
Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320 Glu Thr Glu Leu Glu
Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335 Gln Trp Ser
Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 Gln
Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360
1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala
1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe
Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe
Gln Asp Asn Ser 1400 1405 1410 Ser His Leu Pro Ala Ala Ser Tyr Arg
Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln Glu Ser Ser His Phe Leu
Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440 Ser Leu Ala Ile Leu
Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455 Val Gly Ser
Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470 Lys
Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480
1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys
1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly
His Leu 1505 1510 1515 Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr
Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn Glu Ala Asn Arg Pro Gly
Lys Val Pro Phe Leu Arg 1535 1540 1545 Val Ala Thr Glu Ser Ser Ala
Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560 Pro Leu Ala Trp Asp
Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575 Glu Trp Lys
Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 Lys
Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600
1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu
1610 1615 1620 Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys
Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu
Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln Ser Asp Gln Glu Glu Ile
Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser Val Glu Met Lys Lys Glu
Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680 Glu Asn Gln Ser Pro
Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695 Phe Ile Ala
Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710 Ser
Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720
1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe
1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu
Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
Asn Ile Met Val 1760 1765 1770 Thr Phe Arg Asn Gln Ala Ser Arg Pro
Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu Ile Ser Tyr Glu Glu Asp
Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800 Lys Asn Phe Val Lys
Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815 Val Gln His
His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830 Ala
Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840
1845 Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu
1850 1855 1860 Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe
Ala Leu 1865 1870 1875 Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp
Tyr Phe Thr Glu 1880 1885 1890 Asn Met Glu Arg Asn Cys Arg Ala Pro
Cys Asn Ile Gln Met Glu 1895 1900 1905 Asp Pro Thr Phe Lys Glu Asn
Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920 Tyr Ile Met Asp Thr
Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935 Arg Ile Arg
Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950 His
Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960
1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe
1970 1975 1980 Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp
Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
Met Ser Thr Leu 2000 2005 2010 Phe Leu Val Tyr Ser Asn Lys Cys Gln
Thr Pro Leu Gly Met Ala 2015 2020 2025 Ser Gly His Ile Arg Asp Phe
Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040 Gly Gln Trp Ala Pro
Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055 Ile Asn Ala
Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070 Asp
Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080
2085 Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile
2090 2095 2100 Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg
Gly Asn 2105 2110 2115 Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn
Val Asp Ser Ser 2120 2125 2130 Gly Ile Lys His Asn Ile Phe Asn Pro
Pro Ile Ile Ala Arg Tyr 2135 2140 2145 Ile Arg Leu His Pro Thr His
Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160 Met Glu Leu Met Gly
Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175 Gly Met Glu
Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190 Ser
Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200
2205 Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val
2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys
Thr Met 2225 2230 2235 Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
Ser Leu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys Glu Phe Leu Ile
Ser Ser Ser Gln Asp Gly 2255 2260 2265 His Gln Trp Thr Leu Phe Phe
Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280 Gln Gly Asn Gln Asp
Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295 Pro Pro Leu
Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310 Val
His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320
2325 Gln Asp Leu Tyr 2330 3585PRTHomo Sapiens 3Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40
45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys
Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val
Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe
Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr
Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr
Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu
Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu
Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys
Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp
Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys
Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys
Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420
425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro
Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys
Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe
Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe
Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu
Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val
Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545
550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys
Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585
42043PRTArtificialAmino acid sequence of mature human FVIII with B
domain replacement by human albumin 4Ala Thr Arg Arg Tyr Tyr Leu
Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu
Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro
Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr
Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55
60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His
Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala
Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu
Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr
Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp
Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp
Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu
Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185
190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala
Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly
Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His
Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr
Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu
Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile
Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln
Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310
315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu
Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp
Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys
His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu
Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp
Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg
Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435
440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile
Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg
Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu
Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu
Asp Gly Pro Thr Lys Ser Asp Pro
Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu
Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys
Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met
Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu
Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu
Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln
Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615
620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu
625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr
Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser
Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser
Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser
Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser
Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Arg Ser Thr Arg 740
745 750 Gln Lys Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys
Asp 755 760 765 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala
Phe Ala Gln 770 775 780 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val
Lys Leu Val Asn Glu 785 790 795 800 Val Thr Glu Phe Ala Lys Thr Cys
Val Ala Asp Glu Ser Ala Glu Asn 805 810 815 Cys Asp Lys Ser Leu His
Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 820 825 830 Ala Thr Leu Arg
Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 835 840 845 Gln Glu
Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 850 855 860
Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 865
870 875 880 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu 885 890 895 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe 900 905 910 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu
Cys Cys Gln Ala Ala Asp 915 920 925 Lys Ala Ala Cys Leu Leu Pro Lys
Leu Asp Glu Leu Arg Asp Glu Gly 930 935 940 Lys Ala Ser Ser Ala Lys
Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 945 950 955 960 Phe Gly Glu
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 965 970 975 Arg
Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 980 985
990 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys
995 1000 1005 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu
Asn Gln 1010 1015 1020 Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu 1025 1030 1035 Leu Glu Lys Ser His Cys Ile Ala Glu
Val Glu Asn Asp Glu Met 1040 1045 1050 Pro Ala Asp Leu Pro Ser Leu
Ala Ala Asp Phe Val Glu Ser Lys 1055 1060 1065 Asp Val Cys Lys Asn
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 1070 1075 1080 Met Phe Leu
Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val 1085 1090 1095 Val
Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu 1100 1105
1110 Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val
1115 1120 1125 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn
Leu Ile 1130 1135 1140 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu Tyr Lys Phe 1145 1150 1155 Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro Gln Val 1160 1165 1170 Ser Thr Pro Thr Leu Val Glu
Val Ser Arg Asn Leu Gly Lys Val 1175 1180 1185 Gly Ser Lys Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys 1190 1195 1200 Ala Glu Asp
Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 1205 1210 1215 His
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr 1220 1225
1230 Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val
1235 1240 1245 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
Phe Thr 1250 1255 1260 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile 1265 1270 1275 Lys Lys Gln Thr Ala Leu Val Glu Leu
Val Lys His Lys Pro Lys 1280 1285 1290 Ala Thr Lys Glu Gln Leu Lys
Ala Val Met Asp Asp Phe Ala Ala 1295 1300 1305 Phe Val Glu Lys Cys
Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 1310 1315 1320 Ala Glu Glu
Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu 1325 1330 1335 Gly
Leu Gly Arg Thr Glu Arg Leu Cys Ser Gln Asn Pro Pro Val 1340 1345
1350 Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser
1355 1360 1365 Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val
Glu Met 1370 1375 1380 Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp
Glu Asn Gln Ser 1385 1390 1395 Pro Arg Ser Phe Gln Lys Lys Thr Arg
His Tyr Phe Ile Ala Ala 1400 1405 1410 Val Glu Arg Leu Trp Asp Tyr
Gly Met Ser Ser Ser Pro His Val 1415 1420 1425 Leu Arg Asn Arg Ala
Gln Ser Gly Ser Val Pro Gln Phe Lys Lys 1430 1435 1440 Val Val Phe
Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu 1445 1450 1455 Tyr
Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr 1460 1465
1470 Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn
1475 1480 1485 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile
Ser Tyr 1490 1495 1500 Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg
Lys Asn Phe Val 1505 1510 1515 Lys Pro Asn Glu Thr Lys Thr Tyr Phe
Trp Lys Val Gln His His 1520 1525 1530 Met Ala Pro Thr Lys Asp Glu
Phe Asp Cys Lys Ala Trp Ala Tyr 1535 1540 1545 Phe Ser Asp Val Asp
Leu Glu Lys Asp Val His Ser Gly Leu Ile 1550 1555 1560 Gly Pro Leu
Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 1565 1570 1575 Gly
Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile 1580 1585
1590 Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg
1595 1600 1605 Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro
Thr Phe 1610 1615 1620 Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly
Tyr Ile Met Asp 1625 1630 1635 Thr Leu Pro Gly Leu Val Met Ala Gln
Asp Gln Arg Ile Arg Trp 1640 1645 1650 Tyr Leu Leu Ser Met Gly Ser
Asn Glu Asn Ile His Ser Ile His 1655 1660 1665 Phe Ser Gly His Val
Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys 1670 1675 1680 Met Ala Leu
Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu 1685 1690 1695 Met
Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile 1700 1705
1710 Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr
1715 1720 1725 Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly
His Ile 1730 1735 1740 Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr
Gly Gln Trp Ala 1745 1750 1755 Pro Lys Leu Ala Arg Leu His Tyr Ser
Gly Ser Ile Asn Ala Trp 1760 1765 1770 Ser Thr Lys Glu Pro Phe Ser
Trp Ile Lys Val Asp Leu Leu Ala 1775 1780 1785 Pro Met Ile Ile His
Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys 1790 1795 1800 Phe Ser Ser
Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 1805 1810 1815 Asp
Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr 1820 1825
1830 Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His
1835 1840 1845 Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg
Leu His 1850 1855 1860 Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg
Met Glu Leu Met 1865 1870 1875 Gly Cys Asp Leu Asn Ser Cys Ser Met
Pro Leu Gly Met Glu Ser 1880 1885 1890 Lys Ala Ile Ser Asp Ala Gln
Ile Thr Ala Ser Ser Tyr Phe Thr 1895 1900 1905 Asn Met Phe Ala Thr
Trp Ser Pro Ser Lys Ala Arg Leu His Leu 1910 1915 1920 Gln Gly Arg
Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys 1925 1930 1935 Glu
Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly 1940 1945
1950 Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val
1955 1960 1965 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln
Trp Thr 1970 1975 1980 Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe
Gln Gly Asn Gln 1985 1990 1995 Asp Ser Phe Thr Pro Val Val Asn Ser
Leu Asp Pro Pro Leu Leu 2000 2005 2010 Thr Arg Tyr Leu Arg Ile His
Pro Gln Ser Trp Val His Gln Ile 2015 2020 2025 Ala Leu Arg Met Glu
Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 2030 2035 2040
521DNAArtificialPrimer 5cagcttgagg atccagagtt c
21630DNAArtificialPrimer 6gtgaccggtc ttttgcctag tgctagggtg
30726DNAArtificialPrimer 7gtgaccggta ggactgaaag gctgtg
26823DNAArtificialPrimer 8gattgatccg gaataatgaa gtc
23934DNAArtificialPrimer 9gcgaaccggt caggatgcac acaagagtga ggtt
341030DNAArtificialPrimer 10cgcaccggtt aagcctaagg cagcttgact
301129DNAArtificialPrimer 11ctagcactag gcaaaagcag gatgcacac
291229DNAArtificialPrimer 12gtgtgcatcc tgcttttgcc tagtgctag
291329DNAArtificialPrimer 13ctgccttagg cttaggtagg actgaaagg
291429DNAArtificialPrimer 14cctttcagtc ctacctaagc ctaaggcag
291533DNAArtificialPrimer 15ggactgaaag gctgtcctct caaaacccac cag
331633DNAArtificialPrimer 16ctggtgggtt ttgagaggac agcctttcag tcc
331736DNAArtificialPrimer 17caatgccatt gaaccaagct tctcccagaa ttcaag
361836DNAArtificialPrimer 18cttgaattct gggagaagct tggttcaatg gcattg
36191671PRTArtificialFVIII with human albumin replacing part of the
B-domain 19Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp
Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala
Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr
Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Asp
His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly
Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val
Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu
His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115
120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu
Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser
Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu
Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu
Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr
Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg
Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235
240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe
Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala
Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr
Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile
Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys
Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn
Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser
Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360
365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu
Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr
Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys
Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg
Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu
Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn
Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485
490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr
Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met
Glu Arg Asp Leu Ala 530 535
540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp
545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu
Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val
Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His
Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val
Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile
Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660
665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu
Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr
Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala
Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Ser Phe Ser
Gln Asn Ser Arg His Pro Ser Thr Arg Gln 740 745 750 Lys Gln Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 755 760 765 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 770 775 780
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 785
790 795 800 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 805 810 815 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 820 825 830 Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 835 840 845 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro 850 855 860 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 865 870 875 880 Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Val Thr Lys Asn Gln Val 885 890 895 Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 900 905
910 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
915 920 925 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 930 935 940 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 945 950 955 960 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Ser 965 970 975 Gln Asn Pro Pro Val Leu Lys
Arg His Gln Arg Glu Ile Thr Arg Thr 980 985 990 Thr Leu Gln Ser Asp
Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser 995 1000 1005 Val Glu
Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu 1010 1015 1020
Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe 1025
1030 1035 Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser
Ser 1040 1045 1050 Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser
Val Pro Gln 1055 1060 1065 Phe Lys Lys Val Val Phe Gln Glu Phe Thr
Asp Gly Ser Phe Thr 1070 1075 1080 Gln Pro Leu Tyr Arg Gly Glu Leu
Asn Glu His Leu Gly Leu Leu 1085 1090 1095 Gly Pro Tyr Ile Arg Ala
Glu Val Glu Asp Asn Ile Met Val Thr 1100 1105 1110 Phe Arg Asn Gln
Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu 1115 1120 1125 Ile Ser
Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys 1130 1135 1140
Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 1145
1150 1155 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
Ala 1160 1165 1170 Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp
Val His Ser 1175 1180 1185 Gly Leu Ile Gly Pro Leu Leu Val Cys His
Thr Asn Thr Leu Asn 1190 1195 1200 Pro Ala His Gly Arg Gln Val Thr
Val Gln Glu Phe Ala Leu Phe 1205 1210 1215 Phe Thr Ile Phe Asp Glu
Thr Lys Ser Trp Tyr Phe Thr Glu Asn 1220 1225 1230 Met Glu Arg Asn
Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp 1235 1240 1245 Pro Thr
Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr 1250 1255 1260
Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg 1265
1270 1275 Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
His 1280 1285 1290 Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg
Lys Lys Glu 1295 1300 1305 Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr
Pro Gly Val Phe Glu 1310 1315 1320 Thr Val Glu Met Leu Pro Ser Lys
Ala Gly Ile Trp Arg Val Glu 1325 1330 1335 Cys Leu Ile Gly Glu His
Leu His Ala Gly Met Ser Thr Leu Phe 1340 1345 1350 Leu Val Tyr Ser
Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser 1355 1360 1365 Gly His
Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly 1370 1375 1380
Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 1385
1390 1395 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
Asp 1400 1405 1410 Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr
Gln Gly Ala 1415 1420 1425 Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser
Gln Phe Ile Ile Met 1430 1435 1440 Tyr Ser Leu Asp Gly Lys Lys Trp
Gln Thr Tyr Arg Gly Asn Ser 1445 1450 1455 Thr Gly Thr Leu Met Val
Phe Phe Gly Asn Val Asp Ser Ser Gly 1460 1465 1470 Ile Lys His Asn
Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile 1475 1480 1485 Arg Leu
His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met 1490 1495 1500
Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly 1505
1510 1515 Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser
Ser 1520 1525 1530 Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser
Lys Ala Arg 1535 1540 1545 Leu His Leu Gln Gly Arg Ser Asn Ala Trp
Arg Pro Gln Val Asn 1550 1555 1560 Asn Pro Lys Glu Trp Leu Gln Val
Asp Phe Gln Lys Thr Met Lys 1565 1570 1575 Val Thr Gly Val Thr Thr
Gln Gly Val Lys Ser Leu Leu Thr Ser 1580 1585 1590 Met Tyr Val Lys
Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His 1595 1600 1605 Gln Trp
Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln 1610 1615 1620
Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 1625
1630 1635 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
Val 1640 1645 1650 His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys
Glu Ala Gln 1655 1660 1665 Asp Leu Tyr 1670 20240PRTArtificialAmino
acid sequence of a human immunoglobulin G heavy chain region (240
amino acids 1-19 human IgG signal peptide, 20-35 human IgG hinge
region and 36-240 human IgG heavy chain) 20Met Glu Phe Gly Leu Ser
Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25 30 Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50
55 60 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Val Thr Lys Asn Gln 145 150 155 160 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180
185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 225 230 235 240 21975PRTArtificialmature human
FVIII heavy chain with partial B domain replacement by human
immunoglobulin G heavy chain region 21Ala Thr Arg Arg Tyr Tyr Leu
Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu
Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro
Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr
Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55
60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His
Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala
Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu
Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr
Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp
Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp
Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu
Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185
190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala
Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly
Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His
Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr
Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu
Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile
Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln
Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310
315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu
Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp
Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys
His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu
Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp
Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg
Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435
440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile
Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg
Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu
Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu
Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr
Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly
Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555
560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn
Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu
Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn
Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His
Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln
Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys
His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe
Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680
685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr
Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu
Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Ser Phe Ser Gln Asn Ser
Arg His Pro Ser Thr Arg Gln 740 745 750 Lys Gln Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 755 760 765 Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 770 775 780 Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 785 790 795 800
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 805
810 815 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 820 825 830 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 835 840 845 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro
Ala Pro 850 855 860 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 865 870 875 880 Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Val Thr Lys Asn Gln Val 885 890 895 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 900 905 910 Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 915 920 925 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 930 935 940 Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 945 950
955 960 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
965 970 975 22917PRTArtificialmature human FVIII light chain
attached to human immunoglobulin G heavy chain region 22Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 1 5 10 15 Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 20 25
30 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
35 40 45 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 50 55 60 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 65 70 75 80 Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 85 90 95 Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110 Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125 Thr Leu Pro Pro
Ser Arg Glu Glu Val Thr Lys Asn Gln Val Ser Leu 130 135 140 Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 145 150 155
160 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
165 170 175 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp 180 185 190 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 195 200 205 Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Ser Gln Asn 210 215 220 Pro Pro Val Leu Lys Arg His Gln
Arg Glu Ile Thr Arg Thr Thr Leu 225 230 235 240 Gln Ser Asp Gln Glu
Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu 245 250 255 Met Lys Lys
Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser 260 265 270 Pro
Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val 275 280
285 Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg
290 295 300 Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val
Val Phe 305 310 315 320 Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro
Leu Tyr Arg Gly Glu 325 330 335 Leu Asn Glu His Leu Gly Leu Leu Gly
Pro Tyr Ile Arg Ala Glu Val 340 345 350 Glu Asp Asn Ile Met Val Thr
Phe Arg Asn Gln Ala Ser Arg Pro Tyr 355 360 365 Ser Phe Tyr Ser Ser
Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 370 375 380 Ala Glu Pro
Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr 385 390 395 400
Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp 405
410 415 Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp
Val 420 425 430 His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr
Asn Thr Leu 435 440 445 Asn Pro Ala His Gly Arg Gln Val Thr Val Gln
Glu Phe Ala Leu Phe 450 455 460 Phe Thr Ile Phe Asp Glu Thr Lys Ser
Trp Tyr Phe Thr Glu Asn Met 465 470 475 480 Glu Arg Asn Cys Arg Ala
Pro Cys Asn Ile Gln Met Glu Asp Pro Thr 485 490 495 Phe Lys Glu Asn
Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp 500 505 510 Thr Leu
Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 515 520 525
Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser 530
535 540 Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala
Leu 545 550 555 560 Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu
Met Leu Pro Ser 565 570 575 Lys Ala Gly Ile Trp Arg Val Glu Cys Leu
Ile Gly Glu His Leu His 580 585 590 Ala Gly Met Ser Thr Leu Phe Leu
Val Tyr Ser Asn Lys Cys Gln Thr 595 600 605 Pro Leu Gly Met Ala Ser
Gly His Ile Arg Asp Phe Gln Ile Thr Ala 610 615 620 Ser Gly Gln Tyr
Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr 625 630 635 640 Ser
Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile 645 650
655 Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln
660 665 670 Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe
Ile Ile 675 680 685 Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr
Arg Gly Asn Ser 690 695 700 Thr Gly Thr Leu Met Val Phe Phe Gly Asn
Val Asp Ser Ser Gly Ile 705 710 715 720 Lys His Asn Ile Phe Asn Pro
Pro Ile Ile Ala Arg Tyr Ile Arg Leu 725 730 735 His Pro Thr His Tyr
Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met 740 745 750 Gly Cys Asp
Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 755 760 765 Ala
Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met 770 775
780 Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg
785 790 795 800 Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu
Trp Leu Gln 805 810 815 Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly
Val Thr Thr Gln Gly 820 825 830 Val Lys Ser Leu Leu Thr Ser Met Tyr
Val Lys Glu Phe Leu Ile Ser 835 840 845 Ser Ser Gln Asp Gly His Gln
Trp Thr Leu Phe Phe Gln Asn Gly Lys 850 855 860 Val Lys Val Phe Gln
Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn 865 870 875 880 Ser Leu
Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln 885 890 895
Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu 900
905 910 Ala Gln Asp Leu Tyr 915 2331PRTArtificialLinker 23Asn Asn
Ala Ile Glu Pro Ser Phe Ser Gln Asn Ser Arg His Pro Ser 1 5 10 15
Thr Arg Gln Lys Gln Asp Ala His Lys Ser Glu Val Ala His Arg 20 25
30 2432PRTArtificialLinker 24Asn Asn Ala Ile Glu Pro Arg Ser Phe
Ser Gln Asn Ser Arg His Pro 1 5 10 15 Ser Thr Arg Gln Lys Gln Asp
Ala His Lys Ser Glu Val Ala His Arg 20 25 30
2530PRTArtificialLinker 25Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser
Gln Asn Ser Arg His Pro 1 5 10 15 Ser Thr Arg Gln Asp Ala His Lys
Ser Glu Val Ala His Arg 20 25 30 2628PRTArtificialLinker 26Asn Asn
Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 1 5 10 15
Ser Thr Asp Ala His Lys Ser Glu Val Ala His Arg 20 25
2725PRTArtificialLinker 27Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser
Gln Asn Ser Arg His Asp 1 5 10 15 Ala His Lys Ser Glu Val Ala His
Arg 20 25 2823PRTArtificialLinker 28Asn Asn Ala Ile Glu Pro Arg Ser
Phe Ser Gln Asn Ser Asp Ala His 1 5 10 15 Lys Ser Glu Val Ala His
Arg 20 2920PRTArtificialLinker 29Asn Asn Ala Ile Glu Pro Arg Ser
Phe Ser Asp Ala His Lys Ser Glu 1 5 10 15 Val Ala His Arg 20
3032PRTArtificialLinker 30Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser
Gln Asn Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Gly Ser Asp Ala
His Lys Ser Glu Val Ala His Arg 20 25 30 3127PRTArtificialLinker
31Asn Asn Ala Ile Glu Pro Arg Ser Val Ala Lys Lys His Pro Lys Thr 1
5 10 15 Trp Asp Ala His Lys Ser Glu Val Ala His Arg 20 25
3227PRTArtificialLinker 32Asn Asn Ala Ile Glu Pro Arg Ser Phe Gln
Lys Lys Thr Arg His Tyr 1 5 10 15 Phe Asp Ala His Lys Ser Glu Val
Ala His Arg 20 25 3321PRTArtificialLinker 33Asn Asn Ala Ile Glu Pro
Arg Ala Val Gly Gly Asp Ala His Lys Ser 1 5 10 15 Glu Val Ala His
Arg 20 3436PRTArtificialLinker 34Val Ala Ala Ser Gln Ala Ala Leu
Gly Leu Gly Arg Thr Glu Arg Leu 1 5 10 15 Cys Ser Gln Asn Pro Pro
Val Leu Lys Arg His Gln Arg Glu Ile Thr 20 25 30 Arg Thr Thr Leu 35
3536PRTArtificialLinker 35Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Gly Arg Thr Glu Arg Leu 1 5 10 15 Ser Ser Gln Asn Pro Pro Val Leu
Lys Arg His Gln Arg Glu Ile Thr 20 25 30 Arg Thr Thr Leu 35
3634PRTArtificialLinker 36Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Thr Glu Arg Leu Cys Ser 1 5 10 15 Gln Asn Pro Pro Val Leu Lys Arg
His Gln Arg Glu Ile Thr Arg Thr 20 25 30 Thr Leu
3734PRTArtificialLinker 37Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Thr Glu Arg Leu Ser Ser 1 5 10 15 Gln Asn Pro Pro Val Leu Lys Arg
His Gln Arg Glu Ile Thr Arg Thr 20 25 30 Thr Leu
3831PRTArtificialLinker 38Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Leu Cys Ser Gln Asn Pro 1 5 10 15 Pro Val Leu Lys Arg His Gln Arg
Glu Ile Thr Arg Thr Thr Leu 20 25 30 3931PRTArtificialLinker 39Val
Ala Ala Ser Gln Ala Ala Leu Gly Leu Leu Ser Ser Gln Asn Pro 1 5 10
15 Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu 20
25 30 4029PRTArtificialLinker 40Val Ala Ala Ser Gln Ala Ala Leu Gly
Leu Ser Gln Asn Pro Pro Val 1 5 10 15 Leu Lys Arg His Gln Arg Glu
Ile Thr Arg Thr Thr Leu 20 25 4124PRTArtificialLinker 41Val Ala Ala
Ser Gln Ala Ala Leu Gly Leu Val Leu Lys Arg His Gln 1 5 10 15 Arg
Glu Ile Thr Arg Thr Thr Leu 20 4221PRTArtificialLinker 42Val Ala
Ala Ser Gln Ala Ala Leu Gly Leu Arg His Gln Arg Glu Ile 1 5 10 15
Thr Arg Thr Thr Leu 20 4336PRTArtificialLinker 43Val Ala Ala Ser
Gln Ala Ala Leu Gly Leu Gly Arg Thr Glu Arg Leu 1 5 10 15 Cys Ser
Gln Asn Pro Pro Val Leu Lys Arg His Arg Arg Glu Ile Thr 20 25 30
Arg Thr Thr Leu 35 4429PRTArtificialLinker 44Val Ala Ala Ser Gln
Ala Ala Leu Gly Leu Ser Gln Asn Pro Pro Val 1 5 10 15 Leu Lys Arg
His Arg Arg Glu Ile Thr Arg Thr Thr Leu 20 25
4521PRTArtificialLinker 45Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Arg His Arg Arg Glu Ile 1 5 10 15 Thr Arg Thr Thr Leu 20
4636PRTArtificialLinker 46Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Gly Gly Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Gly Ser Gly Gly
Ser Arg His Arg Arg Glu Ile Thr 20 25 30 Arg Thr Thr Leu 35
4733PRTArtificialLinker 47Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Gly Gly Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Gly Ser Arg His
Arg Arg Glu Ile Thr Arg Thr Thr 20 25 30 Leu
4827PRTArtificialLinker 48Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
Gly Gly Ser Gly Gly Ser 1 5 10 15 Arg His Arg Arg Glu Ile Thr Arg
Thr Thr Leu 20 25 4927PRTArtificialLinker 49Val Ala Ala Ser Gln Ala
Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser 1 5 10 15 Arg His Arg Arg
Glu Ile Thr Arg Thr Thr Leu 20 25
* * * * *
References