U.S. patent application number 15/158060 was filed with the patent office on 2017-04-06 for coagulation factor vii polypeptides.
The applicant listed for this patent is Novo Nordisk HealthCare AG. Invention is credited to Prafull S. Gandhi, Henrik Oestergaard, Ole Hvilsted Olsen.
Application Number | 20170096655 15/158060 |
Document ID | / |
Family ID | 52810172 |
Filed Date | 2017-04-06 |
United States Patent
Application |
20170096655 |
Kind Code |
A1 |
Oestergaard; Henrik ; et
al. |
April 6, 2017 |
COAGULATION FACTOR VII POLYPEPTIDES
Abstract
The present invention relates to modified coagulation Factor VII
polypeptides exhibiting increased resistance to antithrombin
inactivation and enhanced proteolytic activity. The present
invention also relates to polynucleotide constructs encoding such
polypeptides, vectors and host cells comprising and expressing such
polynucleotides, pharmaceutical compositions, uses and methods of
treatment.
Inventors: |
Oestergaard; Henrik;
(Oelstykke, DK) ; Gandhi; Prafull S.; (Ballerup,
DK) ; Olsen; Ole Hvilsted; (Broenshoej, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk HealthCare AG |
Zurich |
|
CH |
|
|
Family ID: |
52810172 |
Appl. No.: |
15/158060 |
Filed: |
May 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14514794 |
Oct 15, 2014 |
9371370 |
|
|
15158060 |
|
|
|
|
61895438 |
Oct 25, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/64 20170801;
A61P 7/00 20180101; C07K 14/745 20130101; C12Y 304/21021 20130101;
A61K 38/00 20130101; A61K 38/36 20130101; A61K 38/4846 20130101;
C12N 9/6437 20130101; A61K 47/60 20170801; A61K 47/61 20170801 |
International
Class: |
C12N 9/64 20060101
C12N009/64; A61K 38/48 20060101 A61K038/48 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2013 |
EP |
13188715.0 |
Feb 12, 2014 |
EP |
14154875.0 |
Claims
1. A Factor VII polypeptide comprising two or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID NO:
1), wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe
(F); and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or
Trp W and/or W201 is replaced by Arg (R), Met (M) or Lys (K) and/or
K337 is replaced by Ala (A) or Gly (G).
2. The Factor VII polypeptide according to claim 1 wherein T293 is
replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 is
replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W).
3. The Factor VII polypeptide according to claim 1, wherein T293 is
replaced by Lys (K) and L288 is replaced by Phe (F).
4. The Factor VII polypeptide according to claim 1, wherein T293 is
replaced by Lys (K) and L288 is replaced by Tyr (Y).
5. The Factor VII polypeptide according to claim 1, wherein T293 is
replaced by Arg (R) and L288 is replaced by Phe (F).
6. The Factor VII polypeptide according to claim 1, wherein T293 is
replaced by Arg (R) and L288 is replaced by Tyr (Y).
7. The Factor VII polypeptide according to claim 1, wherein K337 is
replaced by Ala (A).
8. The Factor VII polypeptide according to claim 1, wherein T293 is
replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and W201 is
replaced by Arg (R), Met (M), or Lys (K).
9. The Factor VII polypeptide according to claim 8, wherein T293 is
replaced by Lys (K) and W201 is replaced by Arg (R).
10. The Factor VII polypeptide according to claim 1, wherein the
Factor VII polypeptide is coupled with at least one half-life
extending moiety.
11. The Factor VII polypeptide according to claim 10, wherein the
half-life extending moiety is selected from biocompatible fatty
acids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g.
Hydroxy Ethyl Starch (HES), Poly Ethylen Glycol (PEG), Poly
(Glyx-Sery)n (HAP), Hyaluronic acid (HA), Heparosan polymers (HEP),
Phosphorylcholine-based polymers (PC polymer), Fleximers, Dextran,
Poly-sialic acids (PSA), Fc domains, Transferrin, Albumin, Elastin
like (ELP) peptides, XTEN polymers, PAS polymers, PA polymers,
Albumin binding peptides, CTP peptides, FcRn binding peptides and
any combination thereof.
12. The Factor VII polypeptide according to claim 11, wherein the
half-life extending moiety is a heparosan polymer.
13. The FVII polypeptide according to claim 1, which has a
proteolytic activity that is at least 110% that of wild type human
Factor VIIa (SEQ ID NO: 1), as measured in an in vitro proteolytic
assay, in the absence of soluble tissue factor; and which has less
than 20% antithrombin reactivity compared to wild type human Factor
VIIa, as measured in an antithrombin inhibition assay, in the
presence of low molecular weight heparin and the absence of soluble
tissue factor.
14. A pharmaceutical composition comprising the Factor VII
polypeptide of claim 1 and a pharmaceutically acceptable
carrier.
15. A method of treating a coagulopathy, comprising administering a
therapeutically or prophylactically effective amount of the Factor
VII polypeptide of claim 1 to a subject in need thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/514,794, filed Oct. 15, 2014, which claims priority under 35
U.S.C. .sctn. 119 of U.S. Provisional Application 61/895438, filed
Oct. 25, 2013; this application further claims priority of European
Application 13188715.0, filed Oct. 15, 2013, and European
Application 14154875.0, filed Feb. 12, 2014; the contents of all
above-named applications are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to coagulation Factor VII
(Factor VII) polypeptides having pro-coagulant activity. It also
relates to pharmaceutical compositions comprising such
polypeptides, methods of treatment and uses of such
polypeptides.
SEQUENCE LISTING
[0003] SEQ ID NO. 1: Wild type human coagulation Factor VII.
[0004] SEQ ID NO. 2: Protease domain of human coagulation Factor
VII.
[0005] SEQ ID NO. 3: Protease domain of hominin (chimpanzee)
coagulation Factor VII.
[0006] SEQ ID NO. 4: Protease domain of canine (dog) coagulation
Factor VII.
[0007] SEQ ID NO. 5: Protease domain of porcine (pig) coagulation
Factor VII.
[0008] SEQ ID NO. 6: Protease domain of bovine (cattle) coagulation
Factor VII.
[0009] SEQ ID NO. 7: Protease domain of murine (mouse) coagulation
Factor VII.
[0010] SEQ ID NO. 8: Protease domain of murine (rat) coagulation
Factor VII.
[0011] SEQ ID NO. 9: Protease domain of lapine (rabbit) coagulation
Factor VII.
[0012] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 16, 2016, is named 8719US05_SeqList and is 22 kilobytes in
size.
BACKGROUND OF INVENTION
[0013] An injury to a blood vessel activates the haemostatic system
that involves complex interactions between cellular and molecular
components. The process that eventually causes the bleeding to stop
is known as haemostasis. An important part of haemostasis is
coagulation of the blood and the formation of a clot at the site of
the injury. The coagulation process is highly dependent on the
function of several protein molecules. These are known as
coagulation factors. Some of the coagulation factors are proteases
which can exist in an inactive zymogen or an enzymatically active
form. The zymogen form can be converted to its enzymatically active
form by specific cleavage of the polypeptide chain catalyzed by
another proteolytically active coagulation factor.
[0014] Factor VII is a vitamin K-dependent plasma protein
synthesized in the liver and secreted into the blood as a
single-chain glycoprotein. The Factor VII zymogen is converted into
an activated form (Factor VIIa) by specific proteolytic cleavage at
a single site, i.e. between R152 and 1153 of SEQ ID NO: 1,
resulting in a two chain molecule linked by a single disulfide
bond. The two polypeptide chains in Factor VIIa are referred to as
light and heavy chain, corresponding to residues 1-152 and 153-406,
respectively, of SEQ ID NO: 1 (wild type human coagulation Factor
VII). Factor VII circulates predominantly as zymogen, but a minor
fraction is in the activated form (Factor VIIa).
[0015] The blood coagulation process can be divided into three
phases: initiation, amplification and propagation. The initiation
and propagation phases contribute to the formation of thrombin, a
coagulation factor with many important functions in haemostasis.
The coagulation cascade starts if the single-layered barrier of
endothelial cells that line the inner surface of blood vessels
becomes damaged. This exposes subendothelial cells and
extravascular matrix proteins to which platelets in the blood will
stick to. If this happens, Tissue Factor (TF) which is present on
the surface of sub-endothelial cells becomes exposed to Factor VIIa
circulating in the blood. TF is a membrane-bound protein and serves
as the receptor for Factor VIIa. Factor VIIa is an enzyme, a serine
protease, with intrinsically low activity. However, when Factor
VIIa is bound to TF, its activity increases greatly. Factor VIIa
interaction with TF also localizes Factor VIIa on the phospholipid
surface of the TF bearing cell and positions it optimally for
activation of Factor X to Xa. When this happens, Factor Xa can
combine with Factor Va to form the so-called "prothombinase"
complex on the surface of the TF bearing cell. The prothrombinase
complex then generates thrombin by cleavage of prothrombin. The
pathway activated by exposing TF to circulating Factor VIIa and
leading to the initial generation of thrombin is known as the TF
pathway. The TF:Factor VIIa complex also catalyzes the activation
of Factor IX to Factor IXa. Then activated Factor IXa can diffuse
to the surface of platelets which are sticking to the site of the
injury and have been activated. This allows Factor IXa to combine
with FVIIIa to form the "tenase" complex on the surface of the
activated platelet. This complex plays a key role in the
propagation phase due to its remarkable efficiency in activating
Factor X to Xa. The efficient tenase catalyzed generation of Factor
Xa activity in turn leads to efficient cleavage of prothrombin to
thrombin catalyzed by the prothrombinase complex.
[0016] If there are any deficiencies in either Factor IX or Factor
VIII, it compromises the important tenase activity, and reduces the
production of the thrombin which is necessary for coagulation.
Thrombin formed initially by the TF pathway serves as a
pro-coagulant signal that encourages recruitment, activation and
aggregation of platelets at the injury site. This results in the
formation of a loose primary plug of platelets. However, this
primary plug of platelets is unstable and needs reinforcement to
sustain haemostasis. Stabilization of the plug involves anchoring
and entangling the platelets in a web of fibrin fibres.
[0017] The formation of a strong and stable clot is dependent on
the generation of a robust burst of local thrombin activity. Thus,
deficiencies in the processes leading to thrombin generation
following a vessel injury can lead to bleeding disorders e.g.
haemophilia A and B. People with haemophilia A and B lack
functional Factor VIIIa or Factor IXa, respectively. Thrombin
generation in the propagation phase is critically dependent on
tenase activity, i.e. requires both Factor VIIIa and FIXa.
Therefore, in people with haemophilia A or B proper consolidation
of the primary platelet plug fails and bleeding continues.
[0018] Replacement therapy is the traditional treatment for
hemophilia A and B, and involves intravenous administration of
Factor VIII or Factor IX. In many cases, however, patients develop
antibodies (also known as inhibitors) against the infused proteins,
which reduce or negate the efficacy of the treatment. Recombinant
Factor VIIa (Novoseven.RTM.) has been approved for the treatment of
hemophilia A or B patients with inhibitors, and also is used to
stop bleeding episodes or prevent bleeding associated with trauma
and/or surgery. Recombinant Factor VIIa has also been approved for
the treatment of patients with congenital Factor VII deficiency. It
has been proposed that recombinant FVIIa operates through a
TF-independent mechanism. According to this model, recombinant
FVIIa is directed to the surface of the activated blood platelets
by virtue of its Gla-domain where it then proteolytically activates
Factor X to Xa thus by-passing the need for a functional tenase
complex. The low enzymatic activity of FVIIa in the absence of TF
as well as the low affinity of the Gla-domain for membranes could
explain the need for supra-physiological levels of circulating
FVIIa needed to achieve haemostasis in people with haemophilia.
[0019] Recombinant Factor VIIa has an in vivo functional half-life
of 2-3 hours which may necessitate frequent administration to
resolve bleedings in patients. Further, patients often only receive
Factor VIIa therapy after a bleed has commenced, rather than as a
precautionary measure, which often impinges upon their general
quality of life. A recombinant Factor VIIa variant with a longer in
vivo functional half-life would decrease the number of necessary
administrations, support less frequent dosing and thus holds the
promise of significantly improving Factor VIIa therapy to the
benefit of patients and care-holders.
[0020] WO02/22776 discloses Factor VIIa variants with enhanced
proteolytic activity compared to wild-type FVIIa. It has been
demonstrated in clinical trials that a Factor VII polypeptide
comprising substitutions disclosed in WO02/22776 shows a favourable
clinical outcome in terms of efficacy of a variant with enhanced
proteolytic activity (de Paula et al (2012) J Thromb Haemost,
10:81-89).
[0021] WO2007/031559 discloses Factor VII variants with reduced
susceptibility to inhibition by antithrombin.
[0022] WO2009/126307 discloses modified Factor VII polypeptides
with altered procoagulant activity.
[0023] In general, there are many unmet medical needs in people
with coagulopathies. The use of recombinant Factor VIIa to promote
clot formation underlines its growing importance as a therapeutic
agent. However, recombinant Factor VIIa therapy still leaves
significant unmet medical needs, a recombinant Factor VIIa
polypeptides having improved pharmaceutical properties, for example
increased in vivo functional half-life and enhanced or higher
activity, would meet some of these needs.
SUMMARY OF THE INVENTION
[0024] The present invention provides Factor VII polypeptides that
are designed to have improved pharmaceutical properties. In one
broad aspect, the invention relates to Factor VII polypeptides
exhibiting increased in vivo functional half-life as compared to
human wild-type Factor VIIa. In another broad aspect, the invention
relates to Factor VII polypeptides with enhanced activity as
compared to human wild-type Factor VIIa. In a further broad aspect,
the invention relates to Factor VII polypeptides exhibiting
increased resistance to inactivation by endogenous plasma
inhibitors, particularly antithrombin, as compared to human
wild-type Factor VIIa.
[0025] Provided herein are Factor VII polypeptides with increased
in vivo functional half-life which comprise a combination of
mutations conferring resistance to antithrombin inactivation and
enhanced or little or no loss of proteolytic activity. In a
particularly interesting aspect of the present invention the Factor
VII polypeptides are coupled to one or more "half-life extending
moieties" to increase the in vivo functional half-life.
[0026] In one aspect, the invention relates to a Factor VII
polypeptide comprising two or more substitutions relative to the
amino acid sequence of human Factor VII (SEQ ID NO:1), wherein T293
is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 is
replaced by Phe (F), Tyr (Y), Asn (N), or Ala (A) and/or W201 is
replaced by Arg (R), Met (M) or Lys (K) and/or K337 is replaced by
Ala (A) or Gly (G).
[0027] The Factor VII polypeptide of the invention may comprise a
substitution of T293 with Lys (K) and a substitution of L288 with
Phe (F). The Factor VII polypeptide may comprise a substitution of
T293 with Lys (K) and a substitution of L288 with Tyr (Y). The
Factor VII polypeptide may comprise a substitution of T293 with Arg
(R) and a substitution of L288 with Phe (F). The Factor VII
polypeptide may comprise a substitution of T293 with Arg (R) and a
substitution of L288 with Tyr (Y). The Factor VII polypeptide may
comprise, or may further comprise, a substitution of K337 with Ala
(A). The Factor VII polypeptide may comprise a substitution of T293
with Lys (K) and a substitution of W201 with Arg (R).
[0028] In an interesting embodiment the invention relates to Factor
VII polypeptides coupled with at least one half-life extending
moiety.
[0029] In another aspect, the invention relates to methods for
producing the Factor VII polypeptides of the invention.
[0030] In a further aspect, the invention relates to pharmaceutical
compositions comprising a Factor VII polypeptide of the
invention.
[0031] The general object of the present invention is to improve
currently available treatment options in people with coagulopathies
and to obtain Factor VII polypeptides with improved therapeutic
utility.
[0032] One object that the present invention has is to obtain
Factor VII polypeptides with prolonged in vivo functional half-life
while maintaining a pharmaceutically acceptable proteolytic
activity. To achieve this, the Factor VII polypeptides of the
present invention comprise a combination of mutations conferring
reduced susceptibility to inactivation by the plasma inhibitor
antithrombin while substantially preserving proteolytic activity;
in particularly interesting embodiments of the present invention
the Factor VII polypeptides are also coupled to one or more
"half-life extending moieties".
[0033] Medical treatment with the modified Factor VII polypeptides
of the present invention offers a number of advantages over the
currently available treatment regimes, such as longer duration
between injections, lower dosage, more convenient administration,
and potentially improved haemostatic protection between
injections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows amino acid sequence alignment of the FVIIa
protease domain from different species.
[0035] FIG. 2 shows the correlation between in vitro antithrombin
reactivity with the in vivo accumulation of FVIIa-antithrombin
complexes.
[0036] FIG. 3 shows the conformation of arginine at position 201 in
the FVIIa variant W201 R T293Y double mutant compared to the
conformation of tryptophan at position 201 in FVIIa WT.
[0037] FIG. 4 shows a hypothetical model of interaction between
tyrosine at position 293 from the FVIIa variant W201 R T293Y double
mutant with the antithrombin. This is based on a theoretical model
of a complex between antithrombin and the FVIIa variant W201 R
T293Y double mutant shown in stick representation. Antithrombin
amino acids are depicted with a prefix "AT"; while, the FVIIa amino
acids are depicted with a prefix "FVIIa".
[0038] FIG. 5A shows structure of heparosan.
[0039] FIG. 5B shows structure of a heparosan polymer with
maleimide functionality at its reducing end.
[0040] FIG. 6A and FIG. 6B show assessment of conjugate purity by
SDS-PAGE. FIG. 6A shows SDS-PAGE analysis of final FVIIa
conjugates. Gel was loaded with HiMark HMW standard (lane 1); FVIIa
(lane 2); 13k-HEP-[C]-FVIIa (lane 3); 27k-HEP-[C]-FVIIa (lane 4);
40k-HEP-[C]-FVIIa (lane 5); 52k-HEP-[C]-FVIIa (lane 6);
60k-HEP-[C]-FVIIa (lane 7); 65k-HEP-[C]-FVIIa (lane 8);
108k-HEP-[C]-FVIIa (lane 9) and 157k-HEP-[C]FVIIa407C (lane 10).
FIG. 6B shows SDS-PAGE of glycoconjugated 52k-HEP-[N]-FVIIa. Gel
was loaded with HiMark HMW standard (lane 1), ST3Gal3 (lane 2),
FVIIa (lane 3), asialo FVIIa (lane 4), and 52k-HEP-[N]-FVIIa (lane
5). [N]-denotes Factor conjugats where HEParosan is attached to the
N-glycan. [C]-Denotes Factor conjugates where Heparosan is attached
to a cystein residue.
[0041] FIG. 7: Analysis of FVIIa clotting activity levels of
heparosan conjugates and glycoPEGylated FVIIa references.
[0042] FIG. 8: Proteolytic activity of heparosan conjugates and
glycoPEGylated FVIIa references.
[0043] FIG. 9: PK results (LOCI) in Sprague Dawley rats. Comparison
of unmodified FVIIa (2 studies), 13k-HEP-[C]FVIIa407C,
27k-HEP-[C]FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]FVIIa407C,
65k-HEP-[C]FVIIa407C, 108k-HEP-[C]FVIIa407C and
157k-HEP-[C]FVIIa407C, glycoconjugated 52k-HEP-[N]-FVIIa and
reference molecules (40 kDa-PEG-[N]FVIIa (2 studies) and 40
kDa-PEG-[C]-FVIIa407C). Data are shown as mean.+-.SD (n =3-6) in a
semilogarithmic plot. [N]-denotes Factor conjugats where HEParosan
is attached to the N-glycan. [C]-Denotes Factor conjugates where
Heparosan is attached to a cystein residue.
[0044] FIG. 10: PK results (Clot Activity) in Sprague Dawley rats.
Comparison of unmodified FVIIa (2 studies), 13k-HEP-[C]FVIIa407C,
27k-HEP-[C]FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C,
65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]FVIIa407C and
157k-HEP-[C]FVIIa407C, glycoconjugated 52k-HEP-[N]-FVIIa and
reference molecules (40 kDa-PEG-[N]FVIIa (2 studies) and 40
kDa-PEG-[C]-FVIIa407C). Data are shown in a semilogarithmic plot.
[N]-denotes Factor conjugats where HEParosan is attached to the
N-glycan. [C]-Denotes Factor conjugates where Heparosan is attached
to a cystein residue.
[0045] FIG. 11: Relationship between HEP-size and mean residence
time (MRT) for a number of HEP-[C]FVIIa407C conjugates. MRT values
from PK studies are plotted against heparosan polymer size of
conjugates. The plot represent values for non-conjugated FVIIa,
13k-HEP-[C]FVIIa407C, 27k-HEP-[C]FVIIa407C, 40k-HEP-[C]FVIIa407C,
52k-HEP-[C]-FVIIa407C, 65k-HEP-[C]FVIIa407C, 108k-HEP-[C]FVIIa407C
and 157k-HEP-[C]-FVIIa407C. MRT (LOCI) was calculated by
non-compartmental methods using Phoenix WinNonlin 6.0 (Pharsight
Corporation). [N]-denotes Factor conjugats where HEParosan is
attached to the N-glycan. [C]-Denotes Factor conjugates where
Heparosan is attached to a cystein residue.
[0046] FIG. 12 Functionalization of glycylsialic acid cytidine
monophosphate (GSC) with a benzaldehyde group. GSC is acylated with
4-formylbenzoic acid and subsequently reacted with heparosan
(HEP)-amine by a reductive aminination reaction.
[0047] FIG. 13: Functionalization of heparosan (HEP) polymer with a
benzaldehyde group and subsequent reaction with glycylsialic acid
cytidine monophosphate (GSC) in a reductive amination reaction.
[0048] FIG. 14: Functionalization of glycylsialic acid cytidine
monophosphate (GSC) with a thio group and subsequent reaction with
a maleimide functionalized heparosan (HEP) polymer.
[0049] FIG. 15: Heparosan (HEP)--glycylsialic acid cytidine
monophosphate (GSC).
[0050] FIG. 16: PK results (LOCI) in Sprague Dawley rats.
Comparison of 2.times.20K-HEP-[N]-FVIIa; 1.times.40K-HEP-[N]FVIIa
and 1.times.40k-PEG-[N]FVIIa in a semilogarithmic plot. Data are
shown as mean .+-.SD (n=3-6).
[0051] FIG. 17: PK results (Clot Activity) in Sprague Dawley rats.
Comparison of 2.times.20K-HEP-[N]-FVIIa; 1.times.40K-HEP-[N]-FVIIa
and 1.times.40k-PEG-[N]-FVIIa in a semilogarithmic plot.
[0052] FIG. 18: Reaction scheme wherein an asialo FVIIa
glycoprotein is reacted with HEP-GSC in the presence of a ST3GaIIII
sialyltransferase.
DETAILED DESCRIPTION
[0053] The present invention relates to the design and use of
Factor VII polypeptides exhibiting improved pharmaceutical
properties.
[0054] In one aspect, the present invention relates to the design
and use of Factor VII polypeptides exhibiting increased in vivo
functional half-life, reduced susceptibility to inactivation by the
plasma inhibitor antithrombin and enhanced or preserved proteolytic
activity. It has been found by the inventors of the present
invention that specific combinations of mutations in human Factor
VII in combination with conjugation to half-life extending moieties
confer the above mentioned properties. The Factor VII polypeptides
of the invention have an extended functional half-life in blood
which is therapeutically useful in situations where a longer
lasting pro-coagulant activity is wanted. Such Factor VII
polypeptides comprise a substitution of T293 with Lys (K), Arg (R),
Tyr (Y) or Phe (F). In this aspect, the invention relates to a
Factor VII polypeptide comprising two or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1), wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe
(F) and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A), or
Trp (W). The invention also relates to a Factor VII polypeptide
comprising two or more substitutions relative to the amino acid
sequence of human Factor VII (SEQ ID NO:1), wherein T293 is
replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and W201 is
replaced by Arg (R), Met (M), or Lys (K). Furthermore, the
invention relates to a Factor VII polypeptide comprising two or
more substitutions relative to the amino acid sequence of human
Factor
[0055] VII (SEQ ID NO:1), whereinT293 is replaced by Lys (K), Arg
(R), Tyr (Y) or Phe (F) and K337 is replaced by Ala (A) or Gly (G).
Optionally, Factor VII polypeptides of the invention may further
comprise substitution of Q176 with Lys (K), Arg (R) or Asn (N).
Optionally, Factor VII polypeptides of the invention may further
comprise substitution of Q286 with Asn (N).
[0056] In another aspect, the present invention relates to the
design and use of Factor VII polypeptides exhibiting enhanced
proteolytic activity. It has been found by the inventors of the
present invention that specific mutations in human Factor VII at
positions L288 and/or W201 confer enhanced proteolytic activity to
Factor VII polypeptides. In this aspect, the invention relates to a
Factor VII polypeptide comprising one or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID NO:
1), wherein L288 is replaced by Phe (F), Tyr(Y), Asn (N), Ala (A)
or Trp (W), with the proviso that the polypeptide does not have the
following pair of substitutions: L288N/R290S or L288N/R290T.
Further according to this aspect, the invention relates to a Factor
VII polypeptide comprising one or more substitutions relative to
the amino acid sequence of human Factor VII (SEQ ID NO:1),
characterized in that one substitution is where W201 is replaced by
Arg (R), Met (M) or Lys (K).
Factor VII Coagulation Factor VII (Factor VII) is a glycoprotein
primarily produced in the liver.
[0057] The mature protein consists of 406 amino acid residues
defined by SEQ ID NO: 1 (also disclosed in, for example, in U.S.
Pat. No. 4,784,950) and is composed of four domains. There is an
N-terminal gamma-carboxyglutamic acid (Gla) rich domain followed by
two epidermal growth factor (EGF)-like domains and a C-terminal
trypsin-like serine protease domain. Factor VII circulates in
plasma, predominantly as a single-chain molecule. Factor VII is
activated to Factor VIIa by cleavage between residues Arg152 and
IIe153, resulting in a two-chain protein held together by a
disulphide bond. The light chain contains the Gla and EGF-like
domains, while the heavy chain is the protease domain. Specific Glu
(E) residues, i.e. E6, E7, E14, E16, E19, E20, E25, E26, E29 and
E35, according to SEQ ID NO: 1 in Factor VII may be
post-translationally gamma-carboxylated. The gamma-carboxyglutamic
acid residues in the Gla domain are required for coordination of a
number of calcium ions, which maintain the Gla domain in a
conformation mediating interaction with phospholipid membranes.
[0058] The terms FVII and "Factor VII" herein refers to the
uncleaved single-chain zymogen, Factor VII, as well as the cleaved,
two-chain and thus activated protease, Factor VIIa. "Factor VII"
includes natural allelic variants of Factor VII that may exist and
differ from one individual to another. A human wild-type Factor VII
sequence is provided in SEQ ID NO: 1. The term "Factor VII
polypeptide" herein refers to the uncleaved single chain zymogen
polypeptide variant of Factor VII (as described herein), as well as
the cleaved, two chain and thus activated protease.
[0059] Factor VII and Factor VII polypeptides may be plasma-derived
or recombinantly produced, using well known methods of production
and purification. The degree and location of glycosylation,
gamma-carboxylation and other post-translational modifications may
vary depending on the chosen host cell and its growth
conditions.
Factor VII Polypeptides
[0060] The terms "Factor VII" or "FVII" denote Factor VII
polypeptides.
[0061] The term "Factor VII polypeptide" encompasses wild type
Factor VII molecules as well as Factor VII variants, Factor VII
conjugates and Factor VII that has been chemically modified. Such,
variants, conjugates and chemically modified Factor VII may exhibit
substantially the same, or improved, activity relative to wild-type
human Factor VIIa.
[0062] The term "activity" of a Factor VII polypeptide, as used
herein, refers to any activity exhibited by wild-type human Factor
VII, and includes, but is not limited to, coagulation or coagulant
activity, pro-coagulant activity, proteolytic or catalytic activity
such as to effect Factor X activation or Factor IX activation;
ability to bind TF, Factor X or Factor IX; and/or ability to bind
to phospholipids. These activities can be assessed in vitro or in
vivo using recognized assays, for example, by measuring coagulation
in vitro or in vivo. The results of such assays indicate that a
polypeptide exhibits an activity that can be correlated to activity
of the polypeptide in vivo, in which in vivo activity can be
referred to as biological activity. Assays to determine activity of
a Factor VII polypeptide are known to those of skill in the art.
Exemplary assays to assess the activity of a FVII polypeptide
include in vitro proteolysis assays, such as those described in the
Examples, below.
[0063] The terms "enhanced, or preserved activity", as used herein,
refer to Factor VIIa polypeptides that exhibit substantially the
same or increased activity compared to wild type human Factor VIIa,
for example i) substantially the same or increased proteolytic
activity compared to recombinant wild type human Factor VIIa in the
presence and/or absence of TF; ii) to Factor VII polypeptides with
substantially the same or increased TF affinity compared to
recombinant wild type human Factor VIIa; iii) to Factor VII
polypeptides with substantially the same or increased affinity for
the activated platelet; or iv) Factor VII polypeptides with
substantially the same or increased affinity/ability to bind to
Factor X or Factor IX compared to recombinant wild type human
Factor VIIa. For example preserved activity means that the amount
of activity that is retained is or is about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100% or more of the activity compared to
wild type human Factor VIIa. For example enhanced activity means
that the amount of activity that is retained is or is about 110%,
120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%,
500%, 1000%, 3000%, 5000%, 10 000%, 30 000% or more of the activity
compared to wild type human Factor VIIa.
[0064] The term "Factor VII variant", as used herein, is intended
to designate a Factor VII having the sequence of SEQ ID NO: 1,
wherein one or more amino acids of the parent protein have been
substituted by another naturally occurring amino acid and/or
wherein one or more amino acids of the parent protein have been
deleted and/or wherein one or more amino acids have been inserted
in the protein and/or wherein one or more amino acids have been
added to the parent protein. Such addition can take place either at
the N- or at the C-terminus of the parent protein or both. In one
embodiment a variant is at least 95% identical with the sequence of
SEQ ID NO: 1. In another embodiment a variant is at least 80, 85,
90, 95, 96, 97, 98 or 99% identical with the sequence of SEQ ID NO:
1. As used herein, any reference to a specific position refers to
the corresponding position in SEQ ID NO: 1.
[0065] In some embodiments, the Factor VII variants of this
invention have an enhanced or preserved activity compared to wild
type human Factor VIIa.
[0066] The terminology for amino acid substitutions used in this
description is as follows. The first letter represents the amino
acid naturally present at a position of SEQ ID NO:1. The following
number represent the position in SEQ ID NO:1. The second letter
represents the different amino acid substituting the natural amino
acid. An example is K197A-Factor VII, wherein the Lysine at
position 197 of SEQ ID NO:1 is replaced by a Alanine.
[0067] In the present context the three-letter or one-letter
abbreviations of the amino acids have been used in their
conventional meaning as indicated in below. Unless indicated
explicitly, the amino acids mentioned herein are L-amino acids.
Abbreviations for Amino Acids:
TABLE-US-00001 [0068] Amino acid Tree-letter code One-letter code
Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu
L Isoleucine Ile I Methionine Met M Cysteine Cys C Phenylalanine
Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lysine Lys K
Arginine Arg R Glutamine Gln Q Asparagine Asn N Glutamic Acid Glu E
Aspartic Acid Asp D Serine Ser S Threonine Thr T
[0069] The term "Factor VII conjugates" as used herein, is intended
to designate a Factor VII polypeptide, in which one or more of the
amino acids and/or one or more of the attached glycan moieties have
been chemically and/or enzymatically modified, such as by
alkylation, glycosylation, acylation, ester formation, disulfide
bond formation, or amide formation.
[0070] In some embodiments, the Factor VII conjugates of the
invention exhibit substantially the same or enhanced biological
activity relative to wild-type Factor VII.
Enhanced Proteolytic Activity
[0071] Factor VII polypeptides with certain mutations of residues
L288 and W201 have, surprisingly, been shown by the inventors to
exhibit enhanced proteolytic activity.
[0072] The Factor VII variant K337A, as described in WO02/22776,
has been described to have enhanced proteolytic activity. The
Factor VII variants L305V and L3051, as described in WO03/027147,
have also been described to have higher intrinsic activity.
[0073] The proteolytic activity may be determined by any suitable
method known in the art as further discussed below.
[0074] For example enhanced proteolytic activity means that the
amount of activity that is retained is or is about 110%, 120%,
130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%,
1000%, 3000%, 5000%, 10 000%, 30 000% or more of the activity
compared to wild type human Factor VIIa.
Half-life--Resistance to Inactivation by Plasma Inhibitors
[0075] Besides in vivo clearance, in vivo functional half-life is
of importance to the period of time during which the compound is
"therapeutically available" in the body. The circulating half-life
of recombinant human wild type Factor VIIa is about 2.3 hours
("Summary Basis for Approval for NovoSeven.COPYRGT.", FDA reference
number 96 0597).
[0076] The term "in vivo functional half-life" is used in its
normal meaning, i.e., the time required for reducing the biological
activity of the Factor VII polypeptide remaining in the body/target
organ with 50% in the terminal phase, or the time at which the
activity of the Factor VII polypeptide is 50% of its initial value.
Alternative terms to in vivo half-life include terminal half-life,
plasma half-life, circulating half-life, circulatory half-life, and
clearance half-life. Half-life may be determined by suitable
methods known in the art, such as that described in Example 17 and
those described in Introduction to Pharmacokinetics and
Pharmacodynamics: The Quantitative Basis of Drug Therapy (Thomas N.
Tozer, Malcolm Rowland).
[0077] The term "increased" as used about the in vivo functional
half-life or plasma half-life is used to indicate that the relevant
half-life of the polypeptide is increased relative to that of a
reference molecule, such as wild-type human Factor VIIa as
determined under comparable conditions.
[0078] In some embodiments, the Factor VII polypeptides of the
invention exhibit increased in vivo functional half-life relative
to wild-type human Factor VIIa. For instance the relevant half-life
may be increased by at least about 25%, such as by at least about
50%, e.g., by at least about 100%, 150%, 200%, 500%, 1000%, 3000%,
5000%, 10 000%, 30 000% or more.
[0079] Despite the detailed understanding of the biochemistry and
pathophysiology of the coagulation cascade, the mechanistic basis
for the clearance of the individual coagulation factors from
circulation remains largely unknown. The marked differences in the
circulating half-lives of Factor VII and its activated form Factor
VIIa compared with zymogen and enzyme forms of other vitamin
K-dependent proteins suggest the existence of specific and distinct
clearance mechanisms for Factor VIIa. Two types of pathways appear
to be operable in the clearance of Factor VIIa--one resulting in
elimination of the intact protein, the other mediated by plasma
inhibitors and leading to proteolytic inactivation.
[0080] Antithrombin III (antithrombin, AT) is an abundant plasma
inhibitor and targets most proteases of the coagulation system,
including Factor VIIa. It is present at micromolar concentrations
in plasma and belongs to the serpin family of serine protease
inhibitors that irreversibly bind and inactivate target proteases
by a suicide substrate mechanism. The inhibition by antithrombin
appears to constitute the predominant clearance pathway of
recombinant Factor VIIa in vivo following intravenous
administration. In a recent study of the pharmacokinetics of
recombinant Factor VIIa in haemophilia patients, about 60% of the
total clearance could be attributed to this pathway (Agerso et al.
(2011) J Thromb Haemost, 9, 333-338).
[0081] In some embodiments, the Factor VII polypeptides of the
invention exhibit increased resistance to inactivation by the
endogenous plasma inhibitors, particularly antithrombin, relative
to wild-type human Factor VIIa.
[0082] It has been found by the inventors of the present invention
that by combining the two groups of mutations mentioned above, i.e.
mutations conferring increased AT resistance and mutations
conferring enhanced proteolytic activity, an increased or preserved
activity is achieved while maintaining high resistance to inhibitor
inactivation. That is, the Factor VII polypeptides of the present
invention comprising a combination of mutations exhibit increased
resistance to antithrombin inactivation as well as substantially
preserved proteolytic activity. When the Factor VII polypeptides of
the invention are conjugated with one or more half-life extending
moieties a surprisingly improved effect on half-life extension is
achieved. Given these properties, such conjugated Factor VII
polypeptides of the invention exhibit increased circulatory
half-life while maintaining a pharmaceutically acceptable
proteolytic activity. Consequently, a lower dose of such conjugated
Factor VII polypeptide may be required to obtain a functionally
adequate concentration at the site of action and thus it will be
possible to administer a lower dose and/or with lower frequency to
the subject having bleeding episodes or needing enhancement of the
normal haemostatic system.
Additional Modifications
[0083] The Factor VII polypeptides of the invention may comprise
further modifications, in particular further modifications which
confer additional advantageous properties to the Factor VII
polypeptide. Thus, in addition to the amino acid substitutions
mentioned above, the Factor VII polypeptides of the invention may
for example comprise further amino acid modification, e.g. one
further amino acid substitution. In one such embodiment, the Factor
VII polypeptide of the invention has an additional mutation or
addition selected from the group R396C, Q250C, and 407C, as
described e.g. in WO2002077218.
[0084] The Factor VII polypeptides of the invention may comprise
additional modifications that are or are not in the primary
sequence of the Factor VII polypeptide. Additional modifications
include, but are not limited to, the addition of a carbohydrate
moiety, the addition of a half-life extending moiety, e.g. the
addition of a, PEG moiety, an Fc domain, etc. For example, such
additional modifications can be made to increase the stability or
half-life of the Factor VII polypeptide.
Half-Life Extending Moieties or Groups
[0085] The term "half-life extending moieties" are herein used
interchangeably and understood to refer to one or more chemical
groups attached to one or more amino acid site chain
functionalities such as --SH, --OH, --COOH, --CONH2, -NH2, and/or
one or more N-and/or O-glycan structures and that can increase in
vivo functional half-life of proteins/polypeptides when
conjugated/coupled to these proteins/polypeptides.
[0086] The in vivo functional half-life may be determined by any
suitable method known in the art as further discussed below
(Example 17).
[0087] Examples of half-life extending moieties include:
Biocompatible fatty acids and derivatives thereof, Hydroxy Alkyl
Starch (HAS) e.g. Hydroxy Ethyl Starch (HES), Poly Ethylen Glycol
(PEG), Poly (Glyx-Sery)n (HAP), Hyaluronic acid (HA), Heparosan
polymers (HEP), Phosphorylcholine-based polymers (PC polymer),
Fleximers, Dextran, Poly-sialic acids (PSA), Fc domains,
Transferrin, Albumin, Elastin like (ELP) peptides, XTEN polymers,
PAS polymers, PA polymers, Albumin binding peptides, CTP peptides,
FcRn binding peptides and any combination thereof.
[0088] In a particularly interesting embodiment, the Factor VII
polypeptide of the invention is coupled with one or more half-life
extending moieties.
[0089] In one embodiment, a cysteine-conjugated Factor VII
polypeptide of the invention have one or more hydrophobic half-life
extending moieties conjugated to a sulfhydryl group of a cysteine
introduced in the Factor VII polypeptide. It is furthermore
possible to link half-life extending moieties to other amino acid
residues.
[0090] In one embodiment, the Factor VII polypeptide of the
invention is disulfide linked to tissue factor, as described e.g.
in WO2007115953.
[0091] In another embodiment, the Factor VII polypeptide of the
invention is a Factor VIIa variant with increased platelet
affinity.
Heparosan Conjugates
[0092] Factor VII polypeptide heparosan conjugates according to the
present invention may have one or more Heparosan polymer (HEP)
molecules attached to any part of the FVII polypeptide including
any amino acid residue or carbohydrate moiety of the Factor VII
polypeptide. Examples of such conjugates are provided in
WO2014/060397, which is herein incorporated by reference. Chemical
and/or enzymatic methods can be employed for conjugating HEP to a
glycan on the Factor VII polypeptide. An example of an enzymatic
conjugation process is described e.g. in WO03031464. The glycan may
be naturally occurring or it may be engineered in, e.g. by
introduction of an N-glycosylation motif (NXT/S where X is any
naturally occurring amino acid) in the amino acid sequence of
Factor VII using methods well known in the art.
[0093] "Cysteine-HEP Factor VII polypeptide conjugates" according
to the present invention have one or more HEP molecules conjugated
to a sulfhydryl group of a cysteine residue present or introduced
in the FVII polypeptide.
[0094] In one interesting embodiment of the invention, the Factor
VII polypeptide is coupled to a HEP polymer. In one embodiment the
HEP polymer coupled to the Factor VII polypeptide has a molecular
weight in a range selected from 13-65 kDa, 13-55 kDa, 25-55 kDa,
25-50 kDa, 25-45 kDa, 30-45 kDa, 36-44 kDa and 38-42 kDa, or a
molecular weight of 40 kDa.
[0095] In one interesting embodiment of the invention, the Factor
VII polypeptide is coupled to a HEP polymer on an N-glycan of the
Factor VII polypeptide.
[0096] In a further embodiment of the invention, two HEP polymers
are coupled to the same Factor VII polypeptide via N-glycans. In
this embodiment each of the HEP polymer coupled to the Factor VII
polypeptide has a molecular weight in a range selected from 13-65
kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa, 36-44
kDa and 38-42 kDa, or a molecular weight of 40 kDa. Preferably, the
polymers have identical molecular weight.
[0097] In a specific embodiment two 20 kDa-HEP polymers are coupled
to the same Factor VII polypeptide via its N-glycans.
[0098] In a specific embodiment two 30 kDa-HEP polymers are coupled
to the same Factor VII polypeptide via its N-glycans.
[0099] In a specific embodiment two 40 kDa-HEP polymers are coupled
to the same Factor VII polypeptide via its N-glycans.
Heparosan Polymers
[0100] Heparosan (HEP) is a natural sugar polymer comprising
(--GlcUA-beta1,4-GlcNAc-alpha1, 4-) repeats (see FIG. 5A). It
belongs to the glycosaminoglycan polysaccharide family and is a
negatively charged polymer at physiological pH. It can be found in
the capsule of certain bacteria but it is also found in higher
vertebrates, where it serves as precursor for the natural polymers
heparin and heparan sulphate. Although not proven in detail,
heparosan is believed to be degraded in the lysosomes. An injection
of a 100 kDa heparosan polymer labelled with Bolton-Hunter reagents
has shown that heparosan is secreted as smaller fragments in body
fluids/waste (U.S. 2010/0036001).
[0101] Heparosan polymers and methods of making such polymers are
described in U.S. 2010/0036001, the content of which is
incorporated herein by reference. In accordance with the present
invention, the heparosan polymer may be any heparosan polymer
described or disclosed in U.S. 2010/0036001.
[0102] For use in the present invention, heparosan polymers can be
produced by any suitable method, such as any of the methods
described in U.S. 2010/0036001 or U.S. 2008/0109236. Heparosan can
be produced using bacterial-derived enzymes. For example, the
heparosan synthase PmHS1 of Pasteurella multocida Type D
polymerises the heparosan sugar chain by transferring both GlcUA
and GlcNAc. The Escherichia coli K5 enzymes KfiA (alpha GlcNAc
transferase) and KfiC (beta GlcUA transferase) can together also
form the disaccharide repeat of heparosan.
[0103] A heparosan polymer for use in the present invention is
typically a polymer of the formula
(-GlcUA-beta1,4-GlcNAc-alpha1,4-).sub.n.
[0104] The size of the heparosan polymer may be defined by the
number of repeats n in this formula. The number of said repeats n
may be, for example, from 2 to about 5000. The number of repeats
may be, for example 50 to 2000 units, 100 to 1000 units or 200 to
700 units. The number of repeats may be 200 to 250 units, 500 to
550 units or 350 to 400 units. Any of the lower limits of these
ranges may be combined with any higher upper limit of these ranges
to form a suitable range of numbers of units in the heparosan
polymer.
[0105] The size of the heparosan polymer may be defined by its
molecular weight. The molecular weight may be the average molecular
weight for a population of heparosan polymer molecules, such as the
weight average molecular mass.
[0106] Molecular weight values as described herein in relation to
size of the heparosan polymer may not, in practise, exactly be the
size listed. Due to batch to batch variation during heparosan
polymer production, some variation is to be expected. To encompass
batch to batch variation, it is therefore to be understood, that a
variation around +/-10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%
around target HEP polymer size should be expected. For example HEP
polymer size of 40 kDa denotes 40 kDa +/-10%, e.g. 40 kDa could for
example in practise mean 38.8 kDa, 41.5 kDa or 43.8 kDa
[0107] The heparosan polymer may have a molecular weight of, for
example, 500 Da to 1,000 kDa. The molecular weight of the polymer
may be 500 Da to 650 kDa, 5 kDa to 750 kDa, 10 kDa to 500 kDa, 15
kDa to 550 kDa or 25 kDa to 250 kDa.
[0108] The molecular weight may be selected at particular levels
within these ranges in order to achieve a suitable balance between
activity of the Factor VII polypeptide and half-life or mean
residence time of the conjugate. For example, the molecular weight
of the polymer may be in a range selected from 15-25 kDa, 25-35
kDa, 35-45 kDa, 45-55 kDa, 55-65 kDa or 65-75 kDa.
[0109] More specific ranges of molecular weight may be selected.
For example, the molecular weight may be 20 kDa to 35 kDa, such as
22 kDa to 32 kDa such as 25 kDa to 30 kDa, such as about 27 kDa.
The molecular weight may be 35 to 65 kDa, such as 40 kDa to 60 kDa,
such as 47 kDa to 57 kDa, such as 50 kDa to 55 kDa such as about 52
kDa. The molecular weight may be 50 to 75 kDa such as 60 to 70 kDa,
such as 63 to 67 kDa such as about 65 kDa.
[0110] In particularly interesting embodiments, the heparosan
polymer of the Factor VII conjugate, of the invention, has a size
in a range selected from 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50
kDa, 25-45 kDa, 30-45 kDa and 38-42 kDa.
[0111] Any of the lower limits of these ranges of molecular weight
may be combined with any higher upper limit from these ranges to
form a a suitable range for the molecular weight of the heparosan
polymer in accordance with the invention.
[0112] The heparosan polymer may have a narrow size distribution
(i.e. be monodisperse) or a broad size distribution (i.e. be
polydisperse). The level of polydispersity (PDI) may be represented
numerically based on the formula Mw/Mn, where Mw=weight average
molecular mass and Mn=number average molecular weight. The
polydispersity value using this equation for an ideal monodisperse
polymer is 1. Preferably, a heparosan polymer for use in the
present invention is monodisperse. The polymer may therefore have a
polydispersity that is about 1, the polydispersity may be less than
1.25, preferably less than 1.20, preferably less than 1.15,
preferably less than 1.10, preferably less than 1.09, preferably
less than 1.08, preferably less than 1.07, preferably less than
1.06, preferably less than 1.05.
[0113] The molecular weight size distribution of the heparosan may
be measured by comparison with monodisperse size standards (HA
Lo-Ladder, Hyalose LLC) which may be run on agarose gels.
[0114] Alternatively, the size distribution of heparosan polymers
may be determined by high performance size exclusion
chromatography-multi angle laser light scattering (SEC-MALLS). Such
a method can be used to assess the molecular weight and
polydispersity of a heparosan polymer.
[0115] Polymer size may be regulated in enzymatic methods of
production. By controlling the molar ratio of heparosan acceptor
chains to UDP sugar, it is possible to select a final heparosan
polymer size that is desired
[0116] The heparosan polymer may further comprise a reactive group
to allow its attachment to a Factor VII polypeptide. A suitable
reactive group may be, for example, an aldehyde, alkyne, ketone,
maleimide, thiol, azide, amino, hydrazide, hydroxylamine, carbonate
ester, chelator or a combination of any thereof. For example, FIG.
5B illustrates a heparosan polymer comprising a maleimide
group.
[0117] As set out in the Examples, maleimide or aldehyde
functionalized heparosan polymers of defined size may be prepared
by an enzymatic (PmHS1) polymerization reaction using the two sugar
nucleotides UDP-GlcNAc and UDP-GlcUA in equimolar amount. A priming
trisaccharide (GlcUA-GlcNAc-GlcUA)NH.sub.2 may be used for
initiating the reaction, and polymerization run until depletion of
sugar nucleotide building blocks. Terminal amine (originating from
the primer) may then be functionalized with suitable reactive
groups such as a reactive group as described above, such as a
maleimide functionality for conjugation to free cysteines or
aldehydes for reductive amination to amino groups. The size of the
heparosan polymers can be pre-determined by variation in sugar
nucleotide: primer stoichiometry. The technique is described in
detail in U.S. 2010/0036001.
[0118] The reactive group may be present at the reducing or
non-reducing termini or throughout the sugar chain. The presence of
only one such reactive group is preferred when conjugating the
heparosan polymer to the polypeptide.
Methods for Preparing FVII-HEP Conjugates
[0119] For example, WO 03/031464 describes methods for remodelling
the glycan structure of a polypeptide, such as a Factor VII or
Factor VIIa polypeptide and methods for the addition of a modifying
group such as a water soluble polymer to such a polypeptide. Such
methods may be used to attach a heparosan polymer to a Factor VII
polypeptide in accordance with the present invention.
[0120] As set out in the Examples, a Factor VII polypeptide may be
conjugated to its glycan moieties using sialyltransferase. For
enablement of this approach, a HEP polymer first need to be linked
to a sialic acid cytidine monophosphate. Glycylsialic acid cytidine
monophosphate (GSC) is a suitable starting point for such
chemistry, but other sialic acid cytidine monophosphate or
fragments of such can be used. Examples set out methods for
covalent linking HEP polymers to GSC molecules. By covalent
attachment, a HEP-GSC (HEP conjugated glycylsialic acid cytidine
monophosphate) molecule is created that can be transferred to
glycan moieties of FVIIa.
[0121] Factor VII-heparosan conjugates may be purified once they
have been produced. For example, purification may comprise affinity
chromatography using immobilised mAb directed towards the Factor
VII polypeptide, such as mAb directed against the calcified
gla-domain on FVIIa. In such an affinity chromatography method,
unconjugated HEP-polymer may be removed by extensive washing of the
column. FVII may be released from the column by releasing the FVII
from the antibody. For example, where the antibody is specific to
the calcified gla-domain, release from the column may be achieved
by washing with a buffer comprising EDTA.
[0122] Size exclusion chromatography may be used to separate Factor
VII-heparosan conjugates from unconjugated Factor VII.
[0123] Pure conjugate may be concentrated by ultrafiltration.
[0124] Final concentrations of Factor VII-heparosan conjugate
resulting from a process of production may be determined by, for
example, HPLC quantification, such as HPLC quantification of the
FVII light chain.
[0125] In connection with the present invention, it is shown that
it is possible to link a carbohydrate polymer such as HEP via a
maleimido group to a thio-modified GSC molecule and transfer the
reagent to an intact glycosyl group on a glycoprotein by means of a
sialyltransferase, thereby creating a linkage that contains a
cyclic succinimide group.
[0126] Succinimide based linkages, however, may undergo hydrolytic
ring opening when the conjugate is stored in aqueous solution for
extended time periods (Bioconjugation Techniques, G. T. Hermanson,
Academic Press, 3.sup.rd edition 2013 p. 309) and while the linkage
may remain intact, the ring opening reaction will add undesirable
heterogeneity in form of regio- and stereo-isomers to the final
conjugate.
[0127] It follows from the above that it is preferable to link the
half-life extending moiety to the glycoprotein in such a way that
1) the glycan residue of the glycoprotein is preserved in intact
form, and 2) no heterogenicity is present in the linker part
between the intact glycosyl residue and the half-life extending
moiety.
[0128] There is a need in the art for methods of conjugating two
compounds, such as a half-life extending moiety such as HEP to a
protein or protein glycan, wherein the compounds are linked such
that a stable and isomer free conjugate is obtained.
[0129] In one aspect the present invention provides a stable and
isomer free linker for use in glycylsialic acid cytidine
monophosphate (GSC) based conjugation of HEP to FVII. The GSC
starting material used in the current invention can be synthesised
chemically (Dufner, G. Eur. J. Org. Chem. 2000, 1467-1482) or it
can be obtained by chemoenzymatic routes as described in
WO07056191. The GSC structure is shown below:
##STR00001##
[0130] In one embodiment conjugates according to the present
invention comprise a linker comprising the following structure:
##STR00002##
--hereinafter also referred to as sublinker or sublinkage - that
connects a HEP-amine and GSC in one of the following ways:
##STR00003##
[0131] The highlighted 4-methylbenzoyl sublinker thus makes up part
of the full linking structure linking the half-life extending
moiety to a target protein. The sublinker is as such a stable
structure compared to alternatives, such as succinimide based
linkers (prepared from maleimide reactions with sulfhydryl groups)
since the latter type of cyclic linkage has a tendency to undergo
hydrolytic ring opening when the conjugate is stored in aqueous
solution for extended time periods (Bioconjugation Techniques, G.
T. Hermanson, Academic Press, 3.sup.rd edition 2013 p. 309). Even
though the linkage in this case (e.g. between HEP and sialic acid
on a glycoprotein) may remain intact, the ring opening reaction
will add heterogeneity in form of regio- and stereo-isomers to the
final conjugate composition.
[0132] One advantage associated with conjugates according to the
present invention is thus that a homogenous composition is
obtained, i.e. that the tendency of isomer formation due to linker
structure and stability is significantly reduced. Another advantage
is that the linker and conjugates according to the invention can be
produced in a simple process, preferably a one-step process.
[0133] Isomers are undesirable since these can lead to a
heterogeneous product and increase the risk for unwanted immune
responses in humans.
[0134] The 4-methylbenzoyl sublinkage as used in the present
invention between HEP and GSC is not able to form sterio- or regio
isomers. HEP polymers can as mentioned earlier be prepared by a
synchronised enzymatic polymerisation reaction (U.S. 20100036001).
This method use heparan synthetase I from Pasturella multocida
(PmHS1) which can be expressed in E.coli as a maltose binding
protein fusion constructs. Purified MBP-PmHS1 is able to produce
monodisperse polymers in a synchronized, stoichiometrically
controlled reaction, when it is added to an equimolar mixture of
sugar nucleotides (GlcNAc-UDP and GlcUA-UDP). A trisaccharide
initiator (GlcUA-GlcNAc-GlcUA) is used to prime the reaction, and
polymer length is determined by the primer:sugar nucleotide ratios.
The polymerization reaction will run until about 90% of the sugar
nucleotides are consumed. Polymers are isolated from the reaction
mixture by anion exchange chromatography, and subsequently
freeze-dried into stable powder.
[0135] Processes for preparation of functional HEP polymers are
described in U.S. 20100036001 which for example lists aldehyde-,
amine- and maleimide functionalized HEP reagents. U.S. 20100036001
is hereby incorporated by reference in its entirety as if fully set
forth herein. A range of other functionally modified HEP
derivatives are available using similar chemistry. HEP polymers
used in certain embodiments of the present invention are initially
produced with a primary amine handle at the reducing terminal
according to methods described in U.S. 20100036001.
[0136] Amine functionalized HEP polymers (i.e. HEP having an
amine-handle) prepared according U.S. 20100036001 can be converted
into a HEP-benzaldehyde by reaction with N-succinimidyl
4-formylbenzoate and subsequently coupled to the glycylamino group
of GSC by a reductive amination reaction. The resulting HEP-GSC
product can subsequently be enzymatically conjugated to a
glycoprotein using a sialyltransferase.
[0137] For example, said amine handle on HEP can be converted into
a benzaldehyde functionality by reaction with N-succinimidyl
4-formylbenzoateaccording to the below scheme:
##STR00004##
[0138] The conversion of HEP amine (1) to the 4-formylbenzamide
compound (2) in the above scheme may be carried out by reaction
with acyl activated forms of 4-formylbenzoic acid.
[0139] N-succinimidyl may be chosen as acyl activation group but a
number of other acyl activation groups are known to the skilled
person. Non-limited examples include 1-hydroxy-7-azabenzotriazole-,
1-hydroxy-benzotriazole-, pentafluorophenyl-esters as know from
peptide chemistry.
[0140] HEP reagents modified with a benzaldehyde functionality can
be kept stable for extended time periods when stored frozen
(-80.degree. C.) in dry form. Alternatively, a benzaldehyde moiety
can be attached to the GSC compound, thereby resulting in a
GSC-benzaldehyde compound suitable for conjugation to an amine
functionalized half-life extending moiety. This route of synthesis
is depicted in FIG. 12.
[0141] For example, GSC can be reacted under pH neutral conditions
with N-succinimidyl 4-formylbenzoate to provide a GSC compound that
contains a reactive aldehyde group (see for example WO2011101267).
The aldehyde derivatized GSC compound (GSC-benzaldehyde) can then
be reacted with HEP-amine and reducing agent to form a HEP-GSC
reagent. The above mentioned reaction may be reversed, so that the
HEP-amine is first reacted with N-succinimidyl 4-formylbenzoate to
form an aldehyde derivatized HEP-polymer, which subsequently is
reacted directly with GSC in the presence of a reducing agent. In
practice this eliminates the tedious chromatographic handling of
GSC-CHO. This route of synthesis is depicted in FIG. 13.
[0142] Thus, in one embodiment of the present invention
HEP-benzaldehyde is coupled to GSC by reductive amination.
[0143] Reductive amination is a two-step reaction which proceeds as
follows: Initially an imine (also known as Schiff-base) is formed
between the aldehyde component and the amine component (in the
present embodiment the glycyl amino group of GSC). The imine is
then reduced to an amine in the second step. The reducing agent is
chosen so that it selectively reduces the formed imine to an amine
derivative.
[0144] A number of suitable reducing reagents are available to the
skilled person. Non-limiting examples include sodium
cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4), pyridin
boran complex (BH3:Py), dimethylsulfide boran complex (Me2S:BH3)
and picoline boran complex.
[0145] Although reductive amination to the reducing end of
carbohydrates (for example to the reducing termini of HEP polymers)
is possible, it has generally been described as a slow and
inefficient reaction (J C. Gildersleeve, Bioconjug Chem. 2008 July;
19(7): 1485-1490). Side reactions, such as the Amadori reaction,
where the initially formed imine rearrange to a keto amine are also
possible, and will lead to heterogenicity which as previously
discussed is undesirable in the present context.
[0146] Aromatic aldehydes such as benzaldehydes derivatives are not
able to form such rearrangement reactions as the imine is unable to
enolize and also lack the required neighbouring hydroxy group
typically found in carbohydrate derived imines. Aromatic aldehydes
such as benzaldehydes derivatives are therefore particular useful
in reductive amination reactions for generating isomer free HEP-GSC
reagent.
[0147] A surplus of GSC and reducing reagent is optionally used in
order to drive reductive amination chemistry fast to completion.
When the reaction is completed, the excess (non-reacted) GSC
reagent and other small molecular components such as excess
reducing reagent can subsequently be removed by dialysis,
tangential flow filtration or size exclusion chromatography.
[0148] Both the natural substrate for sialyltransferases, Sia-CMP,
and the GSC derivatives are multifunctional molecules that are
charged and highly hydrophilic. In addition, they are not stable in
solution for extended time periods especially if pH is below 6.0.
At such low pH, the CMP activation group necessary for substrate
transfer is lost due to acid catalyzed phosphate diester
hydrolysis. Selective modification and isolation of GSC and Sia-CMP
derivatives thus require careful control of pH, as well as fast and
efficient isolation methods, in order to avoid CMP-hydrolysis.
[0149] In the present invention, large half-life extending moieties
are conjugated to GSC using reductive amination chemistry.
Arylaldehydes, such as benzaldhyde modified half-life extending
moieties have been found optimal for this type of modification, as
they efficiently can react with GSC under reductive amination
conditions.
[0150] As GSC may undergo hydrolysis in acid media, it is important
to maintain a near neutral or slightly basic environment during the
coupling to HEP-benzaldehydes. HEP polymers and GSC are both highly
water soluble and aqueous buffer systems are therefore preferable
for maintaining pH at a near neutral level. A number of both
organic and inorganic buffers may be used, however, the buffer
components should preferably not be reactive under reductive
amination conditions. This exclude for instance organic buffer
systems containing primary and--to lesser extend--secondary amino
groups. The skilled person will know which buffers are suitable and
which are not. Some examples of suitable buffers are shown in table
1 below:
TABLE-US-00002 TABLE 1 Buffers Common pKa at Buffer Name 25.degree.
C. Range Full Compound Name Bicine 8.35 7.6-9.0
N,N-bis(2-hydroxyethyl)glycine Hepes 7.48 6.8-8.2
4-2-hydroxyethyl-1-piperazineethanesulfonic acid TES 7.40 6.8-8.2
2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid MOPS 7.20
6.5-7.9 3-(N-morpholino)propanesulfonic acid PIPES 6.76 6.1-7.5
piperazine-N,N'-bis(2-ethanesulfonic acid) MES 6.15 5.5-6.7
2-(N-morpholino)ethanesulfonic acid
[0151] By applying this method, GSC reagents modified with
half-life extending moieties, having isomer free stable linkages
can efficient be prepared, and isolated in a simple process that
minimize the chance for hydrolysis of the CMP activation group.
[0152] By reacting either of said compounds with each other a
HEP-GSC conjugate comprising a 4-methylbenzoyl sublinker moiety may
be created.
[0153] GSC may also be reacted with thiobutyrolactone, thereby
creating a thiol modified GSC molecule (GSC-SH). As demonstrated in
the present invention, such reagents may be reacted with maleimide
functionalized HEP polymers to form HEP-GSC reagents. This
synthesis route is depicted in FIG. 15. The resulting product has a
linkage structure comprising succinimide.
##STR00005##
[0154] However, succinimide based (sub)linkages may undergo
hydrolytic ring opening inter alia when the modified GSC reagent is
stored in aqueous solution for extended time periods and while the
linkage may remain intact, the ring opening reaction will add
undesirable heterogeneity in form of regio- and stereo-isomers.
Methods of Glycoconjugation
[0155] Conjugation of a HEP-GSC conjugate with a (poly)-peptide may
be carried out via a glycan present on residues in the
(poly)-peptide backbone. This form of conjugation is also referred
to as glycoconjugation.
[0156] Methods based on sialyltransferase have over the years
proven to be mild and highly selective for modifying N-glycans or
O-glycans on blood coagulation factors, such as coagulation factor
FVII.
[0157] In contrast to conjugation methods based on cysteine
alkylations, lysine acylations and similar conjugations involving
amino acids in the protein backbone, conjugation via glycans is an
appealing way of attaching larger structures such as polymers of
protein/peptide fragments to bioactive proteins with less
disturbance of bioactivity. This is because glycans being highly
hydrophilic generally tend to be oriented away from the protein
surface and out in solution, leaving the binding surfaces that are
important for the proteins activity free.
[0158] The glycan may be naturally occurring or it may be inserted
via e.g. insertion of an N-linked glycan using methods well known
in the art.
[0159] GSC is a sialic acid derivative that can be transferred to
glycoproteins by the use of sialyltransferases. It can be
selectively modified with substituents such as PEG on the glycyl
amino group and still be enzymatically transferred to glycoproteins
by use of sialyltransferases. GSC can be efficiently prepared by an
enzymatic process in large scale (WO07056191).
Sialyltransferases
[0160] Sialyltransferases are a class of glycosyltransferases that
transfer sialic acid from naturally activated sialic acid
(Sia)--CMP (cytidine monophosphate) compounds to
galactosyl-moieties on e.g. proteins. Many sialyltransferases
(ST3GallII, ST3Gall, ST6GalNAcl) are capable of transfer of sialic
acid--CMP (Sia-CMP) derivatives that have been modified on the C5
acetamido group inter alia with large groups such as 40 kDa PEG
(WO003031464). An extensive, but non-limited list of relevant
sialyltransferases that can be used with the current invention is
disclosed in WO2006094810, which is hereby incorporated by
reference in its entirety.
[0161] In one aspect of the present invention, terminal sialic
acids on glycoproteins can be removed by sialidase treatment to
provide asialo glycoproteins. Asialo glycoproteins and GSC modified
with the half-life extending moiety together will act as substrates
for sialyltransferases. The product of the reaction is a
glycoprotein conjugate having the half-life extending moiety linked
via an intact glycosyl linking group--in this case an intact sialic
acid linker group. A reaction scheme wherein an asialo FVIIa
glycoprotein is reacted with HEP-GSC in the presence of
sialyltransferase is shown in FIG. 18.
[0162] The term "sialic acid" refers to any member of a family of
nine-carbon carboxylated sugars. The most common member of the
sialic acid family is N-acetylneuraminic acid
(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, NeuNAc, or NANA). A
second member of the family is N-glycolyl-neuraminic acid (Neu5Gc
or NeuGc), in which the N-acetyl group of NeuNAc is hydroxylated. A
third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid
(KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557;
Kanamori et al., J. Biol. Chem. 265: 21811-21819 (1990)). Also
included are 9-substituted sialic acids such as a 9-0-C1-C6
acyl-Neu5Ac like 9-O-lactylNeu5Ac or 9-O-acetyl-Neu5Ac. The
synthesis and use of sialic acid compounds in a sialylation
procedure is disclosed in international application WO92/16640,
published Oct. 1, 1992.
[0163] The term "sialic acid derivative" refers to sialic acids as
defined above that are modified with one or more chemical moieties.
The modifying group may for example be alkyl groups such as methyl
groups, azido- and fluoro groups, or functional groups such as
amino or thiol groups that can function as handles for attaching
other chemical moieties. Examples include 9-deoxy-9-fluoro-Neu5Ac
and 9-azido-9-deoxy-Neu5Ac. The term also encompasses sialic acids
that lack one of more functional groups such as the carboxyl group
or one or more of the hydroxyl groups. Derivatives where the
carboxyl group is replaced with a carboxamide group or an ester
group are also encompassed by the term. The term also refers to
sialic acids where one or more hydroxyl groups have been oxidized
to carbonyl groups. Furthermore the term refers to sialic acids
that lack the C9 carbon atom or both the C9-C8 carbon chain for
example after oxidative treatment with periodate.
[0164] Glycyl sialic acid is a sialic acid derivative according to
the definition above, where the N-acetyl group of NeuNAc is
replaced with a glycyl group also known as an amino acetyl group.
Glycyl sialic acid may be represented with the following
structure:
##STR00006##
[0165] The term "CMP-activated" sialic acid or sialic acid
derivatives refer to a sugar nucleotide containing a sialic acid
moiety and a cytidine monophosphate (CMP).
[0166] In the present description, the term "glycyl sialic acid
cytidine monophosphate" is used for describing GSC, and is a
synonym for alternative naming of same CMP activated glycyl sialic
acid. Alternative naming include CMP-5'-glycyl sialic acid,
cytidine-5'-monophospho-N-glycylneuraminic acid,
cytidine-5'-monophospho-N-glycyl sialic acid.
[0167] The term "intact glycosyl linking group" refers to a linking
group that is derived from a glycosyl moiety in which the
saccharide monomer interposed between and covalently attached to
the polypeptide and the HEP moiety is not degraded, e.g., oxidized,
e.g., by sodium metaperiodate during conjugate formation. "Intact
glycosyl linking groups" may be derived from a naturally occurring
oligosaccharide by addition of glycosyl unites or removal of one or
more glycosyl unit from a parent saccharide structure.
[0168] The term "asialo glycoprotein" is intended to include
glycoproteins wherein one or more terminal sialic acid residues
have been removed, e.g., by treatment with a sialidase or by
chemical treatment, exposing at least one galactose or
N-acetylgalactosamine residue from the underlying "layer" of
galactose or N-acetylgalactosamine ("exposed galactose
residue").
[0169] Dotted lines in structure formulas denotes open valence bond
(i.e. bonds that connect the structures to other chemical
moieties).
PEGylated derivatives
[0170] "PEGylated Factor VII polypeptide variants/derivatives"
according to the present invention may have one or more
polyethylene glycol (PEG) molecules attached to any part of the
FVII polypeptide including any amino acid residue or carbohydrate
moiety of the Factor VII polypeptide. Chemical and/or enzymatic
methods can be employed for conjugating PEG or other half-life
extending moieties to a glycan on the Factor VII polypeptide. An
example of an enzymatic conjugation process is described e.g. in
WO03031464. The glycan may be naturally occurring or it may be
engineered as described above for HEP conjugates.
"Cysteine-PEGylated Factor VII polypeptide variants" according to
the present invention have one or more PEG molecules conjugated to
a sulfhydryl group of a cysteine residue present or introduced in
the FVII polypeptide.
Fusion Proteins
[0171] Fusion proteins are proteins created through the in-frame
joining of two or more DNA sequences which originally encode
separate proteins or peptides or fragments thereof. Translation of
the fusion protein DNA sequence will result in a single protein
sequence which may have functional properties derived from each of
the original proteins or peptides. DNA sequences encoding fusion
proteins may be created artificially by standard molecular biology
methods such as overlapping PCR or DNA ligation and the assembly is
performed excluding the stop codon in the first 5'-end DNA sequence
while retaining the stop codon in the 3'-end DNA sequence. The
resulting fusion protein DNA sequence may be inserted into an
appropriate expression vector that supports the heterologous fusion
protein expression in standard host organisms such as bacteria,
yeast, fungi, insect cells or mammalian cells. Fusion proteins may
contain a linker or spacer peptide sequence that separates the
protein or peptide parts which define the fusion protein.
[0172] In one interesting embodiment of the invention, the Factor
VII polypeptide is a fusion protein comprising a Factor VII
polypeptide and a fusion partner protein/peptide, for example an Fc
domain or an albumin.
Fc Fusion Protein
[0173] The term "Fc fusion protein" is herein meant to encompass
Factor VII polypeptides of this invention fused to an Fc domain
that can be derived from any antibody isotype. An IgG Fc domain
will often be preferred due to the relatively long circulatory
half-life of IgG antibodies. The Fc domain may furthermore be
modified in order to modulate certain effector functions such as
e.g. complement binding and/or binding to certain Fc receptors.
Fusion of FVII polypeptides with an Fc domain, which has the
capacity to bind to FcRn receptors, will generally result in a
prolonged circulatory half-life of the fusion protein compared to
the half-life of the wt FVII polypeptides. Mutations in positions
234, 235 and 237 in an IgG Fc domain will generally result in
reduced binding to the Fc.gamma.RI receptor and possibly also the
Fc.gamma.RIIa and the FcyRIII receptors. These mutations do not
alter binding to the FcRn receptor, which promotes a long
circulatory half-life by an endocytic recycling pathway.
Preferably, a modified IgG Fc domain of a fusion protein according
to the invention comprises one or more of the following mutations
that will result in decreased affinity to certain Fc receptors
(L234A, L235E, and G237A) and in reduced C1q-mediated complement
fixation (A330S and P331S), respectively. Alternatively, the Fc
domain may be an IgG4 Fc domain, preferably comprising the S241
P/S228P mutation.
Production of Factor VII Polypeptides
[0174] Factor VII polypeptides, of the current invention, may be
recombinantly produced using well known methods of production and
purification; some examples of these methods are described below;
yet further examples of methods of production and purification are,
inter alia, described in WO2007/031559.
[0175] In one aspect, the invention relates to a method for
producing Factor VII polypeptides. The Factor VII polypeptides
described herein may be produced by means of recombinant nucleic
acid techniques. In general, a cloned human wild-type Factor VII
nucleic acid sequence is modified to encode the desired protein.
This modified sequence is then inserted into an expression vector,
which is in turn transformed or transfected into host cells. Higher
eukaryotic cells, in particular cultured mammalian cells, are
preferred as host cells.
[0176] In a further aspect, the invention relates to a transgenic
animal containing and expressing the polynucleotide construct.
[0177] The complete nucleotide and amino acid sequences for human
wild-type Factor VII are known (see U.S. Pat. No. 4,784,950, where
the cloning and expression of recombinant human Factor VII is
described).
[0178] The amino acid sequence alterations may be accomplished by a
variety of know techniques. Modification of the nucleic acid
sequence may be by site-specific mutagenesis. Techniques for
site-specific mutagenesis are well known in the art and are
described in, for example, Zoller and Smith (DNA 3:479-488, 1984)
or "Splicing by extension overlap", Horton et al., Gene 77, 1989,
pp. 61-68. Thus, using the nucleotide and amino acid sequences of
Factor VII, one may introduce the alteration(s) of choice.
Likewise, procedures for preparing a DNA construct using polymerase
chain reaction using specific primers are well known to persons
skilled in the art (cf. PCR Protocols, 1990, Academic Press, San
Diego, California, USA).
[0179] The nucleic acid construct encoding the Factor VII
polypeptide of the invention may suitably be of genomic or cDNA
origin, The nucleic acid construct encoding the Factor VII
polypeptide may also be prepared synthetically by established
standard methods, e.g. the phosphoamidite method described by
Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869,
The DNA sequences encoding the human Factor VII polypeptides may
also be prepared by polymerase chain reaction using specific
primers, for instance as described in U.S. Pat. No. 4,683,202,
Saiki et al., Science 239 (1988), 487 - 491, or Sambrook et al.,
supra.
[0180] Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed genomic
and cDNA origin prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0181] The nucleic acid construct is preferably a DNA construct.
DNA sequences for use in producing Factor VII polypeptides
according to the present invention will typically encode a pre-pro
polypeptide at the amino-terminus of Factor VII to obtain proper
posttranslational processing (e.g. gamma-carboxylation of glutamic
acid residues) and secretion from the host cell. The pre-pro
polypeptide may be that of Factor VII or another vitamin
K-dependent plasma protein, such as Factor IX, Factor X,
prothrombin, protein C or protein S. As will be appreciated by
those skilled in the art, additional modifications can be made in
the amino acid sequence of the Factor VII polypeptides where those
modifications do not significantly impair the ability of the
protein to act as a coagulant.
[0182] The DNA sequences encoding the human Factor VII polypeptides
are usually inserted into a recombinant vector which may be any
vector, which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e. a vector, which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0183] The vector is preferably an expression vector in which the
DNA sequence encoding the human Factor VII polypeptides is operably
linked to additional segments required for transcription of the
DNA. In general, the expression vector is derived from plasmid or
viral DNA, or may contain elements of both. The term, "operably
linked" indicates that the segments are arranged so that they
function in concert for their intended purposes, e.g. transcription
initiates in a promoter and proceeds through the DNA sequence
coding for the polypeptide.
[0184] Expression vectors for use in expressing Factor VIIa
polypeptide variants will comprise a promoter capable of directing
the transcription of a cloned gene or cDNA. The promoter may be any
DNA sequence, which shows transcriptional activity in the host cell
of choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell.
[0185] Examples of suitable promoters for directing the
transcription of the DNA encoding the human Factor VII polypeptide
in mammalian cells are the SV40 promoter (Subramani et al., Mol.
Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene)
promoter (Palmiter et al., Science 222 (1983), 809 - 814), the CMV
promoter (Boshart et al., Cell 41:521-530, 1985) or the adenovirus
2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol,
2:1304-1319, 1982).
[0186] The DNA sequences encoding the Factor VII polypeptides may
also, if necessary, be operably connected to a suitable terminator,
such as the human growth hormone terminator (Palmiter et al.,
Science 222, 1983, pp. 809-814) or the TPI1 (Alber and Kawasaki, J.
Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The
EMBO J.
[0187] 4, 1985, pp. 2093-2099) terminators. Expression vectors may
also contain a set of RNA splice sites located downstream from the
promoter and upstream from the insertion site for the Factor VII
sequence itself. Preferred RNA splice sites may be obtained from
adenovirus and/or immunoglobulin genes. Also contained in the
expression vectors is a polyadenylation signal located downstream
of the insertion site. Particularly preferred polyadenylation
signals include the early or late polyadenylation signal from SV40
(Kaufman and Sharp, ibid.), the polyadenylation signal from the
adenovirus 5 Elb region, the human growth hormone gene terminator
(DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981) or the
polyadenylation signal from the human Factor VII gene or the bovine
Factor VII gene. The expression vectors may also include a
noncoding viral leader sequence, such as the adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites; and enhancer sequences, such as the SV40 enhancer.
[0188] To direct the Factor VII polypeptides of the present
invention into the secretory pathway of the host cells, a secretory
signal sequence (also known as a leader sequence, prepro sequence
or pre sequence) may be provided in the recombinant vector. The
secretory signal sequence is joined to the DNA sequences encoding
the human Factor VII polypeptides in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA
sequence encoding the peptide. The secretory signal sequence may be
that, normally associated with the protein or may be from a gene
encoding another secreted protein.
[0189] The procedures used to ligate the DNA sequences coding for
the Factor VII polypeptides, the promoter and optionally the
terminator and/or secretory signal sequence, respectively, and to
insert them into suitable vectors containing the information
necessary for replication, are well known to persons skilled in the
art (cf., for instance, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, New York, 1989).
[0190] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in e.g. Kaufman and
Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978),
725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603,
Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al.,
EMBO J. 1 (1982), 841-845.
[0191] Cloned DNA sequences are introduced into cultured mammalian
cells by, for example, calcium phosphate-mediated transfection
(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic
Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology
52d:456-467, 1973) or electroporation (Neumann et al., EMBO J.
1:841-845, 1982).
[0192] To identify and select cells that express the exogenous DNA,
a gene that confers a selectable phenotype (a selectable marker) is
generally introduced into cells along with the gene or cDNA of
interest. Preferred selectable markers include genes that confer
resistance to drugs such as neomycin, hygromycin, and methotrexate.
The selectable marker may be an amplifiable selectable marker. A
preferred amplifiable selectable marker is a dihydrofolate
reductase (DHFR) sequence. Selectable markers may be introduced
into the cell on a separate plasmid at the same time as the gene of
interest, or they may be introduced on the same plasmid. If, on the
same plasmid, the selectable marker and the gene of interest may be
under the control of different promoters or the same promoter, the
latter arrangement producing a dicistronic message. Constructs of
this type are known in the art (for example, Levinson and Simonsen,
U.S. Pat. No. 4,713,339). It may also be advantageous to add
additional DNA, known as "carrier DNA," to the mixture that is
introduced into the cells.
[0193] After the cells have taken up the DNA, they are grown in an
appropriate growth medium, typically 1-2 days, to begin expressing
the gene of interest. As used herein the term "appropriate growth
medium" means a medium containing nutrients and other components
required for the growth of cells and the expression of the Factor
VII polypeptides of interest. Media generally include a carbon
source, a nitrogen source, essential amino acids, essential sugars,
vitamins, salts, phospholipids, protein and growth factors. For
production of gamma-carboxylated proteins, the medium will contain
vitamin K, preferably at a concentration of about 0.1 .mu.g/ml to
about 5 .mu.g/ml. Drug selection is then applied to select for the
growth of cells that are expressing the selectable marker in a
stable fashion. For cells that have been transfected with an
amplifiable selectable marker the drug concentration may be
increased to select for an increased copy number of the cloned
sequences, thereby increasing expression levels. Clones of stably
transfected cells are then screened for expression of the human
Factor VII polypeptide of interest.
[0194] The host cell into which the DNA sequences encoding the
Factor VII polypeptides is introduced may be any cell, which is
capable of producing the posttranslational modified human Factor
VII polypeptides and includes yeast, fungi and higher eucaryotic
cells.
[0195] Examples of mammalian cell lines for use in the present
invention are the Chinese Hamster Ovary (CHO) cells (e.g. ATCC CCL
61), CHO DUKX cells (Urlaub and Chasin,
[0196] Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).), baby
hamster kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen.
Virol. 36:59-72, 1977) cell lines. ,
[0197] The transformed or transfected host cell described above is
then cultured in a suitable nutrient medium under conditions
permitting expression of the Factor VII polypeptide after which all
or part of the resulting peptide may be recovered from the culture.
The medium used to culture the cells may be any conventional medium
suitable for growing the host cells, such as minimal or complex
media containing appropriate supplements. Suitable media are
available from commercial suppliers or may be prepared according to
published recipes (e.g. in catalogues of the American Type Culture
Collection). The Factor VII polypeptide produced by the cells may
then be recovered from the culture medium by conventional
procedures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaqueous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gelfiltration
chromatography, affinity chromatography, or the like, dependent on
the type of polypeptide in question.
[0198] Transgenic animal technology may be employed to produce the
Factor VII polypeptides of the invention. It is preferred to
produce the proteins within the mammary glands of a host female
mammal. Expression in the mammary gland and subsequent secretion of
the protein of interest into the milk overcomes many difficulties
encountered in isolating proteins from other sources. Milk is
readily collected, available in large quantities, and biochemically
well characterized. Furthermore, the major milk proteins are
present in milk at high concentrations (typically from about 1 to
15 g/l).
[0199] From a commercial point of view, it is clearly preferable to
use as the host a species that has a large milk yield. While
smaller animals such as mice and rats can be used (and are
preferred at the proof of principle stage), it is preferred to use
livestock mammals including, but not limited to, pigs, goats, sheep
and cattle. Sheep are particularly preferred due to such factors as
the previous history of transgenesis in this species, milk yield,
cost and the ready availability of equipment for collecting sheep
milk (see, for example, WO 88/00239 for a comparison of factors
influencing the choice of host species). It is generally desirable
to select a breed of host animal that has been bred for dairy use,
such as East Friesland sheep, or to introduce dairy stock by
breeding of the transgenic line at a later date. In any event,
animals of known, good health status should be used.
[0200] To obtain expression in the mammary gland, a transcription
promoter from a milk protein gene is used. Milk protein genes
include those genes encoding caseins (see U.S. Pat. No. 5,304,489),
beta-lactoglobulin, a-lactalbumin, and whey acidic protein. The
beta-lactoglobulin (BLG) promoter is preferred. In the case of the
ovine beta-lactoglobulin gene, a region of at least the proximal
406 by of 5' flanking sequence of the gene will generally be used,
although larger portions of the 5' flanking sequence, up to about 5
kbp, are preferred, such as a -4.25 kbp DNA segment encompassing
the 5' flanking promoter and non-coding portion of the
beta-lactoglobulin gene (see Whitelaw et al., Biochem. J. 286:
31-39 (1992)). Similar fragments of promoter DNA from other species
are also suitable.
[0201] Other regions of the beta-lactoglobulin gene may also be
incorporated in constructs, as may genomic regions of the gene to
be expressed. It is generally accepted in the art that constructs
lacking introns, for example, express poorly in comparison with
those that contain such DNA sequences (see Brinster et al., Proc.
Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc.
Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al.,
Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318, each
of which is incorporated herein by reference). In this regard, it
is generally preferred, where possible, to use genomic sequences
containing all or some of the native introns of a gene encoding the
protein or polypeptide of interest, thus the further inclusion of
at least some introns from, e.g, the beta-lactoglobulin gene, is
preferred. One such region is a DNA segment that provides for
intron splicing and RNA polyadenylation from the 3' non-coding
region of the ovine beta-lactoglobulin gene. When substituted for
the natural 3' non-coding sequences of a gene, this ovine
beta-lactoglobulin segment can both enhance and stabilize
expression levels of the protein or polypeptide of interest. Within
other embodiments, the region surrounding the initiation ATG of the
variant Factor VII sequence is replaced with corresponding
sequences from a milk specific protein gene. Such replacement
provides a putative tissue-specific initiation environment to
enhance expression. It is convenient to replace the entire variant
Factor VII pre-pro and 5' non-coding sequences with those of, for
example, the BLG gene, although smaller regions may be
replaced.
[0202] For expression of Factor VII polypeptides in transgenic
animals, a DNA segment encoding variant Factor VII is operably
linked to additional DNA segments required for its expression to
produce expression units. Such additional segments include the
above-mentioned promoter, as well as sequences that provide for
termination of transcription and polyadenylation of mRNA. The
expression units will further include a DNA segment encoding a
secretory signal sequence operably linked to the segment encoding
modified Factor VII. The secretory signal sequence may be a native
Factor VII secretory signal sequence or may be that of another
protein, such as a milk protein (see, for example, von Heijne,
Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat.
No. 4,873,316, which are incorporated herein by reference).
[0203] Construction of expression units for use in transgenic
animals is conveniently carried out by inserting a variant Factor
VII sequence into a plasmid or phage vector containing the
additional DNA segments, although the expression unit may be
constructed by essentially any sequence of ligations. It is
particularly convenient to provide a vector containing a DNA
segment encoding a milk protein and to replace the coding sequence
for the milk protein with that of a Factor VII variant; thereby
creating a gene fusion that includes the expression control
sequences of the milk protein gene. In any event, cloning of the
expression units in plasmids or other vectors facilitates the
amplification of the variant Factor VII sequence. Amplification is
conveniently carried out in bacterial (e.g. E. coli) host cells,
thus the vectors will typically include an origin of replication
and a selectable marker functional in bacterial host cells. The
expression unit is then introduced into fertilized eggs (including
early-stage embryos) of the chosen host species. Introduction of
heterologous DNA can be accomplished by one of several routes,
including microinjection (e.g. U.S. Pat. No. 4,873,191), retroviral
infection (Jaenisch, Science 240: 1468-1474 (1988)) or
site-directed integration using embryonic stem (ES) cells (reviewed
by Bradley et al., Bio/Technology 10: 534-539 (1992)). The eggs are
then implanted into the oviducts or uteri of pseudopregnant females
and allowed to develop to term. Offspring carrying the introduced
DNA in their germ line can pass the DNA on to their progeny in the
normal, Mendelian fashion, allowing the development of transgenic
herds. General procedures for producing transgenic animals are
known in the art (see, for example, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory,
1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al.,
Biol. Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8:
140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991);
Krimpenfort et al., Bio/Technology 9: 844-847 (1991); Wall et al.,
J. Cell. Biochem. 49: 113-120 (1992); U.S. Pat. No. 4,873,191; U.S.
Pat. No. 4,873,316; WO 88/00239, WO 90/05188, WO 92/11757; and GB
87/00458). Techniques for introducing foreign DNA sequences into
mammals and their germ cells were originally developed in the mouse
(see, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384
(1980); Gordon and Ruddle, Science 214:
[0204] 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345
(1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442
(1985); and Hogan et al. (ibid.)). These techniques were
subsequently adapted for use with larger animals, including
livestock species (see, e.g., WO 88/00239, WO 90/05188, and WO
92/11757; and Simons et al., Bio/Technology 6: 179-183 (1988)). To
summarise, in the most efficient route used to date in the
generation of transgenic mice or livestock, several hundred linear
molecules of the DNA of interest are injected into one of the
pro-nuclei of a fertilized egg according to established techniques.
Injection of DNA into the cytoplasm of a zygote can also be
employed.
Purification
[0205] The Factor VII polypeptides of the invention are recovered
from cell culture medium. The Factor VII polypeptides of the
present invention may be purified by a variety of procedures known
in the art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing (IEF), differential solubility (e.g., ammonium
sulfate precipitation), or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989). Preferably, Factor VII polypeptides may be
purified by affinity chromatography on an anti-Factor VII antibody
column. The use of calcium-dependent monoclonal antibodies, as
described by Wakabayashi et al., J. Biol. Chem. 261:11097-11108,
(1986) and Thim et al., Biochemistry 27: 7785-7793, (1988), is
particularly preferred. Additional purification may be achieved by
conventional chemical purification means, such as high performance
liquid chromatography. Other methods of purification, including
barium citrate precipitation, are known in the art, and may be
applied to the purification of the novel Factor VII polypeptides
described herein (see, for example, Scopes, R., Protein
Purification, Springer-Verlag, N.Y., 1982).
[0206] For therapeutic purposes it is preferred that the Factor VII
polypeptides of the invention are substantially pure. Thus, in a
preferred embodiment of the invention the Factor VII polypeptides
of the invention are purified to at least about 90 to 95%
homogeneity, preferably to at least about 98% homogeneity. Purity
may be assessed by several methods known in the art e.g. HPLC, gel
electrophoresis and amino-terminal amino acid sequencing.
[0207] The Factor VII polypeptide is cleaved at its activation site
in order to convert it to its two-chain form. Activation may be
carried out according to procedures known in the art, such as those
disclosed by Osterud, et al., Biochemistry 11:2853-2857 (1972);
Thomas, U.S. Patent No. 4,456,591; Hedner and Kisiel, J. Clin.
Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, Behring Inst.
Mitt. 73:29-42 (1983). Alternatively, as described by Bjoern et al.
(Research Disclosure, 269 September 1986, pp. 564-565), Factor VII
may be activated by passing it through an ion-exchange
chromatography column, such as Mono Q.RTM. (Pharmacia fine
Chemicals) or the like. The resulting activated Factor VII variant
may then be formulated and administered as described below.
Assays
[0208] Provided herein are suitable in vitro proteolytic and
antithrombin reactivity assays for selecting preferred Factor VII
polypeptides according to the invention. Such assays are described
in detail in Example 5. Briefly, the assays can be performed as
simple preliminary in vitro tests, as follows:
[0209] The proteolytic activity of FVIIa polypeptides can be
measured using the physiological substrate plasma-derived factor X
(X) as substrate at physiological pH and in the presence of calcium
and vesicles composed of phosphatidyl choline (PC) and phosphatidyl
serine (PS) to support the reaction. The assay is performed by
incubating FVIIa with FX at a substrate concentration below Km for
the reaction and for a period sufficient long to allow for the
generation of measurable amounts of FXa while keeping the
conversion of FX below 20%. The generated FXa is quantified after
the addition of a suitable chromogenic substrate such as S-2765 and
reported relative to that of wild-type FVIIa following
normalisation according to the concentration of the FVIIa variant
tested.
[0210] The antithrombin reactivity of the FVIIa polypeptides can be
measured at physiological pH under pseudo-first order conditions in
the presence of excess plasma-derived antithrombin, low molecular
weight (LMW) heparin and calcium. Residual FVIIa activity is
measured discontinuously throughout the time course of the
inhibition reaction using a chromogenic substrate such as S-2288.
The rate of inhibition is obtained by non-linear least-squares
fitting of data to a single exponential decay function and reported
relative to that of wild-type FVIIa following normalisation of
inhibition rates according to the antithrombin concentration used.
The kinetic characterisation of heparin-catalyzed and uncatalyzed
inhibition of blood coagulation proteinases by antithrombinis is
described in Olson et al. (1993), Methods Enzymol. 222,
525-559.
Pharmaceutical Compositions
[0211] In one aspect, the present invention relates to compositions
and formulations comprising a Factor VII polypeptide of the
invention. For example, the invention provides a pharmaceutical
composition that comprises a Factor VII polypeptide of the
invention, formulated together with a pharmaceutically acceptable
carrier.
[0212] Accordingly, one object of the invention is to provide a
pharmaceutical formulation comprising a Factor VII polypeptide
which is present in a concentration from 0.25 mg/ml to 100 mg/ml,
and wherein said formulation has a pH from 2.0 to 10.0. The
formulation may further comprise one or more of a buffer system, a
preservative, a tonicity agent, a chelating agent, a stabilizer, an
antioxidant or a surfactant, as well as various combinations
thereof.
[0213] The use of preservatives, isotonic agents, chelating agents,
stabilizers, antioxidant and surfactants in pharmaceutical
compositions is well-known to the skilled person. Reference may be
made to Remington: The Science and Practice of Pharmacy, 19th
edition, 1995.
[0214] In one embodiment, the pharmaceutical formulation is an
aqueous formulation. Such a formulation is typically a solution or
a suspension, but may also include colloids, dispersions,
emulsions, and multi-phase materials. The term "aqueous
formulation" is defined as a formulation comprising at least 50%
w/w water. Likewise, the term "aqueous solution" is defined as a
solution comprising at least 50% w/w water, and the term "aqueous
suspension" is defined as a suspension comprising at least 50% w/w
water.
[0215] In another embodiment, the pharmaceutical formulation is a
freeze-dried formulation, to which the physician or the patient
adds solvents and/or diluents prior to use.
[0216] In a further aspect, the pharmaceutical formulation
comprises an aqueous solution of a Factor VII polypeptide, and a
buffer, wherein the polypeptide is present in a concentration from
1 mg/ml or above, and wherein said formulation has a pH from about
2.0 to about 10.0.
[0217] In a further aspect, the pharmaceutical formulation may be
any one of those disclosed in WO2014/057069, which is herein
incorporated by reference; or it may be the formulation described
in Example 18.
[0218] A Factor VII polypeptide of the invention may be
administered parenterally, such as intravenously, such as
intramuscularly, such as subcutaneously. Alternatively, a FVII
polypeptide of the invention may be administered via a
non-parenteral route, such as perorally or topically. An
polypeptide of the invention may be administered prophylactically.
An polypeptide of the invention may be administered therapeutically
(on demand).
Therapeutic Uses
[0219] In a broad aspect, a Factor VII polypeptide of the present
invention or a pharmaceutical formulation comprising said
polypeptide, may be used as a medicament.
[0220] In one aspect, a Factor VII polypeptide of the present
invention or a pharmaceutical formulation comprising said
polypeptide, may be used to treat a subject with a
coagulopathy.
[0221] In another aspect, a Factor VII polypeptide of the present
invention or a pharmaceutical formulation comprising said
polypeptide may be used for the preparation of a medicament for the
treatment of bleeding disorders or bleeding episodes or for the
enhancement of the normal haemostatic system.
[0222] In a further aspect, a Factor VII polypeptide of the present
invention or a pharmaceutical formulation comprising said
polypeptide may be used for the treatment of haemophilia A,
haemophilia B or haemophilia A or B with acquired inhibitors.
[0223] In another aspect, a Factor VII polypeptide of the present
invention or a pharmaceutical formulation comprising said
polypeptide may be used in a method for the treatment of bleeding
disorders or bleeding episodes in a subject or for the enhancement
of the normal haemostatic system, the method comprising
administering a therapeutically or prophylactically effective
amount of a Factor VII polypeptide of the present invention to a
subject in need thereof.
[0224] The term "subject", as used herein, includes any human
patient, or non-human vertebrates.
[0225] The term "treatment", as used herein, refers to the medical
therapy of any human or other vertebrate subject in need thereof.
Said subject is expected to have undergone physical examination by
a medical practitioner, or a veterinary medical practitioner, who
has given a tentative or definitive diagnosis which would indicate
that the use of said specific treatment is beneficial to the health
of said human or other vertebrate. The timing and purpose of said
treatment may vary from one individual to another, according to the
status quo of the subject's health. Thus, said treatment may be
prophylactic, palliative, symptomatic and/or curative. In terms of
the present invention, prophylactic, palliative, symptomatic and/or
curative treatments may represent separate aspects of the
invention.
[0226] The term "coagulopathy", as used herein, refers to an
increased haemorrhagic tendency which may be caused by any
qualitative or quantitative deficiency of any pro-coagulative
component of the normal coagulation cascade, or any upregulation of
fibrinolysis. Such coagulopathies may be congenital and/or acquired
and/or iatrogenic and are identified by a person skilled in the
art. Non-limiting examples of congenital hypocoagulopathies are
haemophilia A, haemophilia B, Factor VII deficiency, Factor X
deficiency, Factor XI deficiency, von Willebrand's disease and
thrombocytopenias such as Glanzmann's thombasthenia and
Bernard-Soulier syndrome. . The clinical severity of haemophilia A
or B is determined by the concentration of functional units of
FIX/Factor VIII in the blood and is classified as mild, moderate,
or severe. Severe haemophilia is defined by a clotting factor level
of <0.01 U/ml corresponding to <1% of the normal level, while
people with moderate and mild haemophilia have levels from 1-5% and
>5%, respectively. Haemophilia A with "inhibitors" (that is,
allo-antibodies against factor VIII) and haemophilia B with
"inhibitors" (that is, allo-antibodies against factor IX) are
non-limiting examples of coagulopathies that are partly congenital
and partly acquired.
[0227] A non-limiting example of an acquired coagulopathy is serine
protease deficiency caused by vitamin K deficiency; such vitamin
K-deficiency may be caused by administration of a vitamin K
antagonist, such as warfarin. Acquired coagulopathy may also occur
following extensive trauma. In this case otherwise known as the
"bloody vicious cycle", it is characterised by haemodilution
(dilutional thrombocytopaenia and dilution of clotting factors),
hypothermia, consumption of clotting factors and metabolic
derangements (acidosis). Fluid therapy and increased fibrinolysis
may exacerbate this situation. Said haemorrhage may be from any
part of the body.
[0228] A non-limiting example of an iatrogenic coagulopathy is an
overdosage of anticoagulant medication--such as heparin, aspirin,
warfarin and other platelet aggregation inhibitors--that may be
prescribed to treat thromboembolic disease. A second, non-limiting
example of iatrogenic coagulopathy is that which is induced by
excessive and/or inappropriate fluid therapy, such as that which
may be induced by a blood transfusion.
[0229] In one embodiment of the current invention, haemorrhage is
associated with haemophilia A or B. In another embodiment,
haemorrhage is associated with haemophilia A or B with acquired
inhibitors. In another embodiment, haemorrhage is associated with
thrombocytopenia. In another embodiment, haemorrhage is associated
with von Willebrand's disease. In another embodiment, haemorrhage
is associated with severe tissue damage. In another embodiment,
haemorrhage is associated with severe trauma. In another
embodiment, haemorrhage is associated with surgery. In another
embodiment, haemorrhage is associated with haemorrhagic gastritis
and/or enteritis. In another embodiment, the haemorrhage is profuse
uterine bleeding, such as in placental abruption. In another
embodiment, haemorrhage occurs in organs with a limited possibility
for mechanical haemostasis, such as intracranially, intraaurally or
intraocularly. In another embodiment, haemorrhage is associated
with anticoagulant therapy.
[0230] The invention is further described by the following
non-limiting list of embodiments:
EMBODIMENT 1
[0231] Factor VII polypeptide comprising two or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID NO:
1), wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe
(F) and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or
Trp (W) and/or W201 is replaced by Arg (R), Met (M), or Lys (K)
and/or K337 is replaced by Ala (A) or Gly (G); optionally, where
Q176 is replaced by Lys (K), Arg (R) or Asn (N); or Q286 is
replaced by Asn (N).
EMBODIMENT 1(i)
[0232] Factor VII polypeptide according to embodiment 1, wherein
T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); and L288
is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) and/or
W201 is replaced by Arg (R), Met (M) or Lys (K) and/or K337 is
replaced by Ala (A) or Gly (G).
EMBODIMENT 1(ii)
[0233] Factor VII polypeptide according to embodiment 1, wherein
L288 is replaced by Phe (F), Tyr (Y), Asn (N) or Ala (A).
EMBODIMENT 1(iii)
[0234] Factor VII polypeptide according to embodiment 1, wherein
W201 is replaced by Arg (R), Met (M) or Lys (K).
EMBODIMENT 1(iv)
[0235] Factor VII polypeptide according to embodiment 1, wherein
K337 is replaced by Ala (A) or Gly (G).
EMBODIMENT 2
[0236] Factor VII polypeptide according to embodiment 1, wherein
T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F).
EMBODIMENT 2(i)
[0237] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe
(F) and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or
Trp (W).
EMBODIMENT 2(ii)
[0238] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and L288 is replaced by
Phe (F).
EMBODIMENT 2(iii)
[0239] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and L288 is replaced by
Tyr (Y).
EMBODIMENT 2(iv)
[0240] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and L288 is replaced by
Asn (N).
EMBODIMENT 2(v)
[0241] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and L288 is replaced by
Ala (A).
EMBODIMENT 2(vi)
[0242] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and L288 is replaced by
Trp (W).
EMBODIMENT 2(vii)
[0243] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and L288 is replaced by
Phe (F).
EMBODIMENT 2(viii)
[0244] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and L288 is replaced by
Tyr (Y).
EMBODIMENT 2(ix)
[0245] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and L288 is replaced by
Asn (N).
EMBODIMENT 2(x)
[0246] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and L288 is replaced by
Ala (A).
EMBODIMENT 2(xi)
[0247] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and L288 is replaced by
Trp (W).
EMBODIMENT 2(xii)
[0248] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y); and L288 is replaced by
Phe (F).
EMBODIMENT 2(xiii)
[0249] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y); and L288 is replaced by
Tyr (Y).
EMBODIMENT 2(xiv)
[0250] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y) and L288 is replaced by
Asn (N).
EMBODIMENT 2(xv)
[0251] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y) and L288 is replaced by
Ala (A).
EMBODIMENT 2(xvi)
[0252] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y) and L288 is replaced by
Trp (W).
EMBODIMENT 2(xvii)
[0253] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and L288 is replaced by
Phe (F).
EMBODIMENT 2(xviii)
[0254] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and L288 is replaced by
Tyr (Y).
EMBODIMENT 2(xix)
[0255] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and L288 is replaced by
Asn (N).
EMBODIMENT 2(xx)
[0256] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and L288 is replaced by
Ala (A).
EMBODIMENT 2(xxi)
[0257] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and L288 is replaced by
Trp (W).
EMBODIMENT 2(xxii)
[0258] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and K337 is replaced by
Ala (A).
EMBODIMENT 2(xxiii)
[0259] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and K337 is replaced by
Ala (A).
EMBODIMENT 2(xxiv)
[0260] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y) and K337 is replaced by
Ala (A).
EMBODIMENT 2(xxv)
[0261] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and K337 is replaced by
Ala (A).
EMBODIMENT 2(xxvi)
[0262] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Lys (K) and K337 is replaced by
Gly (G).
EMBODIMENT 2(xxvii)
[0263] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Arg (R) and K337 is replaced by
Gly (G).
EMBODIMENT 2(xxviii)
[0264] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Tyr (Y) and K337 is replaced by
Gly (G).
EMBODIMENT 2(xxix)
[0265] Factor VII polypeptide according to any one of embodiments
1-2, wherein T293 is replaced by Phe (F) and K337 is replaced by
Gly (G).
EMBODIMENT 2(xxx)
[0266] Factor VII polypeptide according to any one of embodiments
2(ii)-2(xxii) wherein K337 is replaced by Ala (A).
EMBODIMENT 3
[0267] Factor VII polypeptide according to embodiment 2, wherein
the polypeptide comprises one of the following groups of
substitutions: L288F/T293K, L288F/T293K/K337A, L288F/T293K/L305V,
L288F/T293K/L3051, L288F/T293R, L288F/T293R/K337A,
L288F/T293R/L305V, L288F/T293R/L305I, L288F/T293Y,
L288F/T293Y/K337A, L288F/T293Y/L305V, L288F/T293Y/L305I,
L288F/T293F, L288F/T293F/K337A, L288F/T293F/L305V,
L288F/T293F/L305I, L288Y/T293K, L288Y/T293K/K337A,
L288Y/T293K/L305V, L288Y/T293K/L305I, L288Y/T293R,
L288Y/T293R/K337A, L288Y/T293R/L305V, L288Y/1293R/L305I,
L288Y/T293Y, L288Y/T293Y/K337A, L288Y/T293Y/L305V,
L288Y/T293Y/L305I, L288Y/T293F, L288Y/T293F/K337A,
L288Y/T293F/L305V, L288Y/T293F/L305I, L288N/T293K,
L288N/T293K/K337A, L288N/T293K/L305V, L288N/T293K/L305I,
L288N/T293R, L288N/T293R/K337A, L288N/T293R/L305V,
L288N/T293R/L305I, L288N/T293Y, L288N/T293Y/K337A,
L288N/T293Y/L305V, L288N/T293Y/L305I, L288N/T293F,
L288N/T293F/K337A, L288N/T293F/L305V, L288N/T293F/L305I,
L288A/T293K, L288A/T293K/K337A, L288A/T293K/L305V,
L288A/T293K/L305I, L288A/T293R, L288A/T293R/K337A,
L288A/T293R/L305V, L288A/T293R/L305I, L288A/T293Y,
L288A/T293Y/K337A, L288A/T293Y/L305V, L288A/T293Y/L305I,
L288A/T293F, L288A/T293F/K337A, L288A/T293F/L305V or
L288A/T293F/L305I.
EMBODIMENT 4
[0268] Factor VII polypeptide according to embodiment 2, wherein
the polypeptide has the following substitutions: L288F/T293K,
L288F/T293K/K337A, L288F/T293R, L288F/T293R/K337A, L288Y/T293K,
L288Y/T293K/K337A, L288Y/T293R, L288Y/T293R/K337A, L288N/T293K,
L288N/T293K/K337A, L288N/T293R or L288N/T293R/K337A.
EMBODIMENT 5
[0269] Factor VII polypeptide according to embodiment 1, wherein
Q176 is replaced by Lys (K), Arg (R), or Asn (N).
EMBODIMENT 6
[0270] Factor VII polypeptide according to embodiment 5, wherein
the polypeptide comprises one of the following groups of
substitutions: L288F/Q176K/K337A, L288Y/Q176K/K337A,
L288N/Q176K/K337A orL288A/Q176K/K337A.
EMBODIMENT 7
[0271] Factor VII polypeptide according to embodiment 1, wherein
Q286 is replaced by Asn (N).
EMBODIMENT 8
[0272] Factor VII polypeptide comprising one or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1), characterized in that one substitution is where L288 is
replaced by Phe (F), Tyr (Y), Asn (N) or Ala (A), with the proviso
that the polypeptide does not have the following pair of
substitutions L288N/R290S or L288N/R290T.
EMBODIMENT 9
[0273] Factor VII polypeptide according to any one of embodiments
1-2(xxx), 5 and 7-8, wherein the Factor VII polypeptide further
comprises one or more of the following substitutions L3051, L305V
or K337A.
EMBODIMENT 10
[0274] Factor VII polypeptide comprising two or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1), wherein W201 is replaced by Arg (R), Met (M), or Lys (K) and
wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F);
wherein Q176 is replaced by Lys (K), Arg (R) or Asn (N); or Q286 is
replaced by Asn (N).
EMBODIMENT 10(i)
[0275] Factor VII polypeptide according to any one of embodiments
1-1(ii) or 10, wherein T293 is replaced by Lys (K), Arg (R), Tyr
(Y) or Phe (F) and wherein W201 is replaced by Arg (R), Met (M) or
Lys (K).
EMBODIMENT 11
[0276] Factor VII polypeptide according to embodiment 10, wherein
T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F).
EMBODIMENT 11(i)
[0277] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Lys (K), Arg (R), Tyr
(Y) or Phe (F) and W201 is replaced by Arg (R), Met (M) or Lys
(K).
EMBODIMENT 11(ii)
[0278] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is
replaced by Arg (R).
EMBODIMENT 11(iii)
[0279] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is
replaced by Met (M).
EMBODIMENT 11(iv)
[0280] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is
replaced by Lys (K).
EMBODIMENT 11(v)
[0281] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is
replaced by Arg (R).
EMBODIMENT 11(vi)
[0282] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is
replaced by Met (M).
EMBODIMENT 11(vii)
[0283] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is
replaced by Lys (K).
EMBODIMENT 11(viii)
[0284] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is
replaced by Arg (R).
EMBODIMENT 11(ix)
[0285] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is
replaced by Met (M).
EMBODIMENT 11(x)
[0286] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is
replaced by Lys (K).
EMBODIMENT 11(xi)
[0287] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Phe (F) and W201 is
replaced by Arg (R).
EMBODIMENT 11(xii)
[0288] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Phe (F) and W201 is
replaced by Met (M).
EMBODIMENT 11(xiii)
[0289] Factor VII polypeptide according to any one of embodiments
1-2, 10 or 11, wherein T293 is replaced by Phe (F) and W201 is
replaced by Lys (K).
EMBODIMENT 12
[0290] Factor VII polypeptide according to embodiment 11, wherein
the polypeptide comprises one of the following groups of
substitutions: W201 R/T293K, W201 R/T293K/K337A, W201
R/T293K/L305V, W201 R/T293K/L3051, W201 R/T293R, W201
R/T293R/K337A, W201 R/T293R/L305V, W201 R/T293R/L3051, W201
R/T293Y, W201 R/T293Y/K337A, W201 R/T293Y/L305V, W201
R/T293Y/L3051, W201 R/T293F, W201 R/T293F/K337A, W201
R/T293F/L305V, W201 R/T293F/L3051, W201 K/T293K, W201
K/T293K/K337A, W201 K/T293K/L305V, W201 K/T293K/L3051, W201
K/T293R, W201 K/T293R/K337A, W201 K/T293R/L305V, W201
K/T293R/L3051, W201 K/T293Y, W201 K/T293Y/K337A, W201
K/T293Y/L305V, W201 K/T293Y/L3051, W201 K/T293F, W201
K/T293F/K337A, W201 K/T293F/L305V, W201 K/T293F/L3051, W201
M/T293K, W201 M/T293K/K337A, W201 M/T293K/L305V, W201
M/T293K/L3051, W201 M/T293R, W201 M/T293R/K337A, W201
M/T293R/L305V, W201 M/T293R/L3051, W201 M/T293Y, W201
M/T293Y/K337A, W201 M/T293Y/L305V, W201 M/T293Y/L3051, W201
M/T293F, W201 M/T293F/K337A, W201 M/T293F/L305V or W201
M/T293F/L3051.
EMBODIMENT 13
[0291] Factor VII polypeptide according to embodiment 11, wherein
the polypeptide has the following substitutions: W201 R/T293K, W201
R/T293K/K337A, W201 R/T293R, W201 R/T293R/K337A, W201 R/T293Y, W201
R/T293F, W201 K/T293K or W201 M/T293K.
EMBODIMENT 14
[0292] Factor VII polypeptide according to embodiment 10, wherein
Q176 is replaced by Lys (K), Arg (R), or Asn (N).
EMBODIMENT 15
[0293] Factor VII polypeptide according to embodiment 14, wherein
the polypeptide comprises one of the following groups of
substitutions W201R/Q176K, W201R/Q176R, W201K/Q176K, W201K/Q176R,
W201M/Q176K, or W201M/Q176R.
Embodiment 16: Factor VII polypeptide according to embodiment 10,
wherein Q286 is replaced by Asn (N).
EMBODIMENT 17
[0294] Factor VII polypeptide according to any one of embodiments
10-11, 14, and 16, wherein the Factor VII polypeptide further
comprises one or more of the following substitutions L3051, L305V
or K337A.
EMBODIMENT 18
[0295] Factor VII polypeptide comprising one or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1), characterized in that one substitution is where W201 is
replaced by Arg (R), Met (M), or Lys (K).
EMBODIMENT 19
[0296] Factor VII polypeptide comprising two or more substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1), wherein L288 is replaced by Phe (F), Tyr (Y), Asn (N), or
Ala (A); wherein W201 is replaced by Arg (R), Met (M), or Lys (K)
and, optionally, wherein T293 is replaced by Lys (K), Arg (R), Tyr
(Y) or Phe (F); Q176 is replaced by Lys (K), Arg (R) or Asn (N); or
Q286 is replaced by Asn (N).
EMBODIMENT 20
[0297] Factor VII polypeptide according to embodiment 19, wherein
the polypeptide comprises one of the following groups of
substitutions L288F/W201K, L288F/W201R, L288F/W201M, L288N/W201 K,
L288N/W201R, L288N/W201M, L288Y/W201 K, L288Y/W201R, L288Y/W201 M,
L288A/W201K, L288A/W201 R, L288A/W201 M, L288F/W201 K/T293K,
L288F/W201K/T293Y, L288F/W201R/T293K, L288F/W201R/T293Y,
L288F/W201M/T293K, L288F/W201M/T293Y, L288N/W201K/T293K,
L288N/W201K/T293Y, L288N/W201R/T293K, L288N/W201R/T293Y,
L288N/W201M/T293K, L288N/W201M/T293Y, L288A/W201K/T293K,
L288A/W201K/T293Y, L288A/W201 R/T293K, L288A/W201 R/T293Y,
L288A/W201M/T293K, L288A/W201M/T293Y, L288Y/W201 K/T293K,
L288Y/W201 K/T293Y, L288Y/W201 R/T293K, L288Y/W201 R/T293Y,
L288Y/W201 M/T293K or L288Y/W201M/T293Y.
EMBODIMENT 21
[0298] Factor VII polypeptide according to any one of the preceding
embodiments, wherein the Factor VII polypeptide further comprises
one or more of the following substitutions R396C, Q250C, or
407C.
EMBODIMENT 22
[0299] Factor VII polypeptide according to any one of the previous
embodiments, wherein said Factor VII polypeptide is a cleaved,
two-chain Factor VIIa polypeptide.
EMBODIMENT 22(i)
[0300] Factor VII polypeptide according to any one of the preceding
embodiments comprising two amino acid substitutions relative to the
amino acid sequence of human Factor VII (SEQ ID NO:1).
EMBODIMENT 22(ii)
[0301] Factor VII polypeptide according to any one of the preceding
embodiments comprising three amino acid substitutions relative to
the amino acid sequence of human Factor VII (SEQ ID NO:1).
EMBODIMENT 22(iii)
[0302] Factor VII polypeptide according to any one of the preceding
embodiments comprising four amino acid substitutions relative to
the amino acid sequence of human Factor VII (SEQ ID NO:1).
EMBODIMENT 22(iv)
[0303] Factor VII polypeptide according to any one of the preceding
embodiments comprising five amino acid substitutions relative to
the amino acid sequence of human Factor VII (SEQ ID NO:1).
EMBODIMENT 22(v)
[0304] Factor VII polypeptide according to any one embodiments
22(i)-(iv) comprising at the most ten amino acid substitutions
relative to the amino acid sequence of human Factor VII (SEQ ID
NO:1).
EMBODIMENT 22(vi)
[0305] Factor VII polypeptide according to any one of the preceding
embodiments, which has a proteolytic activity that is at least
110%, such as at least 120%, such as at least 130%, such as at
least 140%, such as at least 150%, such as at least 160%, such as
at least 170%, such as at least 180%, such as at least 190%, such
as at least 200%, such as at least 300%, such as at least 400%,
such as at least 500%, such as at least 1000%, such as at least
3000%, such as at least 5000%, such as at least 10 000%, such as at
least 30 000% that of wild type human Factor VIIa, as measured in
an in vitro proteolytic assay, in the absence of soluble tissue
factor.
EMBODIMENT 22(vii)
[0306] Factor VII polypeptide according to any one of the preceding
embodiments, which has less than 20%, such as less than 19%, such
as less than 18%, such as less than 17%, such as less than 16%,
such as less than 15%, such as less than 14%, such as less than
13%, such as less than 12%, such as less than 11%, such as less
than 10%, such as less than 9%, such as less than 8%, such as less
than 7%, such as less than 6%, such as less than 5% antithrombin
reactivity compared to that of wild type human Factor VIIa (SEQ ID
NO: 1), as measured in an antithrombin inhibition assay, in the
presence of low molecular weight heparin and the absence of soluble
tissue factor.
EMBODIMENT 23
[0307] Factor VII polypeptide according to any of the preceding
embodiments, wherein the Factor VII polypeptide is coupled with at
least one half-life extending moiety.
EMBODIMENT 24
[0308] Factor VII polypeptide according to embodiment 23, wherein
the half-life extending moiety is selected from biocompatible fatty
acids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g.
Hydroxy Ethyl Starch (HES), Poly Ethylen Glycol (PEG), Poly
(Glyx-Sery)n (HAP), Hyaluronic acid (HA), Heparosan polymers (HEP),
Phosphorylcholine-based polymers (PC polymer), Fleximers, Dextran,
Poly-sialic acids (PSA), Fc domains, Transferrin, Albumin, Elastin
like (ELP) peptides, XTEN polymers, PAS polymers, PA polymers,
Albumin binding peptides, CTP peptides, FcRn binding peptides and
any combination thereof.
EMBODIMENT 25
[0309] Factor VII polypeptide according to embodiment 24, wherein
the half-life extending moiety is a heparosan polymer.
EMBODIMENT 26
[0310] Factor VII polypeptide according to embodiment 25, wherein
the heparosan polymer has a molecular weight in a range selected
from 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45
kDa and 38-42 kDa, or a molecular weight of 40 kDa.
EMBODIMENT 26(i)
[0311] FVII polypeptide according to any one of embodiments 25-26,
comprising the structural fragment shown in Formula I,
##STR00007##
[0312] wherein n is an integer from 95-115.
EMBODIMENT 26(ii)
[0313] Factor VII polypeptide according to any one of the preceding
embodiments, which has a half-life that is increased by at least
100% compared to wild type human Factor VIIa (SEQ ID NO: 1).
EMBODIMENT 27
[0314] Factor VII polypeptide according to any of the preceding
embodiments, wherein said Factor VII polypeptide is disulfide
linked to tissue factor.
EMBODIMENT 28
[0315] Factor VII polypeptide according to any of the preceding
embodiments, wherein said polypeptide has additional amino acid
modifications that increase platelet affinity of the
polypeptide.
EMBODIMENT 29
[0316] Factor VII polypeptide according to any one of embodiments
1-22, wherein said polypeptide is a fusion protein comprising a
Factor VII polypeptide according to any one of embodiments 1-22 and
a fusion partner protein/peptide, for example an Fc domain or an
albumin.
EMBODIMENT 30
[0317] Polynucleotide that encodes a Factor VII polypeptide defined
in any one of embodiments 1-22 and 28-29.
EMBODIMENT 31
[0318] Recombinant host cell comprising the polynucleotide
according to embodiment 30.
EMBODIMENT 32
[0319] Method for producing the Factor VII polypeptide defined in
any of embodiments 1-22 and 28-29, the method comprising
cultivating a cell in an appropriate medium under conditions
allowing expression of the polynucleotide construct and recovering
the resulting polypeptide from the medium.
EMBODIMENT 33
[0320] Pharmaceutical composition comprising a Factor VII
polypeptide as defined in any of embodiments 1-29 and a
pharmaceutically acceptable carrier.
EMBODIMENT 34
[0321] Method for the treatment of bleeding disorders or bleeding
episodes in a subject or for the enhancement of the normal
haemostatic system, the method comprising administering a
therapeutically or prophylactically effective amount of a Factor
VII polypeptide as defined in any of embodiments 1-29 to a subject
in need thereof.
EMBODIMENT 35
[0322] Factor VII polypeptide as defined in any of embodiments 1-26
for use as a medicament.
EMBODIMENT 35(i)
[0323] Factor VII polypeptide as defined in any one of embodiments
1-26 for use in the treatment of a coagulopathy.
EMBODIMENT 36
[0324] Factor VII polypeptide according to embodiment 35(i) for use
as a medicament in the treatment of haemophilia A or B.
[0325] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realising the invention in diverse forms thereof.
EXAMPLES
Proteins
[0326] Human plasma-derived Factor X (FX) and Factor Xa (FXa) were
obtained from Enzyme Research Laboratories Inc. (South Bend, Ind.).
Soluble tissue factor 1-219 (sTF) or 1-209 were prepared according
to published procedures (Freskgard et al., 1996). Expression and
purification of recombinant wild-type FVIIa was performed as
described previously (Thim et al., 1988; Persson and Nielsen,
1996). Human plasma-derived antithrombin (Baxter) was repurified by
heparin sepharose chromatography (GE Healthcare) according to
published procedures (Olson et al., 1993). Bovine serum albumin
(BSA) was obtained from Sigma Aldrich (St. Louis, Mo.).
Example 1
FVIIa Variant Design
[0327] To design FVIIa variants with higher proteolytic activity
towards FX as a substrate, a two-pronged strategy was employed.
FVIIa loops and single amino acids, around the active-site area,
were selected for swapping and for point mutagenesis, respectively,
with corresponding FVII amino acids from different species (FIG.
1). FVIIa proteolytic activity was measured as outlined in example
5. Proteolytic activities for three loop-swapped FVIIa variants are
shown in
[0328] Table 1 where residues at positions 287 and 289 are mutated
to threonine and glutamic acid respectively while changing the
amino acid at position 288. It was observed that changes at
position 288, while maintaining the same amino acids at positions
287 and 289, dramatically affected the proteolytic activity. It was
also observed that substituting the amino acid at position 201 for
either a leucine, carried by rat and rabbit FVII, or an arginine,
carried by bovine FVII, affected the proteolytic activity.
Furthermore, it was observed that substituting the amino acid at
position 337 for either a glutamine carried by horse or a less
bulky amino acid such as alanine affected the proteolytic
activity
[0329] Table 1). These observations suggested that the amino acids
at position 288 and 201 could be involved in FX recognition and
activation. Therefore, positions 288 and 201 were further
investigated by saturation mutagenesis and the representative
results are outlined in Table 2.
TABLE-US-00003 TABLE 1 Proteolytic activity of selected FVIIa
variants. Results are shown in percent (%) of wild-type FVIIa.
Proteolytic Proteolytic activity + activity + PS:PC sTF + PS:PC
FVIIa variant (%) (%) FVIIa 100 100 FVIIa L287T L288F D289E 100
22.1 FVIIa L287T L288H D289E 27.3 4.5 FVIIa L287T L288R D289E 3.2
0.7 FVIIa W201L 66.5 72.8 FVIIa W201R 404.5 177.7 FVIIa K337Q 29.8
63.5 FVIIa K337A 347.3 97.7 FVIIa K337G 317.2 126.1
Example 2
Cloning of FVIIa Variants
[0330] Mutations were introduced into a mammalian expression vector
encoding FVII cDNA using a site directed mutagenesis PCR-based
method using KOD XtremeTM Hot Start DNA Polymerase from Novagen or
QuickChange.RTM. Site-Directed Mutagenesis kit from Stratagene. The
pQMCF expression vector and CHOEBNALT85 from Icosagen Cell Factory
(Estonia) was used as expression system. Introduction of the
desired mutations was verified by DNA sequencing (MWG Biotech.
Germany).
Example 3
FVIIa Expression
[0331] The FVII variants were expressed in CHOEBNALT85 cells from
Icosagen Cell Factory (Estonia). Briefly, CHOEBNALT85 suspension
cells were transiently transfected by electroporation (Gene Pulse
Xcell, Biorad, Copenhagen, DK). Transfected cells were selected
with 700 .mu.g/l Geneticin.RTM. (Gibco by Life Technologies), and
expanded to give a total of 300 ml to 10 litre supernatant. Cells
were cultured in medium according to manufacturer's instructions
supplemented with 5 mg/l Vitamin K1 (Sigma-Aldrich). Depending on
scale, cells were cultured in shake flasks (37.degree. C. 5-8% CO2
and 85-125 rpm) or rocking cultivation bags (37.degree. C. 5% CO2
and 30 rpm). Small scale supernatants were harvested by
centrifugation followed by filtration through a 0.22 .mu.m PES
filter (Corning; Fischer Scientific Biotech, Slangerup, DK) and
larger volumes were harvested by depth filtration followed by 0.22
.mu.m absolute filtration (3 .mu.m Clarigard, Opticap XL10; 0.22 pm
Durapore, Opticap XL10, Merck Millipore, Hellerup, DK).
Example 4
FVIIa Purification and Concentration Determination
[0332] FVII variants were purified by Gla-domain directed antibody
affinity chromatography essentially as described elsewhere (Thim et
al. 1988). Briefly, the protocol comprised of 3 steps. In step 1, 5
mM CaCl.sub.2 was added to the conditioned medium and the sample
was loaded onto the affinity column. After extensive wash with 10
mM His, 2 M NaCl, 5 mM CaCl.sub.2, 0.005% Tween 80, pH 6.0, bound
protein was eluted with 50 mM His, 15 mM EDTA, 0.005% Tween80, pH
6.0 onto (step 2) an anion exchange column (Source 15Q, GE
Healthcare). After wash with 20 mM HEPES, 20 mM NaCl, 0.005%
Tween80, pH 8.0, bound protein was eluted with 20 mM HEPES, 135 mM
NaCl, 10 mM CaCl.sub.2, 0.005% Tween80, pH 8.0 onto (step 3) a
CNBr-Sepharose Fast Flow column (GE Healthcare) to which human
plasma-derived FXa had been coupled at a density of 1 mg/ml
according to manufacturer's instructions. The flow rate was
optimized to ensure essentially complete activation of the purified
zymogen variants to the activated form. For FVIIa variants with
enhanced activity, capable of auto-activation in the conditioned
medium or on the anion exchange column, step 2 and/or step 3 were
omitted to prevent proteolytic degradation. Purified proteins were
stored at -80.degree. C. Protein quality was assessed by SDS-PAGE
analysis and the concentration of functional molecules measured by
active site titration or quantification of the light chain content
by rpHPLC as described below.
Measurement of FVIIa Variant Concentration by Active Site
Titration
[0333] The concentration of functional molecules in the purified
preparations was determined by active site titration from the
irreversible loss of amidolytic activity upon titration with
sub-stoichiometric levels of d-Phe-Phe-Arg-chloromethyl ketone
(FFR-cmk; Bachem) essentially as described elsewhere (Bock P. E.,
1992. J. Biol. Chem. 267. 14963-14973). Briefly, all proteins were
diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM
CaCl.sub.2. 1 mg/mL BSA, and 0.1% w/v PEG8000). A final
concentration of 150 nM FVIIa variant was preincubated with 500 nM
of soluble tissue factor (sTF) for 10 min followed by the addition
of FFR-cmk at final concentrations of 0-300 nM (n=2) in a total
reaction volume of 100 .mu.L in a 96-well plate. The reactions were
incubated over night at room temperature. In an another 96-well
plate, 20 .mu.L of each reaction was diluted 10 times in assay
buffer containing 1 mM S-2288 (Chromogenix, Milano, Italy). The
absorbance increase was measured continuously for 10 min at 405 nM
in a Spectramax 190 microplate spectrophotometer equipped with
SOFTmax PRO software. Amidolytic activity was reported as the slope
of the linear progress curves after blank subtraction. Active site
concentrations were determined by extrapolation, as the minimal
concentration of FFR-cmk needed to completely abolish the
amidolytic activity.
[0334] Measurement of FVIIa variant concentration from the
light-chain content using reversed-phase HPLC--In an alternative
approach, the concentration of functional FVIIa molecules in
purified preparations were determined by quantification of the
FVIIa light chain (LC) content by reversed-phase HPLC (rpHPLC). A
calibration curve with wild-type FVIIa was prepared using FVIIa
concentrations in the range from 0 to 3 .mu.M, while samples of
unknown concentration were prepared in estimated concentrations of
1.5 .mu.M (n=2). All samples were reduced using a 1:1 mixture of
0.5 M tris(2-carboxyethyl)phosphine (TCEP; Calbiochem/Merck KGaA,
Darmstadt, Germany) and formic acid added to the samples to a
concentration of 20% (v/v) followed by heating of samples at
70.degree. C. for 10 min. The reduced FVIIa variants were loaded
onto a C4 column (Vydac. 300 .ANG., particle size 5 .mu.M, 4.6 mm,
250 mm) maintained at 30.degree. C. Mobile phases consisted of
0.09% TFA in water (solvent A) and 0.085% TFA in acetonitrile
(solvent B). Following injection of 80 .mu.L sample, the system was
run isocratically at 25% solvent B for 4 min followed by a linear
gradient from 25-46% B over 10 min. Peaks were detected by
fluorescence using excitation and emission wavelengths of 280 and
348 nm, respectively. Light chain quantification was performed by
peak integration and relative amounts of FVIIa variants were
calculated on basis of the wild-type FVIIa standard curve.
Example 5
Screen for Mutations Conferring Increased Activity
[0335] As outlined in example 1,
[0336] Table 1, and to evaluate the role of FVIIa amino acids at
positions 201 and 288; these positions were subjected to rigorous
site-directed saturation mutagenesis. In order to further identify
FVIIa variants having enhanced proteolytic activity other amino
acid positions, 305 and 337, were also selected for saturation
mutagenesis. Briefly, activity was measured as the ability of each
variant to proteolytically activate the macromolecular substrate
Factor X in the presence of phospholipid vesicles (In vitro
proteolysis assay). Each reaction was performed in the presence or
absence of the co-factor tissue factor (sTF) to mimic the possible
TF dependent and independent mechanisms of action of recombinant
FVIIa. Furthermore, to understand the role of these substitutions
towards FVIIa inhibition by antithrombin; antithrombin inhibition
was quantified under pseudo-first order conditions in the presence
of low molecular weight heparin to mimic the ability of endogenous
heparin-like glycosaminoglycans (GAGs) to accelerate the reaction
in vivo. These results are summarized in
[0337] Table 2. As shown in FIG. 2, the measured in vitro
antithrombin reactivities were found to correlate with the in vivo
accumulation of FVIIa-antithrombin complexes thus validating the
predictiveness of the in vitro screening procedure.
TABLE-US-00004 TABLE 2 Saturation mutagenesis of selected amino
acid positions. Results are shown in percent (%) of wild-type FVIIa
Proteolytic Proteolytic activity + AT AT activity + sTF +
reactivity + reactivity + PS:PC PS:PC LMWH sTF FVIIa variant (%)
(%) (%) (%) FVIIa W201A 99.1 110.9 98.7 73 FVIIa W201D 78 84.2 72.5
72.7 FVIIa W201E 68.4 55.9 70.6 52.1 FVIIa W201F 55 55.5 113.1
152.5 FVIIa W201H 88.8 85.8 111.4 118.9 FVIIa W201I 83.5 85.1 79.3
104.5 FVIIa W201K 149 120.2 160.4 95.6 FVIIa W201L 66.5 72.8 96
32.7 FVIIa W201M 135.3 151.4 114.3 155.8 FVIIa W201N 79.1 56.5 82.9
64 FVIIa W201P 65.5 84.4 104.8 121.6 FVIIa W201Q 115.7 93.3 79.4
62.9 FVIIa W201R 404.5 177.7 160 59.6 FVIIa W201S 120.5 91.5 82.3
74.7 FVIIa W201T 89.5 74.8 71 72 FVIIa W201V 86.1 74.3 81 86.1
FVIIa W201Y 125.7 107.8 115 122.3 FVIIa L288A 206.3 75.1 112.7 87.9
FVIIa L288D 25.9 10.3 FVIIa L288E 91.4 37.7 77.8 91.5 FVIIa L288F
574.6 90.7 156.8 78.6 FVIIa L288G 98.4 44.8 11.6 37.7 FVIIa L288K
10.3 6.2 151.4 69 FVIIa L288M 59.9 47.1 151 91.8 FVIIa L288N 279.6
44.4 85.9 21.7 FVIIa L288Q 62.1 28.9 177.6 67.6 FVIIa L288S 151.2
57.4 214.7 91.7 FVIIa L288T 51.9 29.4 145.7 74.2 FVIIa L288V 35.2
30.8 98.9 71.5 FVIIa L288W 251.3 41.5 221.1 74.5 FVIIa L288Y 530.4
73.4 152.6 89.9 FVIIa L305A 26 18.8 31 28.8 FVIIa L305I 327.5 92.3
201.5 76.6 FVIIa L305T 34.8 85.9 42.5 46.1 FVIIa L305V 164.4 133.2
215.1 56.1 FVIIa K337A 347.3 97.7 157.4 128.7 FVIIa K337D 0 4.2
FVIIa K337E 20.3 39.2 3.8 29.9 FVIIa K337G 317.2 126.1 183.7 208.2
FVIIa K337I 12.3 34 1.8 9.5 FVIIa K337L 8.1 15.8 1.6 13.3 FVIIa
K337N 1.5 12.9 FVIIa K337Q 29.8 63.5 30.3 78.2 FVIIa K337S 49.8
112.3 40.4 144.3 FVIIa K337T 3.9 16 FVIIa K337V 12.4 29.9 7.9 15.5
FVIIa K337Y 8.3 40.7
[0338] Amino acids including glutamine, tyrosine, methionine,
lysine, and arginine at position 201 are required for gaining
proteolytic activity towards FX as substrate in presence of
phospholipids. W201 R provides the most gain in the proteolytic
activity in presence of phopholipids and in either absence or
presence of sTF. On the other hand, amino acids including
phenylalanine, leucine, and asparagine decrease the proteolytic
activity compared to FVIIa WT. In case of position 288, alanine,
asparagine, serine, tryptophan, phenylalanine, and tyrosine provide
gain in the proteolytic activity towards FX as substrate in
presence of phospholipids. L288F and L288Y provide the most gain in
the proteolytic activity in presence of phopholipids. Data
presented in
[0339] Table 2 demonstrates the challenges in predicting the
proteolytic activity and antithrombin reactivity a priori. Our
approach of using saturation mutagenesis is, therefore, justified
in order to explore the full repertoire of influence in activity
that different amino acids bring about in FVIIa variants.
[0340] Measurement of proteolytic activity using factor X as
substrate (in vitro proteolysis assay)--The proteolytic activity of
the FVIIa variants was estimated using plasma-derived factor X (FX)
as substrate. All proteins were diluted in 50 mM HEPES pH 7.4, 100
mM NaCl, 10 mM CaCl.sub.2, 1 mg/mL BSA, and 0.1% w/v PEG8000.
Relative proteolytic activities were determined by incubating 1 to
10 nM of each FVIIa conjugate with 40 nM FX in the presence of 25
.mu.M 75:25 phosphatidyl choline:phosphatidyl serine (PC:PS)
phospholipids (Haematologic technologies, Vermont, USA) for 30 min
at room temperature in a total reaction volume of 100 .mu.L in a
96-well plate (n=2). FX activation in the presence of sTF was
determined by incubating 5 .mu.M of each FVIIa conjugate with 30 nM
FX in the presence of 25 .mu.M PC:PS phospholipids for 20 min at
room temperature in a total reaction volume of 100 .mu.L (n=2).
After incubation, reactions were quenched by adding 100 .mu.L of 1
mM S-2765 (Chromogenix, Milano, Italy) in stop buffer (50 mM HEPES
pH 7.4, 100 mM NaCl, 80 mM EDTA). Immediately after quenching, the
absorbance increase was measured continuously at 405 nM in an
Envision microplate reader (Perkin Elmer, Waltham, Mass.). All
additions, incubations and plate movements were performed by a
Hamilton Microlab Star robot robot (Hamilton, Bonaduz, Switzeland)
on line coupled to an Envision microplate reader. Apparent
catalytic rate values (k.sub.cat/K.sub.m) were estimated by fitting
the data to a simplified form of the Michaelis Menten equation
(v=k.sub.cat*[S]*[E]/K.sub.m) using linear regression since the FX
substrate concentration ([S]) was below K.sub.m for the activation
reaction. The amount of FXa generated was estimated from a standard
curve prepared with human plasma-derived FXa under identical
conditions. Estimated k.sub.cat/K.sub.m values were reported
relative to that of wild-type FVIIa following normalisation of the
measured rate of FXa generation according to the concentration of
the FVIIa variant used. Results are given in
[0341] Table 1,
[0342] Table 2,
[0343] Table 3 and
[0344] Table 7.
[0345] Measurement of FVIIa inhibition by antithrombin--A
discontinuous method was used to measure the in vitro rate of
inhibition by human plasma-derived antithrombin (AT) under
pseudo-first order conditions in the presence of low molecular
weight (LMW) heparin (Calbiochem/Merck KGaA, Darmstadt, Germany).
The assay was performed in a 96-well plate using a buffer
containing 50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM CaCl.sub.2, 1
mg/mL BSA, and 0.1% w/v PEG8000 in a total reaction volume of 200
.mu.L. To a mixture of 200 nM FVIIa and 12 .mu.M LMW heparin was
added 5 .mu.M antithrombin in a final reaction volume of 100 .mu.L.
At different times, the reaction was quenched by transferring 20
.mu.L of the reaction mixture to another microtiter plate
containing 180 .mu.L of sTF (200 nM), polybrene (0.5 mg/mL;
Hexadimethrine bromide, Sigma-Aldrich) and S-2288 (1 mM).
Immediately after transfer at the different times, substrate
cleavage was monitored at 405 nm for 10 min in an Envision
microplate reader. Pseudo-first order rate constants (k.sub.obs)
were obtained by non-linear least-squares fitting of data to an
exponential decay function, and the second-order rate constant (k)
was obtained from the following relationship k=k.sub.obs/[AT]. All
additions, incubations and plate movements were performed by a
Hamilton Microlab Star robot (Hamilton, Bonaduz, Switzeland) on
line coupled to an Envision microplate reader (Perkin Elmer,
Waltham, Mass.). Rates of inhibition were reported relative to that
of wild-type FVIIa. Results are given in
[0346] Table 2,
[0347] Table 3 and
[0348] Table 7.
Example 6
Combining FVIIa Mutations Conferring Increased Activity and
Antithrombin Resistance.
[0349] In order to design FVIIa variants with high proteolytic
activity and antithrombin resistance, a selection of the identified
FVIIa proteolytic activity enhancing variants were combined with
the FVIIa variants that confer antithrombin resistance.
Specifically, FVIIa combination variants were made with
substitutions at positions 293 and 201, 288, 305, 337, 176 and/or
286. Characterization of the combination FVIIa purified protein
preparations using the in vitro proteolysis and antithrombin
inhibition assays described in Example 5 are summarized in
[0350] Table 3.
[0351] Table 3 demonstrates that some combinations resulted in
FVIIa variants exhibiting a desirable high activity while at the
same time having a desirable low antithrombin reactivity. For
example, FVIIa variant L288F T293K displayed 600% proteolytic
activity in presence of phospholipids and just 6% antithrombin
reactivity in presence of low-molecular weight heparin compared to
wild-type FVIIa. Similarly, FVIIa variant L288Y T293K displays
447,8% proteolytic activity in presence of phospholipids and just
5,8% antithrombin reactivity in presence of low-molecular weight
heparin compared to wild-type FVIIa. Furthermore, the W201 R T293K
displayed 609% proteolytic activity in presence of phospholipids
and just 9% antithrombin reactivity in presence of low-molecular
weight heparin compared to wild-type FVIIa.
[0352] Interestingly, combining the two FVIIa mutations L288F and
K337A provides greatly enhanced activity with a measured 2646%
increase in proteolytic activity compared to wild-type FVIIa. Upon
further co-introduction of the mutation T293K, enhanced activity is
retained while a low antithrombin reactivity is achieved. This
variant displays 1310% proteolytic activity in presence of
phospholipids and just 17% antithrombin reactivity in presence of
low-molecular weight heparin compared to wild-type FVIIa.
[0353] Altogether, it can be concluded that the T293K, T293R, and
T293Y mutations when combined with W201 R or L288F effectively
reduce the antithrombin reactivity compared to wild-type FVIIa
while providing higher proteolytic activity compared to wild-type
FVIIa.
TABLE-US-00005 TABLE 3 Proteolytic activities and antithrombin
reactivities of FVIIa combination variants. Results are shown in
percent (%) of wild-type FVIIa. Proteolytic Proteolytic AT AT
activity + activity + sTF + reactivity + reactivity + PS:PC PS:PC
LMWH sTF FVIIa variant (%) (%) (%) (%) FVIIa W201R T293Y 1026.9
202.6 11.1 2.3 FVIIa W201R T293R L305I 1573.6 411.3 46.6 8.9 FVIIa
W201R T293R 217.2 375.5 7.1 9.1 FVIIa W201R T293K L305I 1734.6
446.4 82.5 3.4 FVIIa W201R T293K 590.6 272.2 7.9 7.7 FVIIa W201R
L288F 2542.8 427.2 40.4 33.2 T293R FVIIa W201R L288F 1476 307.2
16.7 18.2 T293K FVIIa W201M T293Y 599.7 145.4 11.6 1.7 FVIIa W201M
T293R 179.2 201.7 3 6.7 FVIIa W201M T293K 146.1 173.5 3.8 5.2 FVIIa
W201K T293Y 617.7 176.7 15.4 2.6 FVIIa W201K T293R 553.5 8.8 11.6
FVIIa W201K T293K 217.2 214.6 6.3 7.3 FVIIa T293Y L305V K337A
1194.6 121 62.4 3.3 FVIIa T293Y K337A 213 162.9 26.5 2 FVIIa T293R
L305V K337A 2552.5 427 37.4 6.9 FVIIa T293R L305V 1325.8 356.1 19.1
4.2 FVIIa T293R L305I 711 229.7 22.1 2.6 FVIIa T293R K337A 690.2
279 10.4 13.4 FVIIa T293K L305V K337A 956.8 170.1 34.2 5.7 FVIIa
T293K L305I 524 152.2 17.9 1.7 FVIIa T293K K337A 773.1 264.9 7.2
6.9 FVIIa L305V T293Y 669.5 110.4 30.4 1.3 FVIIa L305V T293K 792.4
166.6 13.8 1.9 FVIIa L288Y T293R K337A 2530.2 323.8 19.1 10.1 FVIIa
L288Y T293R 1059.7 298.4 7.5 4.8 FVIIa L288Y T293K 676.5 233.7 5.4
4.5 FVIIa L288N T293Y 783.2 116.2 10.6 0.8 FVIIa L288N T293R 209.5
78.7 20.7 3.5 FVIIa L288N T293K 168 69 4.4 0.9 FVIIa L288F T293Y
523.9 48.1 12.2 2 FVIIa L288F T293R L305V 1784.5 101.3 48.6 9.1
FVIIa L288F T293R L305I 1456.4 158.2 41.4 3.5 FVIIa L288F T293R
K337A 2001.9 305 21.5 20.7 FVIIa L288F T293R 259.7 110 8.3 6.7
FVIIa L288F T293K L305V 466.3 181.4 8.2 10.2 FVIIa L288F T293K
L305I 2147.7 147.6 33 2.8 FVIIa L288F T293K K337A 1310.7 133.6 17.1
9 FVIIa L288F T293K 600.6 210.2 6.1 4.3
Example 7
Estimation of FVIIa Potency and Plasma Level
[0354] Potencies were estimated using a commercial FVIIa specific
clotting assay; STACLOT.RTM.VIIa-rTF from Diagnostica Stago. The
assay is based on the method published by J. H. Morrissey et al.
Blood. 81:734-744 (1993). It measures sTF initiated FVIIa
activity-dependent time to fibrin clot formation in FVII deficient
plasma in the presence of phospholipids. Clotting times were
measured on an ACL9000 (ILS) coagulation instrument and results
calculated using linear regression on a bilogarithmic scale based
on a FVIIa calibration curve. The same assay was used for
measurements of FVIIa clotting activity in plasma samples from
animal PK studies. The lower limit of quantification (LLOQ) in
plasma was estimated to 0.25 U/ml. Plasma activity levels were
converted to nM using the specific activity.
Example 8
Crystallographic Analysis of FVIIa Variants
[0355] To explore the mechanism by which the identified
substitutions affect proteolytic activity and antithrombin
recognition, crystal structures of the representative FVIIa
variants L288Y T293K, L288F T293K, W201 R T293K, W201 R T293Y and
L288F T293K K337A were determined.
[0356] When comparing structures on the 3-dimensional level the 1
DAN structure of wild-type (WT) FVIIa, in complex with soluble
Tissue Factor, [Banner, D. W. et al, Nature, (1996), Vol. 380,
41-46] have had its heavy chain residues of FVIIa renumbered
according to the numbering scheme of SEQ ID NO: 1.
[0357] Purified H-.sub.D-Phe-Phe-Arg chloromethyl ketone (FFR-cmk;
Bachem, Switzerland) active-site inhibited FVIIa variants in
complex with soluble Tissue Factor (fragment 1-219) were
crystallized using the hanging drop method in accordance with
[Kirchhofer, D. et al, Proteins Structure Function and Genetics,
(1995), Vol. 22, pages 419-425]. The protein buffer solution was a
mix of 10 mM Tris pH 7.5 at 25 C.degree., 100 mM NaCl, 15 mM
CaCl.sub.2. Protein concentrations together with precipitant
solutions and mixing conditions for the FVIIa variants are shown
in
[0358] Table 4. The hanging drop method using 24-well VDX-plates
and well solution of 1.0 ml was utilized. The drops were set up
with a mix of 1.5 .mu.l of the protein solution and 0.5 .mu.l of
the well solution. Streak seeding was used to initialize
nucleation.
[0359] The cryo conditions are shown in
[0360] Table 4. The crystal was let to soak in the cryo solution
for about 30 seconds after which the crystal was transferred to,
and flash frozen in, liquid nitrogen. Crystallographic data were
processed by the XDS data reduction software [Kabsch, W., Acta
Crystallographica Section D Biological Crystallography, (2010),
Vol. 66, pages 125-132] using resolution cut-off as described by
Karplus et al. [Karplus, P. A. et al, Science (New York, N.Y.),
(2012), Vol. 336, pages 1030-1033].
TABLE-US-00006 TABLE 4 Crystallization and freezing conditions for
the FVIIa variants. Mixing ratio FVIIa Protein protein:precipitant
variant conc. Precipitant solution solution Cryo condition L288Y
2.5 mg/ml 0.1M Cacodylate 3:1 100% TMAO T293K pH 5.1, 13% Peg
(trimethylamine 8000 N-oxide) L288F 2.14 mg/ml 0.1M Na-citrate 3:1
100% TMAO T293K pH 5.6, 17% Peg 3350 and 12% 1- propanol W201R 1.0
mg/ml 0.1M Cacodylate 3:1 As precipitant T293K pH 5.1, 13% Peg
solution but with 8000 35% PEG 8000 W201R 2.93 mg/ml 0.1M
Cacodylate 3:1 As precipitant T293Y pH 5.1, 12% Peg solution but
with 8000 35% PEG 8000 L288F 1.0 mg/ml 0.1M Na-citrate 3:1 As
precipitant T293K pH 5.6, 16% Peg solution but with K337A 3350 and
12% 1- 35% PEG3350. propanol
[0361] In-house generated coordinates (unpublished) based on the
crystallographic coordinates of the 1 DAN entry [Banner, D. W. et
al, Nature, (1996), Vol. 380, pages 41-46] from the Protein Data
Bank (PDB) [Berman, H. M. et al, Nucleic Acids Res., (2000), Vol.
28, pages 235-242], were used as starting model for either
molecular replacement calculations in phenix.phaser [Mccoy, A. J.
et al, J.Appl.Crystallogr., (2007), Vol. 40, pages 658-674] or
straight into refinements with the phenix.refine software [Afonine,
P. V. et al, Acta Crystallogr.Sect.D-Biol.Crystallogr., (2012),
Vol. 68, pages 352-367] of the PHENIX software package [Adams, P.
D. et al, Acta Cryst.D, (2010), Vol. 66, pages 213-221].
Refinements were followed by interactive model corrections in the
computer graphics software COOT [Emsley, P. et al, Acta
Crystallogr.Sect.D-Biol.Crystallogr., (2010), Vol. 66, pages
486-501]. Crystallographic data, refinement and model statistics
for the 5 FVIIa variants are shown in
[0362] Table 5.
TABLE-US-00007 TABLE 5 Data collection, refinement and model
statistics. Statistics for the highest-resolution shell are shown
in parentheses. L288F L288Y L288F W201R W201R T293K FVIIa variant
T293K T293K T293K T293Y K337A Data collection BLI911-3, BLI911-3,
BLI911-3, BLI911-3, X10SA, beamline MAX-lab MAX-lab MAX-lab MAX-lab
SLS Wavelength [.ANG.] 1.0000 1.0000 1.0000 1.0000 0.9999
Resolution range [.ANG.] 35.7-2.01 29.25-2.37 29.5-1.71 29.16-2.5
49.75-2.216 (2.082-2.01) (2.455-2.37) (1.772-1.71) (2.589-2.5)
(2.295-2.216) Space group P 2.sub.1 2.sub.1 2.sub.1 P 2.sub.1
2.sub.1 2.sub.1 P 2.sub.1 2.sub.1 2.sub.1 P 2.sub.1 2.sub.1 2.sub.1
P 2.sub.1 2.sub.1 2.sub.1 Unit cell [.ANG.] 71.34 69.34 68.77 68.86
68.919 82.46 81.57 78.2 81.42 81.545 123.3 125.88 172.21 125.39
125.57 Total reflections 321033 (9043) 103617 (6525) 501348 (14728)
91616 (9007) 234709 (20365) Unique reflections 48292 (4010) 28995
(2424) 96329 (6505) 24966 (2433) 35724 (3412) Multiplicity 6.6
(2.3) 3.6 (2.7) 5.2 (2.3) 3.7 (3.7) 6.6 (6.0) Completeness [%]
98.31 (83.26) 97.71 (83.50) 95.43 (63.97) 99.70 (99.43) 99.60
(96.82) Mean I/sigma (I) 9.33 (0.68) 9.45 (0.79) 11.73 (0.42) 5.09
(0.54) 7.39 (0.53) Wilson B-factor [.ANG.] 22.18 40.30 24.56 25.91
45.71 R-merge 0.2439 (1.316) 0.145 (1.431) 0.1141 (1.968) 0.3456
(2.778) 0.2599 (3.739) R-meas 0.2647 0.1698 0.1266 0.4047 0.2822
CC1/2 0.987 (0.277) 0.992 (0.322) 0.997 (0.219) 0.951 (0.11) 0.992
(0.174) CC* 0.997 (0.659) 0.998 (0.698) 0.999 (0.599) 0.987 (0.446)
0.998 (0.545) Reflections used in 48286 28991 96323 24966 35721
refinement Reflections used for R- 2497 1465 4773 1260 1853 free
R-work 0.2122 (0.3487) 0.2199 (0.3698) 0.2170 (0.5007) 0.2568
(0.3940) 0.2077 (0.4038) R-free 0.2561 (0.3880) 0.2755 (0.3826)
0.2556 (0.5132) 0.3102 (0.4015) 0.2556 (0.4045) Number of non- 5446
4969 5382 4981 4566 hydrogen atoms: Total In Macromolecules 4769
4700 4836 4679 4355 In Ligands 81 51 87 36 39 In Waters 596 218 459
266 172 Protein residues 607 618 622 607 561 RMS(bonds) [.ANG.]
0.007 0.004 0.008 0.002 0.005 RMS(angles) [.degree.] 0.88 0.64 1.05
2.73 0.75 Ramachandran 97 94 94 94 95 favoured [%] Ramachandran
outliers 0 0 0.99 0.51 0 [%] Clashscore 1.69 1.41 3.60 2.19 1.74
Average B-factor [.ANG..sup.2]: 30.70 51.70 60.40 43.70 61.80 Total
For macromolecules 29.70 51.90 61.50 44.80 61.80 For Ligands 56.10
65.60 68.50 43.60 109.30 For Solvent 35.30 44.70 46.70 23.60
50.10
3-Dimensional Structure Analyses
[0363] Generally there are no major differences between the wild
type (WT) FVIIa molecule 1 DAN structure [Banner, D. W. et al,
Nature, (1996), Vol. 380, pages 41-46] and those of the FVIIa
variants. The overall root-mean-square deviation (RMSD), calculated
by gesamt [Krissinel, E., Journal of Molecular Biochemistry,
(2012), Vol. 1, pages 76-85] between the 1 DAN FVIIa heavy chain
and the L288Y T293K, L288F T293K, W201 R T293K, W201 R
[0364] T293Y and L288F T293K K337A FVIIa variants are 0.424, 0.365,
0.451, 0.342 and 0.289 .ANG., respectively. The number of
C.sub..alpha.-atom pairs used in the calculations were 254, 254,
251, 254 and 254, respectively.
The W201R T293Y FVIIa Variant
Mutation FVIIa W201R:
[0365] On the detailed level the heavy chain FVIIa Arg 201 residue
of the double mutant is situated in the "60-loop" (chymotrypsin
numbering). In the likelihood-weighted 2mFo-DFc electron density
map, at 1.0 a cut-off, there are indications of the main chain loop
stretch while that cannot be seen for the side chains of the Arg
201 residue (a Trp residue in the wild type FVIIa), together with
the side chain of the residues before and after (Asn and Arg
residues, respectively). This indicates high flexibility of those
side chains. To aid in the structure interpretation a difference
electron density map was calculated between observed structure
factors from the in-house wild type protein crystals and the
observed structure factors from the FVIIa double mutant
[F.sub.obs(WT FVIIa/sTF)-F.sub.obs(FVIIa W201 R T293Y/sTF)], using
software from the CCP4 software program package [Collaborative
Computational Project, N. Acta crystallographica, Section D,
Biological crystallography, 1994, 50, 760-763]. Using phases from
the wild type data or the double mutant data resulted in similar
difference maps. On the positive side of the difference map the
side chain of the Trp residue from the wild type FVIIa can be
clearly seen (maximum peak at 5.6 a levels using phases from wild
type data) while there is no clear indication of the Arg side chain
on the negative side of the difference map. This also argues for
that the Arg residue is more flexible than the Trp residue of the
wild type protein. It should be noted, however, that neither the
side chains before nor after the Trp residue can be clearly
observed in the 1 DAN structure, using a likelihood-weighted
2mFo-DFc electron density map, which is similar to the results from
the FVIIa W201 R T293Y/sTF crystal structure, while the position of
the Trp 201 is unmistakably seen in the FVIIa WT structure.
[0366] Regarding the main chain orientation of the loop studied,
the likelihood-weighted 2mFo-DFc electron density map and
phenix.refine refinements places the 200, 201 and 202 residues
closer towards the position of the replaced Trp side chain residue,
relatively to the published 1 DAN structure [Banner. D. W., et al.,
Nature, 1996, 380, 41-46]. In particular residue Asn 200 has moved
and its C.sub..alpha. position is 3.1 .ANG. away from its position
in the wild type structure FIG. 3. Also, in the described
[F.sub.obs(WT FVIIa/sTF)-F.sub.obs(FVIIa W201 R T293Y/sTF)]
difference map there are peaks indicating such a movement of
residue Asn 200. One 5.7 a positive peak close to the position of
the wild type loop conformation and another 4.3 a negative peak
slightly on the inside of the refined double mutant conformation.
This supports that the main chain has moved closer towards the
position of the WT Trp side chain and relatively more towards the
center of the FVIIa heavy chain.
[0367] The structural difference seen between the wild type
structure and the double mutated protein for the residues 200, 201
and 202 of the heavy chain FVIIa probably depends on stabilization
by the inward pointing Trp 201 residue side chain in the WT
structure that fills out a primarily hydrophobic volume in the
FVIIa protein and thereby anchors the loop in the wild type
structure. The side chain of the corresponding residue Arg in FVIIa
W201 R T293Y does not form the same rigid structure, with a tightly
bound side chain, but is more flexible, and therefore not anchoring
the loop in the same way as in the WT FVIIa structure.
[0368] FIG. 3 shows a stick representation of a comparison of the
two crystal structures: 1) with light-colored carbon atoms. FVIIa
wild type protein in complex with Tissue Factor, using an in-house
data set from crystals of the same type as the PDB structure 1 DAN
[Banner. D. W., et al., Nature, 1996, 380, 41-46], and 2) with
dark-colored carbon atoms, the FVIIa double mutant W201R T293Y in
complex with Tissue Factor. Some of the residues are labeled with
amino acid one-letter code and ending with "-wt" or "-m" for 1) and
2), respectively. Several side chains have been truncated (atoms
outside of C.sub..beta. have been removed) as likelihood-weighted
2mFo-DFc electron density maps did not show any electron density
for those side chains. For example the residues N200, R201 and R202
of the FVIIa double mutant W201 R T293Y are all truncated for that
reason. The figure was prepared by the molecular graphics software
PyMOL [The PyMOL Molecular Graphics System. Version 1.6.0.0
Schrodinger, LLC].
Mutation FVIIa T293Y:
[0369] The heavy chain FVIIa Tyr 293 residue is situated in the
activation loop 1. The likelihood-weighted 2mFo-DFc electron
density map, at 1.0 a cut-off, clearly show the main chain and side
chain of the Tyr residue in the refined structure. The Tyr side
chain atom C.sub..beta.-C.sub..gamma. follows the same direction as
for the C.sub..beta.-C.sub..gamma.2 atoms in the wild type Thr
residue. The C-C.sub..alpha.-C.sub..beta.-C.sub..gamma. and
C-C.sub..alpha.-C.sub..beta.-C.sub..gamma.2 dihedral angles are 165
and 173.degree. for FVIIa residue 293 of the double mutant and WT
form, respectively. Thereby, the Tyr 293 residue of the double
mutant directs its side chain in the direction of the catalytic
domain and towards the binding site of the FFR-cmk bound inhibitor.
The calculated [F.sub.obs(WT FVIIa/sTF)-F.sub.obs(FVIIa W201 R
T293Y/sTF)] difference map confirms the orientation of the Tyr side
chain with a negative peak (4.7 a height) at the Tyr ring system
and a positive peak (4.2 a height) at the missing Thr
O.gamma..sub.1 atom. To study the possible interactions between
antithrombin and a FVIIa mutated T293Y molecule a superimposition
of the Factor Xa molecular complex with antithrombin, PDB-code 2GD4
[Johnson. D. J. D., et al., Embo J., 2006, 25, 2029-2037], was made
on the FVIIa double mutant. The molecular graphics software PyMOL
[The PyMOL Molecular Graphics System, Version 1.6.0.0 Schrodinger,
LLC] was used for the superimposition of the FXa and FVIIa
molecules and resulted in an RMSD of 0.769 .ANG. for 1194 atoms.
From the riding antithrombin molecule model it is then clear that
the Tyr 293 residue of the FVIIa W201 R T293Y mutant in the
theoretically molecular complex produced (FVIIa W201 R
T293Y/antithrombin III) forms spatial overlap with, in particular,
residue Leu 395 but also Arg 399 of the antithrombin molecule FIG.
4. This is confirmed by distance calculations, performed in the
contacts software of the CCP4 program suite, between Tyr 293 of the
FVIIa double mutant and the riding antithrombin molecule. A cut-off
distance of 3.5 .ANG. was used between the Tyr 293 residue in the
mutant FVIIa molecule and the antithrombin molecule and the results
are shown in
[0370] Table 6. All distances, 3.5 .ANG. or shorter, between the
residue Tyr 293 of the FVIIa W201 R T293Y double mutant and
antithrombin from the Antithrombin-S195A FXa-pentasaccharide
complex, PDB:2GD4, [Johnson. D. J. D., et al., Embo J., 2006, 25,
2029-2037] after the FXa complex has been superimposed on the FVIIa
mutant (W201 R T293Y)/sTF structure, using the FVIIa (W201 R T293Y)
and the FXa as common molecules are summarized in
[0371] Table 6. The spatial overlap will most probably negatively
influence on the possibility for antithrombin to place its reactive
center loop (RCL) into the active site of FVIIa. Thereby a T293Y
mutated FVIIa molecule will be less susceptible to inhibition by
antithrombin. This is in agreement with, and gives an explanation
to, what is observed experimentally showing increased resistance to
inactivation by antithrombin and prolonged half-life.
[0372] FIG. 4 is a stick representation of a theoretical model of a
complex between antithrombin (indicated with light carbon atoms)
and the FVIIa W201 R T293Y double mutant (indicated with dark
carbon atoms). The relative positions of the residues Tyr 293, Gln
255, Lys 341, Gln 286 of the FVIIa mutant W201 R T293Y, and for the
antithrombin molecule residues Leu 395, Arg 399, Glu 295, Tyr 253
and V317 are shown and labeled. The model was constructed based on
the structures of the antithrombin/FXa complex [Johnson. D. J. D.,
et al., Embo J., 2006, 25, 2029-2037], PDB code 2GD4, where the FXa
molecule, with the antithrombin let riding, has been superimposed
on the heavy chain of FVIIa W201 R T293Y variant molecule. Residues
of FVIIa W201 R T293Y and antithrombin have a prefix of "FVIIa" and
"AT" respectively, followed by one-letter amino acid code and
residue number. The figure was prepared by the molecular graphics
software PyMOL [The PyMOL Molecular Graphics System, Version
1.6.0.0 Schrodinger, LLC].
TABLE-US-00008 TABLE 6 All distances. 3.5 .ANG. or shorter between
the residue Tyr 293 of the FVIIa (W201R T293Y) double mutant and
antithrombin amino acids in the described theoretical model between
the two molecules. FVIIa W201R T293Y Antithrombin Res. Res. # and
Atom Res. Res. # and Atom Distance Type Chain name Type Chain name
[.ANG.] Tyr 293H N Tyr 253A OH 3.41 Tyr 293H CD1 Leu 395A CD1 3.07
Tyr 293H CD2 Tyr 253A OH 3.50 Arg 399A NH2 2.54 Arg 399A CZ 3.40
Tyr 293H CE1 Leu 395A CG 2.88 Leu 395A CD1 1.89 Leu 395A CD2 3.40
Tyr 293H CE2 Glu 255A OE2 3.11 Arg 399A NH2 2.24 Leu 395A CD1 2.72
Arg 399A CZ 2.90 Tyr 293H CZ Arg 399A NH2 3.26 Leu 395A CG 2.51 Leu
395A CD1 1.60 Arg 399A CZ 3.43 Leu 395A CD2 2.59 Tyr 293H OH Leu
395A CB 2.78 Leu 395A CG 1.53 Leu 395A CD1 1.54 Leu 395A CD2
1.26
The W201R T293K FVIIa Variant
[0373] The region around residue 201 of FVIIa: On a detailed level
the heavy chain FVIIa Arg 201 residue of the double mutant is
situated in the "60-loop" (chymotrypsin numbering). In the
likelihood-weighted 2mFo-DFc electron density map, at 1.0 a
cut-off, the main chain loop stretch is clearly seen. The side
chain of the Arg 201 residue (a Trp residue in the wild type FVIIa)
is also clearly observed. The outer part, the guanidinium group, of
the Arg 202 residue has, however, missing electron density in the
likelihood-weighted 2mFo-DFc electron density map and at the chosen
cut-off, indicating a higher mobility or disorder. Regarding the
main chain orientation of the loop studied (the "60-loop") it show
transformations between the W201 R T293K and the 1 DAN structure
[Banner, D. W. et al, Nature, (1996), Vol. 380, pages 41-46]. After
superimposing the two structures it is seen that when moving along
the polypeptide from residue 197 towards 203 there are differences
in equivalent C.sub.a positions by 0.64, 2.48, 3.63, 6.41, 4.15 and
0.81 .ANG., respectively. The main chain of the loop has moved
closer towards the position of the in 1 DAN WT Trp side chain
position [Banner, D. W. et al, Nature, (1996), Vol. 380, pages
41-46] and has also moved towards the center of the FVIIa heavy
chain, the catalytic domain. The Arg 201 residue of W201 R T293K
FVIIa is in the superimposed structure placed towards the position
of the replaced WT Trp side chain residue of the published 1 DAN
structure.
[0374] The structural difference seen between the wild type
structure and the W201 R T293K FVIIa variant of the heavy chain
"60-loop" probably depends on stabilization by the inward pointing
Trp 201 residue side chain in the WT structure that fills out a
primarily hydrophobic volume in the FVIIa protein and thereby
anchors the loop in the wild type structure. The side chain of the
corresponding, smaller, residue Arg in FVIIa W201 R T293K does not
anchor the loop in the same way as the Trp in the WT FVIIa
structure.
[0375] The region around residue 293 of FVIIa: The heavy chain
FVIIa Lys 293 residue is situated in the activation loop 1. The
likelihood-weighted 2mFo-DFc electron density map, at 1.0 a
cut-off, clearly show the main chain and side chain of the Lys
residue in the refined structure. The Lys side chain atom
C.sub..beta.-C.sub..gamma. follows the same direction as for the
C.sub..beta.-C.sub..gamma.2 atoms in the wild type Thr residue. The
C-C.sub..alpha.-C.sub..beta.-C.sub..gamma. and
C-C.sub..alpha.-C.sub..beta.-C.sub..gamma.2 dihedral angles are 169
and 173.degree. for FVIIa residue 293 of the double mutant and WT
form, respectively. The Lys 293 show a "mttt" rotamer orientation,
the most common rotamer orientation for Lys [Lovell, S. C. et al,
Proteins, (2000), Vol. 40, pages 389-408] as seen by the computer
graphics software COOT [Emsley, P. et al, Acta
Crystallogr.Sect.D-Biol.Crystallogr., (2010), Vol. 66, pages
486-501]. Moreover, the Lys 293 residue N.sub..zeta. atom of the
W201 R T293K FVIIa variant makes a strong, with a distance of 2.68
.ANG., hydrogen bond with the residue Gln 176 O.sub..epsilon.1 atom
thereby stabilizing the two side chains. Compared to the WT FVIIa 1
DAN structure the Gln 176 residue has therefore altered its side
chain conformation to optimize the hydrogen bond it makes with the
Lys 293 residue in the W201 R T293K FVIIa variant. The rotamer goes
from the "tt0.degree." conformation of the WT structure to a
rotamer conformation which is not among the standard conformations
described in [Lovell, S. C. et al, Proteins, (2000), Vol. 40, pages
389-408]. Thereby, the Lys 293 residue of the double mutant directs
its side chain in the direction of the catalytic domain and towards
the binding site of the FFR-cmk bound inhibitor and is filling out
a prime site of the FVIIa active site cleft.
The L288Y T293K FVIIa variant
The Region Around Residue 288 of FVIIa:
[0376] The region is clearly seen in the crystal structure
likelihood-weighted 2mFo-DFc electron density map, at a 1.0 .sigma.
cut-off. The residues in the loop following the Tyr 288 residue,
residues 289 to 292 in the heavy chain of the FVIIa L288Y T293K
FVIIa variant shows a change in main chain conformation with a
maximum difference at residue Arg 290 where the C.sub..alpha. atoms
differs 2.87 .ANG. between the a superimposed molecules of the
FVIIa L288Y T293K FVIIa variant and the WT structure of FVIIa, 1
DAN [Banner, D. W. et al, Nature, (1996), Vol. 380, pages 41-46].
The C.sub..alpha. atom of the Tyr 288 residue shows a 0.80 .ANG.
difference to the equivalent atom of the Leu 288 residue in the
superimposed WT FVIIa. The side chain rotamer of Tyr 288 in the
FVIIa L288Y T293K FVIIa variant is "p90.degree. " while that of the
Ley side chain rotamer of the WT FVIIa 1 DAN structure show a "mt"
rotamer [Lovell, S. C. et al, Proteins, (2000), Vol. 40, pages
389-408]. That results in that the two equivalent side chains
points in different directions, seen in the difference in the
C-C.sub..alpha.-C.sub..beta.-C.sub..gamma. dihedral angle,
-69.degree. and 157.degree. for the L288Y T293K FVIIa variant and
WT FVIIa, respectively. The hydroxyl group of the Tyr 288 side
chain in the L288Y T293K FVIIa variant interacts favorably with
surrounding water molecules, which are ordered in the crystal
structure and the side chain folds over the loop following the Tyr
288 of the FVIIa L288Y T293K variant. The structural main chain
alteration of the loop following residue 288, and the mutation of
residue 288 itself, might at least partly explain the activity
improvements seen of this FVIIa variant.
The Region Around Residue 293 of FVIIa:
[0377] The 3D structure of this residue and other residues in
contact with it highly similar to what is described for the W201 R
T293K FVIIa variant. Therefore all conclusions drawn for the T293K
mutation of that variant also applies to the of T293K mutation of
the L288Y T293K FVIIa variant.
The L288F T293K FVIIa Variant
The Region Around Residue 288 of FVIIa:
[0378] The region is clearly seen in the crystal structure
likelihood-weighted 2mFo-DFc electron density map, at a 1.0 a
cut-off. The 3D structure of this region is highly similar to the
L288F T293K FVIIa variant. The two variants share same main chain
orientation for example. One thing that differs between the two
FVIIa variants is that the Phe 288 side chain has another preferred
rotamer ("m-85.degree.") for its side chain, actually pointing in
the same orientation as the Leu 288 side chain of the WT FVIIa. An
unusual property of the Phe 288 side chain of the L288F T293K FVIIa
variant is that for Phe residues it is unusually exposed (145
.ANG..sup.2 according to calculations by AREAIMOL of the CCP4
crystallographic program suite [Bailey, S., Acta
Crystallogr.Sect.D-Biol.Crystallogr., (1994), Vol. 50, pages
760-763]) to the surrounding solvent.
The Region Around Residue 293 of FVIIa:
[0379] The 3D structure of this residue and other residues in
contact with it is highly similar to what is described for the W201
R T293K FVIIa variant. Therefore all conclusions drawn for the
T293K mutation of that variant also applies to the of T293K
mutation of the L288F T293K FVIIa variant.
The L288F T293K K337A FVIIa Variant
The Region Around Residue 288 of FVIIa:
[0380] The region is clearly seen in the crystal structure
likelihood-weighted 2mFo-DFc electron density map, at a 1.0 .sigma.
cut-off. The 3D structure of this region is highly similar to the
L288F T293K FVIIa variant. The two variants share same main chain
orientation for example. One thing that differs between the two
FVIIa variants is that the Phe 288 side chain has another preferred
rotamer ("m-85.degree.") for its side chain, actually pointing in
the same orientation as the Leu 288 side chain of the WT FVIIa and
the L288F T293K FVIIa variant. Therefore all conclusions drawn for
the L288F mutation of that variant also applies to the of L288F
mutation of the L288F T293K K337A FVIIa variant.
The Region Around Residue 293 of FVIIa:
[0381] The 3D structure of this residue and other residues in
contact with it highly similar to what is described for the W201 R
T293K FVIIa variant. Therefore all conclusions drawn for the T293K
mutation of that variant also applies to the of T293K mutation of
the L288F T293K K337A FVIIa variant.
The Region Around Residue 337 of FVIIa:
[0382] The region is clearly observed in the crystal structure
likelihood-weighted 2mFo-DFc electron density map, at a 1.0 a
cut-off. The 3D structure of this region is similar to the WT
structure of FVIIa, 1 DAN [Banner, D. W. et al, Nature, (1996),
Vol. 380, pages 41-46], and to the other FVIIa variants of this
Example. The overall main- and side-chain orientations are close to
the WT FVIIa 1 DAN structure and the other FVIIa variant structures
there are, however, small differences, slightly larger than the by
phenix.refine maximum-likelihood based calculation of the
coordinate error of 0.40 .ANG. of the crystal structure. The
equivalent C.sub..beta. atoms of residue 337 are 0.8 .ANG. apart in
WT structure of FVIIa, 1 DAN, and the L288F T293K K337A FVIIa
variant. The C.sub.a atoms of the same residues are 0.4 .ANG.
apart. The equivalent C.sub..alpha. atoms of residue 336 are 0.6
.ANG. apart in the superimposed WT structure of FVIIa, 1 DAN, and
the L288F T293K K337A FVIIa variant. For the Phe 332 residue the
side chain in shifted approximately 0.5 .ANG. towards the Ala 337
residue of the L288F T293K K337A FVIIa variant compared to the WT
structure of FVIIa, 1 DAN [Banner, D. W. et al, Nature, (1996),
Vol. 380, pages 41-46]. It can also be concluded that the other
FVIIa variants of this Example show approximately the same
deviation to the L288F T293K K337A FVIIa variant as the WT
structure of FVIIa 1 DAN does. Moreover the other FVIIa variants
cluster much closer to the WT FVIIa 1 DAN structure than the L288F
T293K K337A FVIIa variant does. This might explain at least in part
the altered properties of this variant.
Examples 9-14
Chemical Modification of FVIIa Variants
[0383] Abbreviations used in Examples 9-14: [0384] AUS:
Arthrobacter ureafaciens Sialidase [0385] CMP-NAN:
Cytidine-5'-monophosphate-N-acetyl neuraminic acid [0386] CV:
Column volume [0387] GlcUA: Glucuronic acid [0388] GlcNAc:
N-Acetylglucosamine [0389] GSC: 5'-Glycylsialic acid cytidine
monophosphate [0390] GSC-SH: 5'-[(4-Mercaptobutanoyl)glycyl]sialic
acid cytidine monophosphate [0391] HEP: HEParosan polymer [0392]
HEP-GSC: GSC-functionalized heparosan polymer [0393] HEP-[N]-FVIIa:
HEParosan conjugated via N-glycan to FVIIa. [0394] HEPES:
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid [0395] His:
Histidine [0396] PABA: p-Aminobenzamidine [0397] ST3GaIIII N-glycan
specific a2,3-sialyltransferase [0398] TCEP:
tris(2-carboxyethyl)phosphine [0399] UDP: Uridine diphosphate
Quantification method used in Examples 9-14:
[0400] The conjugates of the invention were analysed for purity by
HPLC. HPLC was also used to quantify amount of isolated conjugate
based on a FVIIa reference molecule. Samples were analysed either
in non-reduced or reduced form. A Zorbax 300SB-C3 column
(4.6.times.50 mm; 3.5 .mu.m Agilent, Cat. No.: 865973-909) was
used. Column was operated at 30.degree. C. 5 .mu.g sample was
injected, and column eluted with a water (A)--acetonitrile (B)
solvent system containing 0.1% trifluoroacetic acid. The gradient
program was as follows: 0 min (25% B); 4 min (25% B); 14 min (46%
B); 35 min (52% B); 40 min (90% B); 40.1 min (25% B). Reduced
samples were prepared by adding 10 .mu.l TCEP/formic acid solution
(70 mM tris(2-carboxyethyl)phosphine and 10% formic acid in water)
to 25 .mu.l/30 .mu.g FVIIa (or conjugate). Reactions were left for
10 minutes at 70CC, before they were analysed on HPLC (5 .mu.l
injection).
Starting Materials Used in Examples 9-14:
HEP-Maleimide and HEP-Benzaldehyde Polymers
[0401] Maleimide and aldehyde functionalized HEP polymers of
defined size is prepared by an enzymatic polymerization reaction as
described in U.S. 2010/0036001. Two sugar nucleotides (UDP-GlcNAc
and UDP-GlcUA) and a priming trisaccharide
(GlcUA-GlcNAc-GlcUA)NH.sub.2 for initiating the reaction is used,
and polymerization is run until depletion of sugar nucleotide
building blocks. The process produced HEP polymers with a single
terminal amino group. The size of HEP polymer is determined by the
sugar nucleotide to primer ratio. The terminal amine (originating
from the primer) is then functionalized with either a maleimide
functionality for conjugation to GSC-SH, or a benzaldehyde
functionality for reductive amination chemistry to the glycyl
terminal of GSC.
[0402] HEP-benzaldehydes can be prepared by reacting amine
functionalized HEP polymers with a surplus of
N-succinimidyl-4-formylbenzoic acid (Nano Letters (2007), 7(8),
2207-2210) in aqueous neutral solution. The benzaldehyde
functionalized polymers may be isolated by ion-exchange
chromatography, size exclusion chromatography, or HPLC.
HEP-maleimides can be prepared by reacting amine functionalized HEP
polymers with a surplus of N-maleimidobutyryl-oxysuccinimide ester
(GMBS; Fujiwara, K., et al. (1988), J. Immunol. Meth. 112,
77-83).
[0403] The benzaldehyde or maleimide functionalized polymers may be
isolated by ion-exchange chromatography, size exclusion
chromatography, or HPLC. Any HEP polymer functionalized with a
terminal primary amine functionality (HEP-NH.sub.2) may be used in
the present examples. Two options are shown below:
##STR00008##
[0404] The terminal sugar residue in the non-reducing end of the
polysaccharide can be either N-acetylglucosamine or glucuronic acid
(glucuronic acid is drawn above). Typically a mixture of both sugar
residues are to be expected in the non-reducing end, if equimolar
amount of UDP-GlcNAc and UDP-GlcUA has been used in the
polymerization reaction. 5'-Glycylsialic acid cytidine
monophosphate (GSC):
[0405] The GSC starting material used in the current invention can
be synthesised chemically (Dufner, G. Eur. J. Org. Chem. 2000,
1467-1482) or it can be obtained by chemoenzymatic routes as
described in WO07056191. The GSC structure is shown below:
##STR00009##
Example 9
[0406] Preparation of 38.8k-HEP-[N]-FVIIa L288F T293K
Step 1
[0407] Synthesis of [(4-Mercaptobutanoyl)Glycyl]Sialic Acid
cytidine monophosphate (GSC-SH)
##STR00010##
[0408] Glycyl sialic acid cytidine monophosphate (200 mg; 0.318
mmol) was dissolved in water (2 ml), and thiobutyrolactone (325 mg;
3.18 mmol) was added. The two phase solution was gently mixed for
21 h at room temperature. The reaction mixture was then diluted
with water (10 ml) and applied to a reverse phase HPLC column (C18,
50 mm.times.200 mm). Column was eluted at a flow rate of 50 ml/min
with a gradient system of water (A), acetonitrile (B) and 250 mM
ammonium hydrogen carbonate (C) as follows: 0 min (A: 90%, B: 0%,
C:10%); 12 min (A: 90%, B: 0%, C:10%); 48 min (A: 70%, B: 20%,
C:10%). Fractions (20 ml size) were collected and analysed by
LC-MS. Pure fractions were pooled, and passed slowly through a
short pad of Dowex 50W.times.2 (100-200 mesh) resin in sodium form,
before lyophilized into dry powder. Content of title material in
freeze dried powder was then determined by HPLC using absorbance at
260 nm, and glycyl sialic acid cytidine monophosphate as reference
material. For the HPLC analysis, a Waters X-Bridge phenyl column (5
.mu.m 4.6mm.times.250mm) and a water acetonitrile system (linear
gradient from 0-85% acetonitrile over 30 min containing 0.1%
phosphoric acid) was used. Yield: 61.6 mg (26%). LCMS: 732.18
(MH+); 427.14 (MH.sup.+-CMP). Compound was stable for extended
periods (>12 months) when stored at -80.degree. C.
Step 2
[0409] Synthesis of 38.8 kDa HEP-GSC Reagent with Succinimide
Linkage
##STR00011##
[0410] The HEP-GSC reagent was prepared by coupling GSC-SH
([(4-mercaptobutanoyl)glycyl]sialic acid cytidine monophosphate)
from step 1 with HEP-maleimide in a 1:1 molar ratio as follows:
GSC-SH (0.68 mg) dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (50
.mu.l) was added 35 mg of the 38.8 k-HEP-maleimide dissolved in 50
mM Hepes, 100 mM NaCl, pH 7.0 (1.35 ml). The clear solution was
left for 2 hours at 25.degree. C. Unreacted GSC-SH was removed by
dialysis using a Slide-A-Lyzer cassette (Thermo Scientific) with a
cut-off of 10 kD. The dialysis buffer was 50 mM Hepes, 100 mM NaCl,
10 mM CaCl.sub.2, pH 7.0. The reaction mixture was dialyzed twice
for 2.5 hours. The recovered material was used as such in step 4
below, assuming a quantitative reaction between GSC-SH and
HEP-maleimide. The HEP-GSC reagent made by this procedure will
contain a HEP polymer attached to sialic acid cytidine
monophosphate via a succinimide linkage.
Step 3
Desialylation of FVIIa L288F T293K
[0411] FVIIa L288F T293K (30 mg) was added sialidase (AUS, 100 ul,
20 U) in 10 mM His, 100 mM NaCl, 60 mM CaCl.sub.2, 10 mM PABA pH
5.9 (10 ml), and left for 1 hour at room temperature. The reaction
mixture was then diluted with 50 mM HEPES, 100 mM NaCl, 1 mM EDTA,
pH 7.0 (30 ml), and cooled on ice. 250 mM EDTA solution (2.6 ml)
was added in small portions, keeping pH at neutral by sodium
hydroxide addition. The EDTA treated sample was then applied to a
2.times.5 ml HiTrap Q FF ion-exchange columns (Amersham
Biosciences, GE Healthcare) equilibrated with 50 mM HEPES, 100 mM
NaCl, 1 mM EDTA, pH 7.0. Unbound protein was eluted with 50 mM
HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0 (4 CV), followed by 50 mM
HEPES, 150 mM NaCl, pH 7.0 (8 CV), before eluting asialo FVIIa
L288F T293K with 50 mM HEPES, 100 mM NaCl, 10 mM CaCl.sub.2, pH 7.0
(20 CV). Asialo FVIIa L288F T293K was isolated in 50 mM Hepes, 150
mM NaCl, 10 mM CaCl.sub.2, pH 7.0. Yield (19.15 mg) was determined
by quantifying the FVIIa L288F T293K light chain content against a
FVIIa standard after tris(2-carboxyethyl)phosphine reduction using
reverse phase HPLC.
Step 4
[0412] Synthesis of 38.8 kDa HEP-[N]-FVIIa L288F T293K with
Succinimide Linkage
[0413] To asialo FVIIa L288F T293K (19.2 mg) in 50 mM Hepes, 100 mM
NaCl, 10 mM CaCl.sub.2, 10 mM PABA, pH 7.0 (18.0 ml) was added 38.8
kDa-HEP-GSC (35 mg from step 2) in 50 mM Hepes, 100 mM NaCl, 10 mM
CaCl.sub.2, pH 7.0 (2.3 ml), and rat ST3GaIIII enzyme (5 mg; 1.1
unit/mg) in 20 mM Hepes, 120 mM NaCl, 50% glycerol, pH 7.0 (7.2
ml). The reaction mixture was incubated over night at 32.degree. C.
under slow rotation. The reaction mixture was then applied to a
FVIIa specific affinity column (CV=24 ml) modified with a
Gla-domain specific antibody and step eluted first with 2 column
volumes of buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl.sub.2, pH
7.4) then 2 column volumes of buffer B (50 mM Hepes, 100 mM NaCl,
10 mM EDTA, pH 7.4). The method essentially follows the principle
described by Thim, L et al. Biochemistry (1988) 27, 7785-779. The
product with unfolded Gla-domain was collected and directly applied
to a 2.times.5 ml HiTrap Q FF ion-exchange columns (Amersham
Biosciences, GE Healthcare). Column was washed with 10 mM His, 100
mM NaCl, 0.01% Tween80, pH 7.5 (3 column volumes), and 10 mM His,
100 mM NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 7.5 (for 3.5
column volume). The pH was then lowered to 6.0 with 10 mM His, 100
mM NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 6.0 (3 column
volumes), and the HEPylated material eluted with 5 column volumes
of a buffer mixture composed of 60% buffer A (10 mM His, 100 mM
NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 6.0) and 40% buffer B (10
mM His, 1 M NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 6.0). The
recovered asialo FVIIa L288F T293K (unmodified) was recycled, ie.
was HEPylated once more as described in step 4 and purified in the
same way as just described. The combined fractions from two
hepylation runs were pooled and concentrated by ultrafiltration
(Millipore Amicon Ultra, cut off 10 kD).
Step 5
[0414] Capping of Mono Glycoconjugated Heparosan 38.8k-HEP-R-FVIIa
L288F T293K
[0415] Non-sialylated N-glycanes of 38.8k-HEP-[N]-FVIIa L288F T293K
were finally capped (i.e. sialylated) with ST3GaIIII enzyme and
CMP-NAN as follows: 38.8k-HEP-[N]-FVIIa L288F T293K (5.85 mg) was
incubated with ST3GaIIII (0.18 mg/ml); CMP-NAN (4.98 mM) in 8.4 ml
of 10 mM His, 100 mM NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 6.0
for 1 h at 32.degree. C. The reaction mixture was then applied to a
FVIIa specific affinity column modified with a Gla-domain specific
antibody and step eluted first with 2 column volumes of buffer A
(50 mM Hepes, 100 mM NaCl, 10 mM CaCl.sub.2, pH 7.4) then 2 column
volumes of buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4).
The pooled fractions containing 38.8k-HEP-[N]-FVIIa L288F T293K
were combined and dialyzed using a Slide-A-Lyzer cassette (Thermo
Scientific) with a cut-off of 10 kD. The dialysis buffer was 10 mM
His, 100 mM NaCl, 10 mM CaCl.sub.2, 0.01% Tween80, pH 6.0. The
protein concentration was determined by light-chain HPLC analysis
after TCEP reduction. The overall yield of 38.8k-HEP-[N]-FVIIa
L288F T293K was 2.46 mg (13%).
Example 10
[0416] Preparation of 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A
Step 1
[0417] Synthesis of 41.5 kDa HEP-GSC Reagent with 4-Methylbenzoyl
Linkage
##STR00012##
[0418] Glycyl sialic acid cytidine monophosphate (GSC) (20 mg; 32
.mu.mol) in 5.0 ml 50 mM Hepes, 100 mM NaCl, 10 mM CaCl.sub.2
buffer, pH 7.0 was added 41.5 kDa HEP-benzaldehyde (99.7 mg; 2.5
.mu.mol). The mixture was gently rotated until all HEP-benzaldehyde
had dissolved. During the following 2 hours, a 1M solution of
sodium cyanoborohydride in MilliQ water was added in portions
(5.times.50 .mu.l), to reach a final concentration of 48 mM.
Reaction mixture was left at room temperature overnight. Excess of
GSC was then removed by dialysis as follows: the total reaction
volume (5250 pl) was transferred to a dialysis cassette
(Slide-A-Lyzer Dialysis Cassette, Thermo Scientific Prod No. 66810
with cut-off 10 kDa capacity: 3 -12 ml). Solution was dialysed for
2 hours against 2000 ml of 25 mM Hepes buffer (pH 7.2) and once
more for 17 h against 2000 ml of 25 mM Hepes buffer (pH 7.2).
Complete removal of excess GSC from inner chamber was verified by
HPLC on Waters X-Bridge phenyl column (4.6mm.times.250mm, 5 .mu.m)
and a water acetonitrile system (linear gradient from 0-85%
acetonitrile over 30 min containing 0.1% phosphoric acid) using GSC
as reference. Inner chamber material was collected and freeze dried
to give 41.5 kDa HEP-GSC as white powder. The HEP-GSC reagent was
analysed by NMR and on SEC chromatography. The HEP-GSC reagent made
by this procedure contains a HEP polymer attached to sialic acid
cytidine monophosphate via a 4-methylbenzoyl linkage.
Step 2
Desialylation of FVIIa L288F T293K K337A:
[0419] To a solution of FVIIa L288F T293K K337A (43.5 mg) in 21 ml
of 10 mM His, 100 mM NaCl, 60 mM CaCl.sub.2 , 10 mM PABA, pH 6.7
buffer was added sialidase (Arthrobacter ureafaciens, 9 units/ml).
The reaction mixture was incubated for 1 hour at room temperature.
The reaction mixture was then cooled on ice and added 14 ml of 10
mM His, 100 mM NaCl pH 7.7. 50 ml of a 100 mM EDTA solution was
then added while maintaining neutral pH. The reaction mixture was
then diluted with 50 ml of MilliQ water, and applied to 4.times.5
ml interconnected HiTrap Q FF ion-exchange columns (Amersham
Biosciences, GE Healthcare) equilibrated in 50 mM HEPES, 50 mM
NaCl, pH 7.0. Unbound protein including sialidase was eluted with 5
CV of 50 mM HEPES, 150 mM NaCl, pH 7.0. Desialylated protein was
eluted with 12 CV of 50 mM HEPES, 150 mM NaCl, 30 mM CaCl2, pH 7.0.
Fractions containing protein were combined and added 0.5 M PABA to
reach a final concentration of 10 mM. Protein yield was determined
by quantifying the FVIIa L288F T293K K337A light chain against a
FVIIa standard after tris(2-carboxyethyl)phosphine reduction using
reverse phase HPLC as described above. 32.5 mg asialo FVIIa L288F
T293K K337A (2.83 mg/ml) was in this way isolated in 11.5 ml of 50
mM Hepes, 150 mM NaCl, 30 mM CaCl.sub.2, 10 mM PABA, pH 7.0.
[0420] To asialo FVIIa L288F T293K K337A (16.3 mg) in 5.75 ml of 50
mM Hepes, 150 mM NaCl, 30 mM CaCl.sub.2, 10 mM PABA, pH 7.0 was
added 41.5 kDa HEP-GSC (3 equivalents, 41.5 mg) and rat ST3GaIIII
enzyme (2.93 mg; 1.1 unit/mg) in 4.2 ml of 20 mM Hepes, 120 mM
NaCl, 50% glycerol, pH 7.0. PABA concentration was then adjusted to
10 mM with a 0.5M aqueous PABA solution, and pH was adjusted to 6.7
with 1N NaOH. The reaction mixture was incubated overnight at
32.degree. C. under slow stirring. A solution 157 mM CMP-NAN in 50
mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0 (356 .mu.l) was then
added, and the reaction was incubated at 32.degree. C. for an
additional hour. HPLC analysis showed a product distribution
containing un-reacted FVIIa L288F T293K K337A (68%), mono HEPylated
FVIIa (25%) and various polyHEPylated forms (7%).
[0421] The entire reaction mixture was then applied to a FVIIa
specific affinity column (CV=24 ml) modified with a Gla-domain
specific antibody and step eluted first with 2 column volumes of
buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl.sub.2, pH 7.4) then
2 column volumes of buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA,
pH 7.4). The method essentially follows the principle described by
Thim, L et al. Biochemistry (1988) 27, 7785-779.
[0422] The products with unfolded Gla-domain was collected and
directly applied to 3.times.5ml interconnected HiTrap Q FF
ion-exchange columns (Amersham Biosciences, GE Healthcare)
equilibrated with a buffer containing 10 mM His, 100 mM NaCl, pH
6.0. The column was washed with 4 column volumes of 10 mM His, 100
mM NaCl, pH 6.0. Unmodified FVIIa L288F T293K K337A was eluted with
12 CV of 10 mM His, 100 mM NaCl, 10 mM CaCl.sub.2, pH 6.0 (elution
buffer A). 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was then eluted
with 15 CV of 10 mM His, 325 mM NaCl, 10 mM CaCl.sub.2, pH 6.0.
Pure fractions were combined, and protein concentration was
determined by HPLC quantification method previously described. 3.42
mg (21%) pure 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was
isolated. The combined fractions containing 41.5 kDa-HEP-[N]-FVIIa
L288F T293K K337A was finally dialyzed against 10 mM His, 100 mM
NaCl, 10 mM CaCl.sub.2, pH 6.0 using a Slide-A-Lyzer cassette
(Thermo Scientific) with a cut-off of 10 kD, and concentration
adjusted to (0.40 mg/ml) by adding dialysis buffer.
Example 11
Preparation of 41.5 kDa HEP-[N]-FVIIa W201 T293K
[0423] This material was prepared essentially as described in
example 10. FVIIa W201 R T293K (40 mg) was initially desialylated
and asialo FVIIa W201 R T293K (27.2 mg) was isolated by the
Gla-specific ion-exchange method. The desialylated analogue was
then incubated with 41.5 kDa HEP-GSC (produced as described in
example 10) and ST3GaIIII.
[0424] The conjugation product was then isolated by ion-exchange
chromatography. Final buffer exchange afforded 2.9 mg (7.5%) of
41.5 kDa HEP-[N]-FVIIa W201 T293K in 10 mM His, 100 mM NaCl, 10 mM
CaCl.sub.2, pH 6.0.
Example 12
Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293K
[0425] This material was prepared essentially as described in
example 10. FVIIa L288Y T293K (19.9 mg) was initially desialylated
and asialo FVIIa L288Y T293K (16.9 mg) was isolated by the
Gla-specific ion-exchange method. The desialylated analogue was
then incubated with 41.5 kDa HEP-GSC (produced as described in
example 10) and ST3GaIIII. The conjugation product was then
isolated by ion-exchange chromatography. Final buffer exchange
afforded 1.95 mg (11.5%) of 41.5 kDa HEP-[N]-FVIIa L288Y T293K in
10 mM His, 100 mM NaCl, 10 mM CaCl.sub.2, pH 6.0.
Example 13
Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293R
[0426] This material was prepared essentially as described in
example 10. After desialylation asialo FVIIa L288Y T293R (30 mg)
was reacted with 41.5 kDa HEP-GSC (produced as described in example
10) and ST3GaIIII. The conjugation product was then isolated by
ion-exchange chromatography. Final buffer exchange afforded 4.33 mg
(14.4%) of 41.5 kDa HEP-[N]-FVIIa L288Y T293R was obtained in 10 mM
His, 100 mM NaCl, 10 mM CaCl.sub.2, pH 6.0.
Example 14
Preparation of 41.5 kDa HEP-[N]-FVIIa T293K K337A
[0427] This material was prepared essentially as described in
example 10. After desialylation asialo FVIIa L288Y T293R (8 mg) was
reacted with 41.5 kDa HEP-GSC (produced as described in example 10)
and ST3GaIIII. The conjugation product was isolated by ion-exchange
chromatography. Final buffer exchange afforded 1.72 mg (15%) of
41.5 kDa HEP-[N]-FVIIa T293K K337A in 10 mM His, 100 mM NaCl, 10 mM
CaCl.sub.2, pH 6.0.
Example 15
Functional Properties of Modified Combination FVIIa Variants
[0428] FVIIa combination variants glycoconjugated to either PEG or
heparosan (HEP), as described in examples 9-14, were characterized
for proteolytic activity and antithrombin reactivity as described
in example 5. Results are summarized in
[0429] Table 7. These data show that chemical modifications of
FVIIa, in the cases with PEG or HEP, decreases FVIIa variant
proteolytic activity but for some variants allows to retain higher
than wild-type FVIIa proteolytic activity and further allows to
retain antithrombin resistance.
TABLE-US-00009 TABLE 7 Functional properties of modified FVIIa
variants. Results are shown in percent (%) of wild-type FVIIa.
Proteolytic Proteolytic activity + ATIII ATIII activity + sTF +
Inhibition + Inhibition + PS:PC PS:PC LMWH sTF IUPAC name (% WT) (%
WT) (% WT) (% WT) 40k-PEG-[N]-FVIIa W201R T293Y 266.9 88.8 5.9 1.9
40k-HEP-[N]-FVIIa W201R T293K 155.3 236.7 0.2 1.2 40k-HEP-[N]-FVIIa
L288Y T293R 407 200.3 5.2 4.4 40k-HEP-[N]-FVIIa L288Y T293K 264.9
147.4 3.7 2.9 40k-HEP-[N]-FVIIa L288F T293K 555.5 115 K337A
40K-HEP-[N]-FVIIa L288F T293K 279.3 116.4 4.4 3.6
Example 16
Evaluation in Haemophilia A-Like Whole Blood Thrombelastography
[0430] Thrombelastography (TEG) assay was chosen to evaluate
activity of FVIIa variants in a heamophilia A-like whole blood by
comparison to FVIIa. TEG assay provides a profile of the entire
coagulation process--initiation, propagation and final clot
strength measurements. Apart from the possible influence of shear
forces in flowing blood and the vasculature, TEG assay mimics the
in vivo conditions of coagulation as the method measures the visco
elastic properties of clotting whole blood (Viuff, E. et al,
Thrombosis Research, (2010), Vol. 126, pages 144-149). Each TEG
assay was initiated by using kaolin and TEG parameters clotting
time (R) and maximum thrombus generation rate (MTG) were recorded
and reported in
[0431] Table 8. The clotting time (R) denotes the latency time from
placing blood in the sample cup until the clot starts to form (2 mm
amplitude); whereas, the maximum thrombus generation (MTG) denotes
the velocity of clot formation. The clotting time (R) in seconds is
determined using standard TEG software; whereas, MTG is calculated
as the first derivative of the TEG track multiplied with 100
(100.times.mm/second).
[0432] Blood samples were obtained from normal, healthy donors who
were members of the Danish National Corps of Voluntary Blood Donors
and met the criteria for blood donation. Blood was sampled in 3.2%
citrate vacutainers (Vacuette ref. 455322, Greiner bio-one, Lot
A020601 2007-02) and assayed within 60 minutes. Haemophilia A-like
blood was prepared from normal human whole blood by addition of
anti-human FVIII (Sheep anti-human FVIII, Lot AA11-01, Haematologic
Technologies, VT, USA) antibody to a final concentration of 10
Bethesda Units (BU) per ml (final 0.1 mg/ml) and rotated gently at
2 rpm/min for 30 min at room temperature. The test compounds were
added at 0.1, 1, 10 and 25 nM final concentrations besides FVIIa
L288Y T293K that was tested in 0.069, 0.69, 6.9, 17.3 nM and FVIIa
W201R T293K that was tested in 0.076, 0.76, 7.6, 19.1 nM.
[0433] Data from the kaolin-induced TEG showed that all compounds
dose-dependently decreased clotting time (R-time) and increases
maximum thrombus generation (MTG) in haemophilia A-like blood
[0434] Table 8). All 40k-HEP-M-FVIIa-variants showed shorter or
similar clot time compared with FVIIa when evaluated in the highest
test concentration. Also maximum thrombus generation of the
variants was as similar or increased relative to FVIIa. Moreover,
the data showed that 40k-HEPylation of FVIIa variants reduced the
activity of the 40k-HEPylated compounds when compared to their
corresponding FVIIa variants (with no 40k-EPylation).
[0435] Table 8 shows the R-time (clot time) and MTG (maximum
thrombus generation) of test compounds in kaolin-induced TEG in
Haemophila A-like whole blood. The highest concentration of test
compound was 25 nM besides FVIIa L288Y T293K that was tested in
17.3 nM and FVIIa W201R T293K that was tested in 19.1 nM. FVIIa,
40k-PEG-[N]FVIIa and 40k-HEP-[N]-FVIIa was tested in four
individual donors (n=4) whereas the remaining compounds were tested
in two individual donors (n=2). Data in square brackets indicate
the range for the parameter from the four individual donors.
TABLE-US-00010 TABLE 8 Thromboelastography parameters for selected
FVIIa variants in Haemophilia A-like whole blood. R-time Test
compound mean MTG (at highest concentration) (sec) (.times.100
mm/sec) FVIIa 526 21.6 [480; 580] [19.4; 26.1] 40k-PEG-[N]-FVIIa
753 17.9 [680; 835] [16.1; 19.9] 40k-HEP-[N]-FVIIa 668 19.0 [580;
835] [15.8; 22.4] FVIIa L288Y T293K 345 24.5 [320; 370] [24.1;
24.8] 40k-HEP-[N]-FVIIa L288Y T293K 485 21.5 [465; 505] [20.3;
22.7] FVIIa L288Y T293R 313 25.5 [305; 320] [24.3; 26.8]
40k-HEP-[N]-FVIIa L288Y T293R 398 23.4 [370; 425] [23.1; 23.7]
FVIIa L288F T293K 400 25.9 [375; 425] [23.3; 28.5]
40k-HEP-[N]-FVIIa L288F T293K 498 21.6 [425; 570] [19.0; 24.1]
FVIIa L288F T293K K337A 280 27.2 [255; 305] [26.9; 27.6]
40k-HEP-[N]-FVIIa L288F T293K 348 25.7 K337A [335; 360] [23.4;
28.0] FVIIa W201R T293K 390 25.2 [335; 445] [22.4; 28.1]
40k-HEP-[N]-FVIIa W201R T293K 345 25.7 [295; 395] [23.7; 27; 7]
FVIIa T293K K337A 355 25.5 [350-360] [24.3; 26.6] 40k-HEP-[N]-FVIIa
T293K K337A 423 22.2 [420; 425] [21.7; 22.8]
Example 17
Assessment of PK in Rat
[0436] A pharmacokinetic analysis of identified FVIIa variants in
an unmodified form or glycoconjugated with either PEG or heparosan
(HEP) was performed in rats to assess their effect on the in vivo
survival of FVIIa. Sprague Dawley rats (three per group) were dosed
intravenously. Stabylite.TM. (TriniLize Stabylite Tubes; Tcoag
Ireland Ltd, Ireland) stabilized plasma samples were collected as
full profiles at appropriate time points and frozen until further
analysis. Plasma samples were analysed for clot activity (as
described in Example 7) and by an ELISA quantifying
FVIIa-antithrombin complexes. Pharmacokinetic analysis was carried
out by non-compartmental methods using Phoenix WinNonlin 6.0
(Pharsight Corporation). The following parameters were estimated:
Cmax (maximum concentration) of FVIIa-antithrombin complex, T1/2
(the functional terminal half-life) and MRT (the functional mean
residence time) for clot activity.
[0437] Briefly, FVIIa--antithrombin complexes were measured by use
of an enzyme immunoassay (EIA). A monoclonal anti-FVIIa antibody
that binds to the N-terminal of the EGF-domain and does not block
antithrombin binding is used for capture of the complex (Dako
Denmark A/S, Glostrup; product code 09572). A polyclonal anti-human
AT antibody peroxidase conjugate was used for detection (Siemens
Healthcare Diagnostics ApS, Ballerup/Denmark; product code OWMG15).
A preformed purified complex of human wild-type or variant FVIIa
and plasma-derived human antithrombin was used as standard to
construct EIA calibration curves. Plasma samples were diluted and
analysed and mean concentration of duplicate measurements
calculated. The intra-assay precision of the EIA was between
1-8%.
[0438] Pharmacokinetic estimated parameters are listed in Table 9.
Relative to wild-type FVIIa, the tested variants exhibited reduced
accumulation of FVIIa-antithrombin complexes (Rat AT complex) with
plasma levels approaching the detection level. Furthermore, a
significantly prolonged functional half-life of 40k-HEP-[N]-FVIIa
L288F T293K (18.4 hrs in rat) was observed compared to
40k-PEG-[N]-FVIIa (7.4 hrs in rat).
[0439] In conclusion, the presence of Lys at position 293 increases
the T1/2 in rat and reduces the FVIIa-antithrombin complex
formation. Furthermore, introduction of glycoconjugated heparason
substantially improves the T1/2 in rat.
TABLE-US-00011 TABLE 9 Pharmacokinetic estimated parameters for
selected FVIIa variants in rat. Rat AT complex T1/2 in rat MRT in
rat Cmax/dose FVIIa variant (hrs) (hrs) (kg/l) FVIIa 0.8 .+-. 0.01
1.1 .+-. 0.03 0.6 .+-. 0.08 40k-PEG-[N]-FVIIa 7.4 .+-. 0.20 8.3
.+-. 0.30 0.7 .+-. 0.05 40k-HEP-[N]-FVIIa L288Y T293K 15.9 .+-. 0.5
20.6 .+-. 1.0 0.04 .+-. 0.004 40k-HEP-[N]-FVIIa L288Y T293R 11.5
.+-. 0.5 13.9 .+-. 0.6 0.05 .+-. 0.004 FVIIa L288F T293K 1.2 .+-.
0.02 1.6 .+-. 0.30 0.07 .+-. 0.01 40k-HEP-[N]-FVIIa L288F T293K
18.4 .+-. 3.4 20.5 .+-. .6 0.04 .+-. 0.000 40k-HEP-[N]-FVIIa W201R
T293K 21.1 .+-. 0.5 24.8 .+-. 0.8 Not measured 40k-HEP-[N]-FVIIa
L288F T293K 11.0 .+-. 1.6 11.5 .+-. 0.8 0.14 .+-. 0.01 K337A
40k-HEP-[N]-FVIIa T293K K337A 12.4 .+-. 0.1 15.4 .+-. 0.3 0.05 .+-.
0.004 T1/2: Terminal half-life of the active molecule following IV
administration MRT: Mean residence time of the active molecule
following IV administration. AT complex Cmax/dose: Maximum measured
level of compound-antithrombin complex divided by the dose.
Example 18
[0440] Liquid Formulation of FVIIa L288Y T293K through Active Site
Stabilization
[0441] The stability of FVIIa in solution is limited by a number of
modifications to the polypeptide chain occurring as a result of
autoproteolysis, oxidation, deamidation, isomerization, etc.
Previous studies have identified three sites on the heavy chain
that are susceptible to autoproteolytic attack; these are
Arg290-Gly291, Arg315-Lys316, and Lys316-Va1317 (Nicolaisen et al.,
FEBS, 1993, 317:245-249). Calcium-free conditions further promote
proteolytic release of the first 38 residues of the light chain
encompassing the .gamma.-carboxyglutamic acid (Gla) domain.
[0442] Here we have used the small molecule, PCI-27483-S
(2-{2-[5-(6-Carbamimidoyl-1H-benzoimidazol-2-yl)-6,2'-dihydroxy-5'-sulfam-
oyl-biphenyl-3-yl]acetylamino}-succinic acid), which stabilize the
active site of FVIIa through non-covalent interactions and to
prevent autoproteolysis of the heavy chain in a liquid formulation
(See WO2014/057069 for further details on PCI-27483-S).
[0443] Quantification of heavy chain cleavage has been assessed by
analysis of reduced FVIIa L288Y T293K with reversed phase HPLC
(RP-HPLC). The assay solution was reduced in 127 mM dithiothreitol
(DTT) and 3 M guanidinium hydrochloride, which were incubated for
60.degree. C. in 15 min, followed by the addition of 1 .mu.L
concentrated acetic acid (per 50 .mu.L of original assay solution)
and cooling to 25.degree. C. 25 .mu.g reduced FVIIa L288Y T293K
were then injected on a ACE 3 .mu.M C4 column (300 .ANG.,
4.6.times.100 mm; Advanced Chromatography Technologies LTD,
Scotland) which were temperature equilibrated at 40.degree. C. The
protein fragments were separated with a linear gradient having a
mobile phase A consisting of 0.05% trifluoroacetic acid (TFA) in
water and going from 35-80% of mobile phase B consisting of 0.045%
TFA in 80% acetonitrile. The gradient time was 30 min with a flow
rate of 0.7 mL/min and peak elution were detected with absorbance
at 215 nm.
[0444] The formulation of FVIIa L288Y T293K were made up by 1.47
mg/mL CaCl.sub.2, 7.5 mg/mL NaCl, 1.55 mg/mL L-Histidine, 1.32
mg/mL Glycylglycine, 0.5 mg/mL L-Methionine, 0.07 mg/mL Polysorbate
80, 0.021 mg/ml PCI-27483-S, a protein concentration of 1 mg/ml
(i.e. a protein inhibitor mole ratio of 1:1.75) and a final pH of
6.8. The solution was incubated for 1 month at 30.degree. C. under
quiescent conditions and away from light. As seen in
[0445] Table 10 the presence of PCI-27483-S, led to near-complete
inhibition of heavy chain cleavage of FVIIa L288Y T293K; whereas,
no addition of PCI-27483-S led to a prominent increase in the
cleavage at the positions 315-316 and 290-291.
TABLE-US-00012 TABLE 10 Percentage increase of the peak areas
relative to day zero of heavy chain fragments corresponding to two
different cleavage sites as determined in the RP-HPLC chromatograms
upon 28 days of incubation with and without PCI-27483-S inhibitor.
Cleavage site With PCI-27483-S Without PCI-27483-S 315-316 20% 287%
290-291 17% 675%
Example 19
In Silico Assessment of Immunogenicity Risk
[0446] The in-silico study investigated whether the novel peptides
sequences that results from protein engineering to generate FVIIa
analogues could result in peptide sequences capable of binding to
major histocompatibility complex class II (MHC-II), also known as
HLA-II in humans. Such binding is pre-requisite for the presence of
T-cell epitopes. The peptide/HLA-II binding prediction software
used in this study was based on two algorithms, NetMHCIIpan 2.1
(Nielsen et al. 2010), performing HLA-DR predictions, and NetMHCII
2.2 (Nielsen et al. 2009) performing HLA-DP/DQ predictions.
[0447] An Immunogenicity Risk Score (IRS) was calculated in order
to be able to compare the different FVIIa analogues with regard to
immunogenicity risk potential. The calculation was performed as
follows: FVIIa wild-type was used as reference and only predicted
15 mers not in the reference (FVIIa wild-type) which had a
predicted Rank of 10 or less were included in the analysis. The
HLA-II alleles were classified into three classes: Class 1
(Rank.rarw.1) with weight of 2. Class 2 (1>Rank.rarw.3) with
weight of 0.5 and Class 3 (3>Rank.rarw.10) with weight of 0.2.
The class weight (2. 0.5 or 0.2) was multiplied by the allele
frequency (for each population) to give the IRS. The sum of IRS was
calculated for each population and each HLA-II (DRB1, DP and
DQ).
[0448] The calculated risk scores for select single and combination
variants are given in
[0449] Table 11. Particularly favourable combinations include
L288F/T293K, L288F/T293K/K337A, L288Y/T293K and L288Y/T293K/K337A
which at the same time exhibit a high proteolytic activity as well
as reduced susceptibility to inhibition by antithrombin.
TABLE-US-00013 TABLE 11 Calculated risk scores for select single
and combination FVIIa variants FVIIa variant Risk score FVIIa L288F
T293K 0.10 FVIIa L288F T293K K337A 0.10 FVIIa L288F T293K L305I
0.40 FVIIa L288F T293K L305V 0.35 FVIIa L288F T293R 0.12 FVIIa
L288F T293R K337A 0.12 FVIIa L288F T293R L305I 0.42 FVIIa L288F
T293R L305V 0.37 FVIIa L288F T293Y 0.32 FVIIa L288F T293Y K337A
0.32 FVIIa L288N T293K 0.06 FVIIa L288N T293R 0.08 FVIIa L288N
T293Y 0.26 FVIIa L288Y T293K 0.06 FVIIa L288Y T293K K337A 0.06
FVIIa L288Y T293R 0.09 FVIIa L288Y T293R K337A 0.09 FVIIa L305V
T293K 0.28 FVIIa L305V T293Y 0.49 FVIIa T293K K337A 0.02 FVIIa
T293K L305I 0.32 FVIIa T293K L305V K337A 0.28 FVIIa T293R K337A
0.06 FVIIa T293R L305I 0.36 FVIIa T293R L305V 0.32 FVIIa T293R
L305V K337A 0.32 FVIIa T293Y K337A 0.23 FVIIa T293Y L305V K337A
0.49 FVIIa W201K T293K 0.19 FVIIa W201K T293R 0.23 FVIIa W201K
T293Y 0.39 FVIIa W201M T293K 1.03 FVIIa W201M T293R 1.06 FVIIa
W201M T293Y 1.23 FVIIa W201R L288F T293K 0.32 FVIIa W201R L288F
T293R 0.34 FVIIa W201R T293K 0.25 FVIIa W201R T293K L305I 0.55
FVIIa W201R T293R 0.29 FVIIa W201R T293R L305I 0.58 FVIIa W201R
T293Y 0.45
Sequence CWU 1
1
91406PRTHomo sapiens 1Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro Gly
Ser Leu Glu Arg Glu 1 5 10 15 Cys Lys Glu Glu Gln Cys Ser Phe Glu
Glu Ala Arg Glu Ile Phe Lys 20 25 30 Asp Ala Glu Arg Thr Lys Leu
Phe Trp Ile Ser Tyr Ser Asp Gly Asp 35 40 45 Gln Cys Ala Ser Ser
Pro Cys Gln Asn Gly Gly Ser Cys Lys Asp Gln 50 55 60 Leu Gln Ser
Tyr Ile Cys Phe Cys Leu Pro Ala Phe Glu Gly Arg Asn 65 70 75 80 Cys
Glu Thr His Lys Asp Asp Gln Leu Ile Cys Val Asn Glu Asn Gly 85 90
95 Gly Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser Cys
100 105 110 Arg Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser
Cys Thr 115 120 125 Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile Pro Ile
Leu Glu Lys Arg 130 135 140 Asn Ala Ser Lys Pro Gln Gly Arg Ile Val
Gly Gly Lys Val Cys Pro 145 150 155 160 Lys Gly Glu Cys Pro Trp Gln
Val Leu Leu Leu Val Asn Gly Ala Gln 165 170 175 Leu Cys Gly Gly Thr
Leu Ile Asn Thr Ile Trp Val Val Ser Ala Ala 180 185 190 His Cys Phe
Asp Lys Ile Lys Asn Trp Arg Asn Leu Ile Ala Val Leu 195 200 205 Gly
Glu His Asp Leu Ser Glu His Asp Gly Asp Glu Gln Ser Arg Arg 210 215
220 Val Ala Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn
225 230 235 240 His Asp Ile Ala Leu Leu Arg Leu His Gln Pro Val Val
Leu Thr Asp 245 250 255 His Val Val Pro Leu Cys Leu Pro Glu Arg Thr
Phe Ser Glu Arg Thr 260 265 270 Leu Ala Phe Val Arg Phe Ser Leu Val
Ser Gly Trp Gly Gln Leu Leu 275 280 285 Asp Arg Gly Ala Thr Ala Leu
Glu Leu Met Val Leu Asn Val Pro Arg 290 295 300 Leu Met Thr Gln Asp
Cys Leu Gln Gln Ser Arg Lys Val Gly Asp Ser 305 310 315 320 Pro Asn
Ile Thr Glu Tyr Met Phe Cys Ala Gly Tyr Ser Asp Gly Ser 325 330 335
Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro His Ala Thr His Tyr 340
345 350 Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gly
Cys 355 360 365 Ala Thr Val Gly His Phe Gly Val Tyr Thr Arg Val Ser
Gln Tyr Ile 370 375 380 Glu Trp Leu Gln Lys Leu Met Arg Ser Glu Pro
Arg Pro Gly Val Leu 385 390 395 400 Leu Arg Ala Pro Phe Pro 405
2254PRTHomo sapiens 2Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu
Cys Pro Trp Gln Val 1 5 10 15 Leu Leu Leu Val Asn Gly Ala Gln Leu
Cys Gly Gly Thr Leu Ile Asn 20 25 30 Thr Ile Trp Val Val Ser Ala
Ala His Cys Phe Asp Lys Ile Lys Asn 35 40 45 Trp Arg Asn Leu Ile
Ala Val Leu Gly Glu His Asp Leu Ser Glu His 50 55 60 Asp Gly Asp
Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser 65 70 75 80 Thr
Tyr Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu 85 90
95 His Gln Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro
100 105 110 Glu Arg Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe
Ser Leu 115 120 125 Val Ser Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala
Thr Ala Leu Glu 130 135 140 Leu Met Val Leu Asn Val Pro Arg Leu Met
Thr Gln Asp Cys Leu Gln 145 150 155 160 Gln Ser Arg Lys Val Gly Asp
Ser Pro Asn Ile Thr Glu Tyr Met Phe 165 170 175 Cys Ala Gly Tyr Ser
Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser 180 185 190 Gly Gly Pro
His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly 195 200 205 Ile
Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val 210 215
220 Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg
225 230 235 240 Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe
Pro 245 250 3254PRTUnknownChimpanzee 3Ile Val Gly Gly Lys Val Cys
Pro Lys Gly Glu Cys Pro Trp Gln Val 1 5 10 15 Leu Leu Leu Val Asn
Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn 20 25 30 Thr Ile Trp
Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn 35 40 45 Trp
Arg Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu Ser Glu His 50 55
60 Asp Gly Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser
65 70 75 80 Thr Tyr Ile Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu
Arg Leu 85 90 95 His Gln Pro Val Val Leu Thr Asp His Val Val Pro
Leu Cys Leu Pro 100 105 110 Glu Arg Ala Phe Ser Glu Arg Thr Leu Ala
Phe Val Arg Phe Ser Leu 115 120 125 Val Ser Gly Trp Gly Gln Leu Leu
Asp Arg Gly Ala Thr Ala Leu Glu 130 135 140 Leu Met Val Leu Asn Val
Pro Arg Leu Met Thr Gln Asp Cys Leu Gln 145 150 155 160 Gln Ser Arg
Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe 165 170 175 Cys
Ala Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser 180 185
190 Gly Gly Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly
195 200 205 Ile Val Ser Trp Gly Gln Gly Cys Ala Ser Val Gly His Phe
Gly Val 210 215 220 Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln
Lys Leu Met Arg 225 230 235 240 Ser Glu Pro Arg Pro Gly Val Leu Leu
Arg Ala Pro Phe Pro 245 250 4254PRTUnknownDog 4Ile Val Gly Gly Lys
Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 Ala Val Lys
Val Asp Gly Lys Leu Leu Cys Gly Gly Thr Leu Ile Asp 20 25 30 Ala
Ala Trp Val Val Ser Ala Ala His Cys Phe Glu Arg Ile Lys Asn 35 40
45 Trp Lys Asn Leu Thr Val Val Leu Gly Glu His Asp Leu Ser Glu Asp
50 55 60 Asp Gly Asp Glu Gln Glu Arg His Val Ala Arg Val Ile Val
Pro Asp 65 70 75 80 Lys Tyr Ile Pro Leu Lys Thr Asn His Asp Ile Ala
Leu Leu His Leu 85 90 95 Arg Thr Pro Val Ala Tyr Thr Asp His Val
Val Pro Leu Cys Leu Pro 100 105 110 Glu Lys Thr Phe Ser Glu Arg Thr
Leu Ala Phe Ile Arg Phe Ser Ala 115 120 125 Val Ser Gly Trp Gly Arg
Leu Leu Asp Arg Gly Ala Lys Ala Arg Val 130 135 140 Leu Met Ala Ile
Gln Val Pro Arg Leu Met Thr Gln Asp Cys Leu Glu 145 150 155 160 Gln
Ala Arg Arg Arg Pro Gly Ser Pro Ser Ile Thr Asp Asn Met Phe 165 170
175 Cys Ala Gly Tyr Leu Asp Gly Ser Lys Asp Ala Cys Gln Gly Asp Ser
180 185 190 Gly Gly Pro His Ala Thr Lys Phe Gln Gly Thr Trp Tyr Leu
Thr Gly 195 200 205 Val Val Ser Trp Gly Glu Gly Cys Ala Ala Glu Gly
His Phe Gly Val 210 215 220 Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp
Leu Arg Gln Leu Met Val 225 230 235 240 Ser Ser His Thr Leu Arg Gly
Leu Leu Arg Ala Pro Leu Pro 245 250 5255PRTUnknownPorcine 5Ile Val
Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Ala 1 5 10 15
Met Leu Lys Leu Lys Gly Ala Leu Leu Cys Gly Gly Thr Leu Leu Asn 20
25 30 Thr Ser Trp Val Val Ser Ala Ala His Cys Phe Asp Arg Ile Arg
Ser 35 40 45 Trp Lys Asp Leu Thr Val Val Leu Gly Glu His Asp Leu
Ser Lys Asp 50 55 60 Glu Gly Asp Glu Gln Glu Arg Pro Val Ala Gln
Val Phe Val Pro Asp 65 70 75 80 Lys Tyr Val Pro Gly Lys Thr Asp His
Asp Leu Ala Leu Val Arg Leu 85 90 95 Ala Arg Pro Val Ala Leu Thr
Asp His Val Val Pro Leu Cys Leu Pro 100 105 110 Glu Arg Ser Phe Ser
Glu Arg Thr Leu Ala Phe Ile Arg Phe Ser Ala 115 120 125 Val Ser Gly
Trp Gly Arg Leu Leu Asp Arg Gly Ala Lys Ala Arg Val 130 135 140 Leu
Met Ala Ile Gln Val Pro Arg Leu Met Thr Gln Asp Cys Leu Glu 145 150
155 160 Gln Ala Arg Arg Arg Pro Gly Ser Pro Ser Ile Thr Asp Asn Met
Phe 165 170 175 Cys Ala Gly Tyr Leu Asp Gly Ser Lys Asp Ala Cys Lys
Gly Asp Ser 180 185 190 Gly Gly Pro His Ala Thr Arg Phe Arg Gly Thr
Trp Phe Leu Thr Gly 195 200 205 Val Val Ser Trp Gly Glu Gly Cys Ala
Ala Thr Gly Arg Phe Gly Val 210 215 220 Tyr Thr Arg Val Ser Arg Tyr
Thr Ala Trp Leu Leu Gly Leu Met Ser 225 230 235 240 Ala Pro Pro Pro
Pro Ser Glu Gly Leu Leu Arg Ala Pro Leu Pro 245 250 255
6255PRTUnknownBovine 6Ile Val Gly Gly His Val Cys Pro Lys Gly Glu
Cys Pro Trp Gln Ala 1 5 10 15 Met Leu Lys Leu Asn Gly Ala Leu Leu
Cys Gly Gly Thr Leu Val Gly 20 25 30 Pro Ala Trp Val Val Ser Ala
Ala His Cys Phe Glu Arg Leu Arg Ser 35 40 45 Arg Gly Asn Leu Thr
Ala Val Leu Gly Glu His Asp Leu Ser Arg Val 50 55 60 Glu Gly Pro
Glu Gln Glu Arg Arg Val Ala Gln Ile Ile Val Pro Lys 65 70 75 80 Gln
Tyr Val Pro Gly Gln Thr Asp His Asp Val Ala Leu Leu Gln Leu 85 90
95 Ala Gln Pro Val Ala Leu Gly Asp His Val Ala Pro Leu Cys Leu Pro
100 105 110 Asp Pro Asp Phe Ala Asp Gln Thr Leu Ala Phe Val Arg Phe
Ser Ala 115 120 125 Val Ser Gly Trp Gly Gln Leu Leu Glu Arg Gly Val
Thr Ala Arg Lys 130 135 140 Leu Met Val Val Leu Val Pro Arg Leu Leu
Thr Gln Asp Cys Leu Gln 145 150 155 160 Gln Ser Arg Gln Arg Pro Gly
Gly Pro Val Val Thr Asp Asn Met Phe 165 170 175 Cys Ala Gly Tyr Ser
Asp Gly Ser Lys Asp Ala Cys Lys Gly Asp Ser 180 185 190 Gly Gly Pro
His Ala Thr Arg Phe Arg Gly Thr Trp Phe Leu Thr Gly 195 200 205 Val
Val Ser Trp Gly Glu Gly Cys Ala Ala Ala Gly His Phe Gly Ile 210 215
220 Tyr Thr Arg Val Ser Arg Tyr Thr Ala Trp Leu Arg Gln Leu Met Gly
225 230 235 240 His Pro Pro Ser Arg Gln Gly Phe Phe Gln Val Pro Leu
Leu Pro 245 250 255 7253PRTMus musculus 7Ile Val Gly Gly Asn Val
Cys Pro Lys Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 Val Leu Lys Ile
Asn Gly Leu Leu Leu Cys Gly Ala Val Leu Leu Asp 20 25 30 Ala Arg
Trp Ile Val Thr Ala Ala His Cys Phe Asp Asn Ile Arg Tyr 35 40 45
Trp Gly Asn Ile Thr Val Val Met Gly Glu His Asp Phe Ser Glu Lys 50
55 60 Asp Gly Asp Glu Gln Val Arg Arg Val Thr Gln Val Ile Met Pro
Asp 65 70 75 80 Lys Tyr Ile Arg Gly Lys Ile Asn His Asp Ile Ala Leu
Leu Arg Leu 85 90 95 His Arg Pro Val Thr Phe Thr Asp Tyr Val Val
Pro Leu Cys Leu Pro 100 105 110 Glu Lys Ser Phe Ser Glu Asn Thr Leu
Ala Arg Ile Arg Phe Ser Arg 115 120 125 Val Ser Gly Trp Gly Gln Leu
Leu Asp Arg Gly Ala Thr Ala Leu Glu 130 135 140 Leu Met Ser Ile Glu
Val Pro Arg Leu Met Thr Gln Asp Cys Leu Glu 145 150 155 160 His Ala
Lys His Ser Ser Asn Thr Pro Lys Ile Thr Glu Asn Met Phe 165 170 175
Cys Ala Gly Tyr Met Asp Gly Thr Lys Asp Ala Cys Lys Gly Asp Ser 180
185 190 Gly Gly Pro His Ala Thr His Tyr His Gly Thr Trp Tyr Leu Thr
Gly 195 200 205 Val Val Ser Trp Gly Glu Gly Cys Ala Ala Ile Gly His
Ile Gly Val 210 215 220 Tyr Thr Arg Val Ser Gln Tyr Ile Asp Trp Leu
Val Arg His Met Asp 225 230 235 240 Ser Lys Leu Gln Val Gly Val Phe
Arg Leu Pro Leu Leu 245 250 8253PRTUnknownRat 8Ile Val Gly Gly Tyr
Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 Val Leu Lys
Phe Asn Glu Ala Leu Leu Cys Gly Ala Val Leu Leu Asp 20 25 30 Thr
Arg Trp Ile Val Thr Ala Ala His Cys Phe Asp Lys Phe Gly Lys 35 40
45 Leu Val Asn Ile Thr Val Val Leu Gly Glu His Asp Phe Ser Glu Lys
50 55 60 Glu Gly Thr Glu Gln Val Arg Leu Val Glu Gln Val Ile Met
Pro Asn 65 70 75 80 Lys Tyr Thr Arg Gly Arg Thr Asp His Asp Ile Ala
Leu Val Arg Leu 85 90 95 His Arg Pro Val Thr Phe Thr Asp Tyr Val
Val Pro Leu Cys Leu Pro 100 105 110 Glu Arg Ala Phe Ser Glu Asn Thr
Leu Ala Ser Ile Arg Phe Ser Arg 115 120 125 Val Ser Gly Trp Gly Gln
Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu 130 135 140 Leu Met Val Ile
Glu Val Pro Arg Leu Met Thr Gln Asp Cys Leu Glu 145 150 155 160 His
Ala Lys His Ser Ala Asn Thr Pro Arg Ile Thr Glu Asn Met Phe 165 170
175 Cys Ala Gly Tyr Met Asp Gly Thr Lys Asp Ala Cys Lys Gly Asp Ser
180 185 190 Gly Gly Pro His Ala Thr His Tyr His Gly Thr Trp Tyr Leu
Thr Gly 195 200 205 Val Val Ser Trp Gly Glu Gly Cys Ala Ala Ile Gly
His Ile Gly Val 210 215 220 Tyr Thr Arg Val Ser Gln Tyr Ile Asp Trp
Leu Val Lys Tyr Met Asp 225 230 235 240 Ser Lys Leu Arg Val Gly Ile
Ser Arg Val Ser Leu Leu 245 250 9253PRTUnknownRabbit 9Ile Val Gly
Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 Ala
Leu Met Asn Gly Ser Thr Leu Leu Cys Gly Gly Ser Leu Leu Asp 20 25
30 Thr His Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Leu Ser Ser
35 40 45 Leu Arg Asn Leu Thr Ile Val Leu Gly Glu His Asp Leu Ser
Glu His 50 55 60 Glu Gly Asp Glu Gln Val Arg His Val Ala Gln Leu
Ile Met Pro Asp 65 70 75 80 Lys Tyr Val Pro Gly Lys Thr Asp His Asp
Ile Ala Leu Leu Arg Leu 85 90 95 Leu Gln
Pro Ala Ala Leu Thr Asn Asn Val Val Pro Leu Cys Leu Pro 100 105 110
Glu Arg Asn Phe Ser Glu Ser Thr Leu Ala Thr Ile Arg Phe Ser Arg 115
120 125 Val Ser Gly Trp Gly Gln Leu Leu Tyr Arg Gly Ala Leu Ala Arg
Glu 130 135 140 Leu Met Ala Ile Asp Val Pro Arg Leu Met Thr Gln Asp
Cys Val Glu 145 150 155 160 Gln Ser Glu His Lys Pro Gly Ser Pro Glu
Val Thr Gly Asn Met Phe 165 170 175 Cys Ala Gly Tyr Leu Asp Gly Ser
Lys Asp Ala Cys Lys Gly Asp Ser 180 185 190 Gly Gly Pro His Ala Thr
Ser Tyr His Gly Thr Trp Tyr Leu Thr Gly 195 200 205 Val Val Ser Trp
Gly Glu Gly Cys Ala Ala Val Gly His Val Gly Val 210 215 220 Tyr Thr
Arg Val Ser Arg Tyr Thr Glu Trp Leu Ser Arg Leu Met Arg 225 230 235
240 Ser Lys Leu His His Gly Ile Gln Arg His Pro Phe Pro 245 250
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