U.S. patent application number 09/969358 was filed with the patent office on 2002-10-17 for factor vii glycoforms.
Invention is credited to Klausen, Niels Kristian, Pingel, Hans Kurt.
Application Number | 20020151471 09/969358 |
Document ID | / |
Family ID | 27439823 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151471 |
Kind Code |
A1 |
Pingel, Hans Kurt ; et
al. |
October 17, 2002 |
Factor VII glycoforms
Abstract
The present invention provides preparations of Factor VIIa
polypeptides or Factor VIIa-related polypeptides that exhibit
predetermined glycoform patterns. The preparations of the invention
exhibit improved functional properties and are useful for treating
Factor VII-mediated conditions.
Inventors: |
Pingel, Hans Kurt; (Farum,
DK) ; Klausen, Niels Kristian; (Gentofte,
DK) |
Correspondence
Address: |
Reza Green, Esq.
Novo Nordisk of North America, Inc.
Suite 6400
405 Lexington Avenue
New York
NY
10174-6401
US
|
Family ID: |
27439823 |
Appl. No.: |
09/969358 |
Filed: |
October 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60238944 |
Oct 10, 2000 |
|
|
|
60271581 |
Feb 26, 2001 |
|
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60276322 |
Mar 16, 2001 |
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Current U.S.
Class: |
514/1.5 ;
514/13.5; 514/13.6; 514/14.3; 514/20.9; 530/384 |
Current CPC
Class: |
A61K 38/4846 20130101;
G01N 2400/02 20130101; C12P 21/02 20130101; A61P 7/04 20180101;
C12Y 304/21021 20130101; A61K 38/00 20130101; Y10S 514/834
20130101; Y10S 514/802 20130101; C12N 9/6437 20130101 |
Class at
Publication: |
514/8 ;
530/384 |
International
Class: |
A61K 038/36; C07K
014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2000 |
DK |
PA 2000 01456 |
Feb 16, 2001 |
DK |
PA 2001 00262 |
Mar 14, 2001 |
DK |
PA 2001 00430 |
May 14, 2001 |
DK |
PA 2001 00751 |
Claims
What is claimed is:
1. A preparation comprising a plurality of Factor VII or Factor
VII-related polypeptides, wherein the polypeptides comprise
asparagine-linked oligosaccharide chains and wherein the
oligosaccharides exhibit a pattern selected from the group
consisting of: (a) between about 94-99% of the oligosaccharide
chains comprise at least one sialic acid moiety; (b) between about
1-7% of the oligosaccharide chains have a neutral charge; (c)
between about 6-16% of the oligosaccharide chains comprise at least
one terminal galactose residue; (d) between about 6-9% of the
oligosaccharide chains comprise at least one terminal
N-acetylgalactosamine residue; (e) between about 11-23% of the
oligosaccharide chains comprise at least one terminal galactose or
N-acetylgalactosamine residue; and (f) combinations of any of the
foregoing.
2. A preparation as defined in claim 1, wherein all sialic residues
in the oligosaccharide chains are linked to galactose via an
.alpha.2.fwdarw.3 linkage.
3. A preparation as defined in claim 1, wherein the sialic acid
residues comprise N-acetyl neuraminic acid (Neu5Ac) and N-glycol
neuraminic acid (Neu5Gc).
4. A preparation as defined in claim 1, wherein the
oligosaccharides comprise fucose linked .alpha.1.fwdarw.6 to a core
N-acetylglucosamine.
5. A preparation as defined in claim 1, wherein between about
87-92% of the oligosaccharide chains contain at least one sialic
acid residue.
6. A preparation as defined in claim 1, wherein between about 2-4%
of the oligosaccharide chains have a neutral charge.
7. A preparation as defined in claim 1, wherein between about 8-12%
of the oligosaccharide chains contain at least one terminal
galactose residue.
8. A preparation as defined in claim 1, wherein wherein between
about 7-8% of the oligosaccharide chains contain at least one
terminal N-acetylgalactosamine residue.
9. A preparation as defined in claim 1, wherein between about
12-18% of the oligosaccharide chains contain at least one terminal
galactose or N-acetylgalactosamine residue.
10. A preparation comprising a plurality of Factor VII or Factor
VII-related polypeptides, wherein the polypeptides comprise
asparagine-linked oligosaccharide chains and wherein (i) between
about 94-100% of the oligosaccharide chains comprise at least one
sialic acid moiety and (ii) the oligosaccharide chains comprise
fucose linked .alpha.1.fwdarw.6 to a core N-acetylglucosamine.
11. A preparation as defined in claim 1, wherein the Factor VII
polypeptides have the amino acid sequence of wild-type Factor
VII.
12. A preparation as defined in claim 1, wherein the Factor VII
polypeptides are selected from the group consisting of: S52A-Factor
VII, S60A-Factor VII, Factor VII that has been proteolytically
cleaved between residues 290 and 291; Factor VII that has been
proteolytically cleaved between residues 315 and 316; and Factor
VII that has been oxidized.
13. A preparation as defined in claim 1, wherein the Factor
VII-related polypeptides are selected from the group consisting of:
R152E-Factor VII, S344A-Factor VII, FFR-Factor VII, and Factor VIIa
lacking the Gla domain.
14. A preparation as defined in claim 1, wherein the polypeptides
are produced in a host cell selected from the group consisting of
fungal, insect, and vertebrate cells.
15. A preparation as defined in claim 14, wherein said host cell is
a mammalian cell.
16. A preparation as defined in claim 15, wherein said mammalian
cell is derived from a hamster.
17. A preparation as defined in claim 16, wherein said hamster cell
is selected from the group consisting of CHO cells and BHK
cells.
18. A preparation as defined in claim 1, wherein the preparation
exhibits a bioavailability that is at least about 110% of the
bioavailability of a reference preparation, wherein less than about
93% of the oligosaccharide chains in said reference preparation
comprise at least one sialic acid moiety.
19. A preparation as defined in claim 18, wherein the preparation
exhibits a bioavailability that is at least about 120% of the
bioavailability of the reference preparation.
20. A preparation as defined in claim 19, wherein the preparation
exhibits a bioavailability that is at least about 130% of the
bioavailability of the reference preparation.
21. A preparation as defined in claim 20, wherein the preparation
exhibits a bioavailability that is at least about 140% of the
bioavailability of the reference preparation.
22. A method for determining the glycoform pattern of Factor VII
and Factor VII-related polypeptides, said method comprising: (a)
culturing a cell expressing Factor VII or Factor VII-related
polypeptides under a first set of predetermined culture conditions;
(b) recovering Factor VII or Factor VII-related polypeptides from
the culture to obtain a preparation comprising the polypeptides;
and (c) analyzing the structure of the oligosaccharides linked to
the polypepides to determine the glycoform pattern of said
preparation.
23. A method as defined in claim 22, further comprising (d1)
altering the culture conditions of step (a) to achieve a second set
of predetermined culture conditions; (e1) repeating steps (b)-(d1)
until a desired glycoform pattern is achieved.
24. A method as defined in claim 22, further comprising: (d2)
treating the preparation chemically or enzymatically to alter the
oligosaccharide structure; and (e2) repeating steps (b)-(d2) until
a desired glycoform pattern is achieved.
25. A method for producing a preparation comprising Factor VII
polypeptides or Factor VII-related polypeptides having a
predetermined pattern of N-linked glycosylation, said method
comprising culturing a cell expressing the polypeptides under
conditions in which at least about 94% of the asparagines-linked
oligosaccharides present on the polypeptides comprise at least one
sialic acid residue.
26. A pharmaceutical formulation comprising a preparation as
defined in claim 1 and a pharmaceutically acceptable carrier or
adjuvant.
27. A method for treating or preventing a Factor VII-responsive
syndrome, said method comprising administering a pharmaceutical
formulation as defined in claim 26 to a patient in need of such
treatment, under conditions that result in a decrease in bleeding
and/or an increase in blood clotting, wherein said formulation
comprises Factor VII polypeptides.
28. A method as defined in claim 30, wherein said syndrome is
selected from the group consisting of haemophilia A, haemophilia B,
Factor XI deficiency, Factor VII deficiency, presence of a clotting
factor inhibitor, surgery, trauma, and anticoagulant therapy.
29. A method for preventing unwanted blood clotting, said method
comprising administering a pharmaceutical formulation as defined in
claim 26 to a patient in need of such treatment, under conditions
effective for inhibiting coagulation, wherein said formulation
comprises Factor VII-related polypeptides.
30. A method as defined in claim 29, wherein said unwanted blood
clotting is a result of a condition selected from the group
consisting of: angioplasty, deep vein thrombosis, pulmonary
embolism, stroke, disseminated intravascular coagulation (DIC),
fibrin deposition in lungs and kidneys associated with
gram-negative endotoxemia, and myocardial infarction.
31. A method for preventing tissue factor-mediated reactions, said
method comprising administering a pharmaceutical formulation as
defined in claim 26 to a patient in need of such treatment, under
conditions effective for said prevention, wherein said formulation
comprises Factor VII-related polypeptides.
32. A method as defined in claim 31, wherein said tissue-factor
mediated reaction is associated with a condition selected from the
group consisting of: Acute Respiratory Distress Syndrome (ARDS),
Systemic Inflammatory Response Syndrome (SIRS), Hemolytic Uremic
Syndrome (HUS), Multiple Organ Failure (MOF), and thrombocytopenia
purpura (TTP).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119 of
Danish application no. PA 2000 01456 filed on Oct. 2, 2000, and U.S
provisional application no. 60/238,944 filed on Oct. 10, 2000, the
contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions comprising
Factor VII and other blood clotting factors having altered patterns
of asparagine-linked glycosylation.
BACKGROUND OF THE INVENTION
[0003] The proteins involved in the clotting cascade, including,
e.g., Factor VII, Factor VIII, Factor IX, Factor X, and Protein C,
are proving to be useful therapeutic agents to treat a variety of
pathological conditions. Accordingly, there is an increasing need
for formulations comprising these proteins that are
pharmaceutically acceptable and exhibit a uniform and predetermined
clinical efficacy.
[0004] Because of the many disadvantages of using human plasma as a
source of pharmaceutical products, it is preferred to produce these
proteins in recombinant systems. The clotting proteins, however,
are subject to a variety of co- and post-translational
modifications, including, e.g., asparagine-linked (N-linked)
glycosylation; O-linked glycosylation; and .gamma.-carboxylation of
glu residues. These modifications may be qualitatively or
quantitatively different when heterologous cells are used as hosts
for large-scale production of the proteins. In particular,
production in heterologous cells often results in a different array
of glycoforms, which are identical polypeptides having different
covalently linked oligosaccharide structures.
[0005] In different systems, variations in the oligosaccharide
structure of therapeutic proteins have been linked to, inter alia,
changes in immunogenicity and in vivo clearance. Thus, there is a
need in the art for compositions and methods that provide clotting
protein preparations, particularly preparations comprising
recombinant human Factor VII, modified Factor VII, or Factor
VII-related polypeptides, that contain predetermined glycoform
patterns.
SUMMARY OF THE INVENTION
[0006] The present invention relates to preparations comprising
Factor VII polypeptides or Factor VII-related polypeptides that
exhibit predetermined glycoform patterns. As used herein, a Factor
VII or Factor VII-related preparation refers to a plurality of
Factor VII or Factor VII-related polypeptides, including variants
and chemically modified forms, as well as forms that have been
proteolytically activated (e.g., Factor VIIa), that have been
separated from the cell in which they were synthesized. A glycoform
pattern refers to the distribution within the preparation of
oligosaccharide chains having varying structures that are
covalently linked to Factor VII polypeptides or Factor VII-related
polypeptides.
[0007] In one aspect, the invention provides a preparation
comprising a plurality of Factor VII polypeptides or Factor
VII-related polypeptides, wherein the polypeptides comprise
asparagine-linked oligosaccharide chains and wherein one or more of
the following applies: (i) between about 94-100% of the
oligosaccharide chains comprise at least one sialic acid moiety;
(ii) between about 0-7% of the oligosaccharide chains have a
neutral charge; (iii) less than about 16%, such as, e.g., between
about 6-16% of the oligosaccharide chains comprise at least one
terminal galactose residue; (iv) less than about 25%, such as,
e.g., between about 6-9% of the oligosaccharide chains comprise at
least one terminal N-acetylgalactosamine residue; or (v) less than
about 30%, such as, e.g., between about 11-23% of the
oligosaccharide chains comprise at least one terminal galactose or
N-acetylgalactosamine residue. In some embodiments, in addition to
one or more of (i)-(v): all of the sialic acid residues in the
oligosaccharide chains are linked to galactose via an
.alpha.2.fwdarw.3 linkage; at least some of the sialic acid
residues comprise N-glycolylneuraminic acid (Neu5Gc) in addition to
N-acetyineuraminic acid (Neu5Ac); and/or the oligosaccharide chains
comprise fucose residues linked .alpha.1.fwdarw.6 to a core
N-acetylglucosamine. In one embodiment, the invention encompasses a
preparation comprising wild-type Factor VIIa in which between about
94-100% of the oligosaccharide chains have at least one sialic acid
residue and all of the sialic acid residues are linked to galactose
via an .alpha.2.fwdarw.3 linkage. In another embodiment, the
invention encompasses a preparation comprising wild-type Factor
VIIa in which between about 94-100% of the oligosaccharide chains
have at least one sialic acid residue and at least some of the
sialic acid residues are N-glycolylneuraminic acid. In yet another
embodiment, the invention encompasses a preparation comprising
wild-type Factor VIIa in which between about 94-100% of the
oligosaccharide chains have at least one sialic acid residue and at
least some of the chains contain N-acetylgalactosamine. The
preparations of the present invention thus do not encompass
wild-type Factor VII or Factor VIIa that has been isolated from
human plasma and has not been modified ex vivo by glycosidase
treatment.
[0008] In another aspect, the invention provides a preparation
comprising a plurality of Factor VII polypeptides or Factor
VII-related polypeptides, wherein the polypeptides comprise
asparagines-linked oligosaccharide chains and wherein at least
about 2% of the oligosaccharide chains contain at least one fucose
linked .alpha.1.fwdarw.3 to an antennary N-acetylglucosamine
residue (i.e., an N-acetylglucosamine residue that is linked
.alpha.1.fwdarw.2,4, or 6 to a Man residue). Preferably, at least
about 5% of the oligosaccharide chains contain at least one such
antennary fucose residue; more preferably, at least about 10% or
20%; and most preferably, at least about 40%.
[0009] The preparations according to invention may comprise one or
more of unmodified wild-type Factor VII; wild-type Factor VII that
has been subjected to chemical and/or enzymatic modification; and
Factor VII variants having one or more alterations in amino acid
sequence relative to wild-type Factor VII. The preparations of the
invention may be derived from human cells expressing Factor VII
from an endogenous Factor VII gene or from cells programmed to
express Factor VII or a Factor VII-related polypeptide from a
recombinant gene.
[0010] In another aspect, the invention provides preparations
comprising Factor VII or Factor VII-related polypeptides that
exhibit one or more improved functional properties, including,
without limitation, increased storage stability, bioavailability,
and/or half-life.
[0011] In another aspect, the invention encompasses methods for
determining and/or optimizing the glycoform pattern of Factor VII
and Factor VII-related polypeptides, which are carried out by the
steps of:
[0012] (a) culturing a cell expressing Factor VII or Factor
VII-related polypeptides under a first set of predetermined culture
conditions;
[0013] (b) recovering Factor VII or Factor VII-related polypeptides
from the culture to obtain a preparation comprising the
polypeptides; and
[0014] (c) analyzing the structure of the oligosaccharides linked
to the polypeptides to determine the glycoform pattern of the
preparation.
[0015] The methods may further comprise altering the culture
conditions of step (a) to achieve a second set of predetermined
culture conditions; and repeating the steps until a desired
glycoform pattern is achieved. Alternatively, the methods may
further comprise treating the preparation chemically or
enzymatically to alter the oligosaccharide structure; and repeating
the steps until a desired glycoform pattern is achieved.
Furthermore, the methods may comprise the additional steps of
subjecting preparations having predetermined glycoform patterns to
at least one test of bioactivity or other functionality (such as,
e.g., pharmacokinetic profile or stability), and correlating
particular glycoform patterns with particular bioactivity or
functionality profiles.
[0016] In another aspect, the invention provides methods for
producing a preparation comprising Factor VII polypeptides or
Factor VII-related polypeptides having a predetermined pattern of
N-linked glycosylation. In some embodiments, the methods are
carried out by culturing a cell expressing the polypeptides under
conditions in which at least about 94% of the asparagine-linked
oligosaccharides linked to the Factor VII polypeptides or Factor
VII-related polypeptides comprise at least one sialic acid residue,
e.g., one, two, three, or four sialic acid residues. In some
embodiments, the methods are carried out by culturing a cell
expressing the polypeptides under conditions in which at least
about 5% of the oligosaccharide chains contain at least one fucose
linked .alpha.1.fwdarw.3 to an antennary N-acetylglucosamine
residue. In some embodiments, Factor VII polypeptides or Factor
VII-related polypeptides, irrespective of their source, are
subjected to enzymatic treatments to achieve the desired glycoform
patterns.
[0017] In another aspect, the invention provides pharmaceutical
formulations comprising the preparations of the invention and
methods of preventing and/or treating syndromes that are responsive
to Factor VII polypeptides or Factor VII-related polypeptides. The
methods comprise administering the pharmaceutical formulations to a
patient in need of treatment, under conditions that result in
either an enhancement or inhibition in blood clotting. In one
series of embodiments, Factor VII preparations are administered
when it is desired to enhance blood clotting, such as, e.g., in
haemophilia A, haemophilia B, Factor XI deficiency, Factor VII
deficiency, thrombocytopenia, or von Willebrand's disease; in
syndromes accompanied by the presence of a clotting factor
inhibitor; before, during, or after surgery or anticoagulant
therapy; or after trauma. In another series of embodiments,
preparations of Factor VII-related polypeptides (i.e., preparations
having reduced or modified bioactivity relative to wild-type Factor
VII) are administered to reduce blood clotting, such as, e.g., in
patients undergoing angioplasty or those suffering from deep vein
thrombosis, pulmonary embolism, stroke, disseminated intravascular
coagulation (DIC), fibrin deposition in lungs and kidneys
associated with gram-negative endotoxemia, or myocardial
infarction. According to the invention, preparations of Factor
VII-related polypeptides may also be administered when it is
desired to modify, such as, e.g., reduce, intracellular signalling
via a tissue factor (TF)-mediated pathway, to treat conditions such
as, e.g., Acute Respiratory Distress Syndrome (ARDS), Systemic
Inflammatory Response Syndrome (SIRS), Hemolytic Uremic Syndrome
(HUS), Multiple Organ Failure (MOF), and thrombocytopenia purpura
(TTP).
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present inventors have discovered that preparations of
coagulation proteins having predetermined glycoform patterns
exhibit improved functional properties. Accordingly, the present
invention relates to methods and compositions that provide these
protein preparations. In particular, the invention relates to
preparations comprising Factor VII polypeptides and Factor
VII-related polypeptides having specific predetermined patterns of
asparagine-linked (N-linked) oligosaccharides. The preparations of
the invention exhibit altered properties, including, without
limitation, improved pharmacokinetic properties and improved
clinical efficacy. The invention also encompasses pharmaceutical
formulations that comprise these preparations, as well as
therapeutic methods that utilize the formulations.
[0019] Factor VII Polypeptides and Factor VII-Related
Polypeptides
[0020] The present invention encompasses human Factor VII
polypeptides, such as, e.g., those having the amino acid sequence
disclosed in U.S. Pat. No. 4,784,950 (wild-type Factor VII). As
used herein, "Factor VII" or "Factor VII polypeptide" encompasses
wild-type Factor VII, as well as variants of Factor VII exhibiting
substantially the same or improved biological activity relative to
wild-type Factor VII. The term "Factor VII" is intended to
encompass Factor VII polypeptides in their uncleaved (zymogen)
form, as well as those that have been proteolytically processed to
yield their respective bioactive forms, which may be designated
Factor VIIa. Typically, Factor VII is cleaved between residues 152
and 153 to yield Factor VIIa.
[0021] As used herein, "Factor VII-related polypeptides"
encompasses polypeptides, including variants, in which the Factor
VIIa biological activity has been substantially modified or reduced
relative to the activity of wild-type Factor VIIa. These
polypeptides include, without limitation, Factor VII or Factor VIIa
that has been chemically modified and Factor VII variants into
which specific amino acid sequence alterations have been introduced
that modify or disrupt the bioactivity of the polypeptide.
[0022] The biological activity of Factor VIIa in blood clotting
derives from its ability to (i) bind to tissue factor (TF) and (ii)
catalyze the proteolytic cleavage of Factor IX or Factor X to
produce activated Factor IX or X (Factor IXa or Xa, respectively).
For purposes of the invention, Factor VIIa biological activity may
be quantified by measuring the ability of a preparation to promote
blood clotting using Factor VII-deficient plasma and
thromboplastin, as described, e.g., in U.S. Pat. No. 5,997,864. In
this assay, biological activity is expressed as the reduction in
clotting time relative to a control sample and is converted to
"Factor VII units" by comparison with a pooled human serum standard
containing 1 unit/ml Factor VII activity. Alternatively, Factor
VIIa biological activity may be quantified by (i) measuring the
ability of Factor VIIa to produce of Factor Xa in a system
comprising TF embedded in a lipid membrane and Factor X. (Persson
et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) measuring
Factor X hydrolysis in an aqueous system (see, Example 5 below);
(iii) measuring its physical binding to TF using an instrument
based on surface plasmon resonance (Persson, FEBS Letts.
413:359-363, 1997) (iv) measuring hydrolysis of a synthetic
substrate (see, Example 4 below); and (v) measuring generation of
thrombin in a TF-independent in vitro system.
[0023] Factor VII variants having substantially the same or
improved biological activity relative to wild-type Factor VIIa
encompass those that exhibit at least about 25%, preferably at
least about 50%, more preferably at least about 75% and most
preferably at least about 90% of the specific activity of wild-type
Factor VIIa that has been produced in the same cell type, when
tested in one or more of a clotting assay, proteolysis assay, or TF
binding assay as described above. Factor VII variants having
substantially reduced biological activity relative to wild-type
Factor VIIa are those that exhibit less than about 25%, preferably
less than about 10%, more preferably less than about 5% and most
preferably less than about 1% of the specific activity of wild-type
Factor VIIa that has been produced in the same cell type when
tested in one or more of a clotting assay, proteolysis assay, or TF
binding assay as described above. Factor VII variants having a
substantially modified biological activity relative to wild-type
Factor VII include, without limitation, Factor VII variants that
exhibit TF-independent Factor X proteolytic activity and those that
bind TF but do not cleave Factor X.
[0024] Variants of Factor VII, whether exhibiting substantially the
same or better bioactivity than wild-type Factor VII, or,
alternatively, exhibiting substantially modified or reduced
bioactivity relative to wild-type Factor VII, include, without
limitation, polypeptides having an amino acid sequence that differs
from the sequence of wild-type Factor VII by insertion, deletion,
or substitution of one or more amino acids. Non-limiting examples
of Factor VII variants having substantially the same biological
activity as wild-type Factor VII include S52A-FVIIa, S60A-FVIIa
(lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa
variants exhibiting increased proteolytic stability as disclosed in
U.S. Pat. No. 5,580,560; Factor VIIa that has been proteolytically
cleaved between residues 290 and 291 or between residues 315 and
316 (Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); and
oxidized forms of Factor VIIa (Kornfelt et al., Arch. Biochem.
Biophys. 363:43-54, 1999). Non-limiting examples of Factor VII
variants having substantially reduced or modified biological
activity relative to wild-type Factor VII include R152E-FVIIa
(Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa (Kazama
et al., J. Biol. Chem. 270:66-72, 1995), FFR-FVIIa (Holst et al.,
Eur. J. Vasc. Endovasc. Surg. 15:515-520, 1998), and Factor VIIa
lacking the Gla domain, (Nicolaisen et al., FEBS Letts.
317:245-249, 1993). Non-limiting examples of chemically modified
Factor VII polypeptides and sequence variants are described, e.g.,
in U.S. Pat. No. 5,997,864.
[0025] Asparagine-Linked Glycosylation
[0026] The present invention provides preparations of Factor VII
polypeptides or Factor VII-related polypeptides that comprise a
particular spectrum of Factor VII glycoforms, i.e., Factor VII
polypeptides or Factor VII-related polypeptides having
predetermined patterns of asparagine-linked (N-linked)
oligosaccharide chains.
[0027] As used herein, a "pattern" of N-linked glycosylation or a
glycoform "pattern", "distribution", or "spectrum" refers to the
representation of particular oligosaccharide structures within a
given population of Factor VII polypeptides or Factor VII-related
polypeptides. Non-limiting examples of such patterns include the
relative proportion of oligosaccharide chains that (i) have at
least one sialic acid residue; (ii) lack any sialic acid residues
(i.e., are neutral in charge); (iii) have at least one terminal
galactose residue; (iv) have at least one terminal
N-acetylgalactosamine residue; (v) have at least one "uncapped"
antenna, i.e., have at least one terminal galactose or
N-acetylgalactosamine residue; or (vi) have at least one fucose
linked .alpha.1.fwdarw.3 to an antennary N-acetylglucosamine
residue.
[0028] As used herein, an oligosaccharide chain refers to the
entire oligosaccharide structure that is covalently linked to a
single asparagine residue. Factor VII is normally glycosylated at
Asn 145 and Asn 322. An N-linked oligosaccharide chain present on
Factor VII produced in a human in situ may be bi-, tri, or
tetraantennary, with each antenna having the structure
Neu5Ac(.alpha.2.fwdarw.3 or .alpha.2.fwdarw.6)Gal(.b-
eta.1.fwdarw.4) GlcNAc linked (.beta.1.fwdarw.2,4 or 6) to a Man
residue which is linked (.alpha.1.fwdarw.3 or 6) to
Man(.beta.1.fwdarw.4)GlcNAc(.- beta.1.fwdarw.4)GlcNAc-Asn. (Neu5Ac
signifies N-acetyineuraminic acid (sialic acid), Gal signifies
galactose, GlcNAc signifies N-acetylglucosamine, and Man signifies
mannose). The oligosaccharide chains may also comprise fucose
residues, which may be linked .alpha. 1.fwdarw.6 to GlcNAc. When
Factor VII is produced in a human in situ, some of the
oligosaccharide chains lack core fucose residues; all of the chains
lack antennary fucose residues; and all of the chains are almost
completely sialylated, i.e., the terminal sugar of each antenna is
N-acetylneuraminic acid linked to galactose via an
.alpha.2.fwdarw.3 or .alpha.2.fwdarw.6 linkage.
[0029] When produced in other circumstances, however, Factor VII
may contain oligosaccharide chains having different terminal
structures on one or more of their antennae, such as, e.g., lacking
sialic acid residues; containing N-glycolylneuraminic acid (Neu5Gc)
residues; containing a terminal N-acetylgalactosamine (GalNAc)
residue in place of galactose; and the like. When produced in,
e.g., BHK cells cultured in the presence of calf serum, Factor VII
preparations exhibit the following oligosaccharide patterns:
[0030] 87-93% of the oligosaccharide chains contain at least a
single sialic acid residue;
[0031] 7-13% are neutral (lack any sialic acid);
[0032] 9-16% contain at least one terminal galactose residue;
[0033] 19-29% contain at least one terminal N-acetylgalactosamine
residue; and
[0034] 30-39% contain at least one uncapped antenna, i.e., contain
at least one terminal galactose or N-acetylgalactosamine
residue.
[0035] The present inventors have produced Factor VII preparations
containing specific predetermined oligosaccharide patterns that
differ from those previously described. The present invention
encompasses preparations comprising Factor VII polypeptides or
Factor VII-related polypeptides exhibiting one or more of the
following glycoform patterns:
[0036] (i) Between about 94-100% of the oligosaccharide chains
contain at least one sialic acid residue, such as, e.g., between
about 94-99%, between about 95-98%, or between about 96-97%. In
different embodiments, at least about 94%, 95%, 96%, or 97% of the
oligosaccharide chains contain at least one sialic acid
residue.
[0037] (ii) 6% or less of the oligosaccharide chains are neutral,
such as, e.g., between about 1.5-6% or between about 2-4%.
[0038] (iii) Less than about 16%, preferably, less than about 10%
of the oligosaccharide chains contain at least one terminal
galactose, such as, e.g., between about 6-10% or between about
8-9%;
[0039] (iv) Less than about 25%, preferably, less than about 10% of
the oligosaccharide chains contain at least one terminal GaINAc
residue, such as, e.g., between about 6-9% or between about
7-8%;
[0040] (v) Less than about 30, preferably, less than about 25% of
the oligosaccharide chains contain at least one uncapped antenna,
such as, e.g., between about 11-23% or between about 12-18%;
and
[0041] (vi) At least about 2%, preferably, at least about 5%, more
preferably, at least about 10% or 20%; and most preferably, at
least about 40%, of the oligosaccharide chains contain at least one
fucose linked .alpha.1.fwdarw.3 to an antennary N-acetylglucosamine
residue (i.e., an N-acetylglucosamine residue that is linked
.beta.1.fwdarw.2,4, or 6 to a Man residue).
[0042] It will be understood that each of (i)-(vi) may represent a
distinct glycoform pattern that is encompassed by the present
invention, i.e., a preparation according to the invention may be
described by only one of (i)-(vi). Alternatively, depending on the
particular glycoform pattern, a preparation encompassed by the
invention may be described by more than one of (i)-(vi).
[0043] Furthermore, a preparation encompassed by the invention may
be described by one or more of (i)-(vi) in combination with one or
more other structural features. For example, the invention
encompasses preparations comprising Factor VII polypeptides or
Factor VII-related polypeptides in which the sialic acid residues
(Neu5Ac or Neu5Gc) are linked to galactose exclusively in an
.alpha.2.fwdarw.3 configuration. The invention also encompasses
preparations comprising Factor VII polypeptides or Factor
VII-related polypeptides that contain fucose linked .alpha.
1.fwdarw.6 to a core N-acetylglucosamine and/or fucose linked
.alpha.1.fwdarw.3 to an antennary N-acetylglucosamine. In one
series of embodiments, the preparations of the invention encompass
Factor VII or Factor VII-related polypeptides in which more than
99% of the oligosaccharide chains containg least one sialic acid
residue and (a) the sialic acid residues are linked exclusively in
an .alpha.2.fwdarw.3 configuration and/or (b) there are fucose
residues linked to core N-acetylglucosamines and/or (c) a
detectable number of antenna terminate in N-acetylgalactosamine. In
one embodiment, the invention encompasses preparations comprising
wild-type Factor VIIa in which more than 99% of the oligosaccharide
chains contain at least one sialic acid residue and the sialic acid
residues are linked to galactose exclusively in an
.alpha.2.fwdarw.3 configuration. In another embodiment, the
invention encompasses preparations comprising wild-type Factor VIIa
in which more than 99% of the oligosaccharide chains contain at
least one sialic acid residue and at least some of the
oligosaccharide chains comprise N-acetylgalactosamine. The present
invention does not encompass wild-type Factor VII or wild-type
Factor VIIa that is isolated from human plasma and is not modified
ex vivo by treatment with glycosidases.
[0044] In one embodiment, the Factor VIIa preparation comprises
oligosaccharide chains having a single fucose linked
.alpha.1.fwdarw.3 to one antennary N-acetylglucosamine. In another
embodiment, the Factor VIIa preparation comprises oligosaccharide
chains having fucose residues linked .alpha.1.fwdarw.3 to each
antennary N-acetylglucosamine of a biantennary oligosaccharide
(Sialyl Lewis X structure). In another embodiment, the Factor VIIa
preparation comprises oligosaccharide chains having (i) a fucose
linked to a core N-acetylglucosamine and (ii) a single fucose
linked .alpha.1.fwdarw.3 to one antennary N-acetylglucosamine. In
another embodiment, the Factor VIIa preparation comprises
oligosaccharide chains having (i) a fucose linked to a core
N-acetylglucosamine and (ii) fucose residues linked
.alpha.1.fwdarw.3 to each antennary N-acetylglucosamine of a
biantennary oligosaccharide.
[0045] In practicing the present invention, the pattern of N-linked
oligosaccharides may be determined using any method known in the
art, including, without limitation: high-performance liquid
chromatography (HPLC); capillary electrophoresis (CE); nuclear
magnetic resonance (NMR); mass spectrometry (MS) using ionization
techniques such as fast-atom bombardment, electrospray, or
matrix-assisted laser desorption (MALDI); gas chromatography (GC);
and treatment with exoglycosidases in conjunction with
anion-exchange (AIE)-HPLC, size-exclusion chromatography (SEC), or
MS. See, e.g., Weber et al., Anal. Biochem. 225:135 (1995); Klausen
et al., J. Chromatog. 718:195 (1995); Morris et al., in Mass
Spectrometry of Biological Materials, McEwen et al., eds., Marcel
Dekker, (1990), pp 137-167; Conboy et al., Biol. Mass Spectrom.
21:397, 1992; Hellerqvist, Meth. Enzymol. 193:554 (1990); Sutton et
al., Anal. Biohcem. 318:34 (1994); Harvey et al., Organic Mass
Spectrometry 29:752 (1994).
[0046] Following resolution of Factor VII-derived oligosaccharide
chains using any of the above methods (or any other method that
resolves oligosaccharide chains having different structures), the
resolved species are assigned, e.g., to one of groups (i)-(v). The
relative content of each of (i)-(v) is calculated as the sum of the
oligosaccharides assigned to that group relative to the total
content of oligosaccharide chains in the sample.
[0047] For example, using AIE-HPLC, 13 or more N-linked
oligosaccharide peaks can be resolved from a recombinant Factor VII
preparation produced in BHK cells. See, e.g., Klausen et al., Mol.
Biotechnol. 9:195, 1998. Five of the peaks (designated 1-5 in
Klausen et al.) do not contain sialic acid, while eight of the
peaks (designated 6, 7, and 10-15) do contain sialic acid.
[0048] It will be understood that, in a given analysis, the number
and distribution of sialic acid-containing and sialic acid-lacking
chains may depend upon (a) the polypeptide being expressed; (b) the
cell type and culture conditions; and (c) the method of analysis
that is employed, and that the resulting patterns may vary
accordingly.
[0049] In any case, once the sialic acid-containing
oligosaccharides have been resolved from the non-sialic
acid-containing oligosaccharides, conventional data analysis
programs are used to calculate the area under each peak; the total
peak area; and the percentage of the total peak area represented by
a particular peak. In this manner, for a given preparation, the sum
of the areas of sialic acid-containing peaks/total peak area X 100
yields the % sialylation value for the preparation according to the
present invention (i.e., the proportion of oligosaccharide chains
having at least one sialic acid residue). In a similar manner, the
% of chains having no sialic acid or at least one galactose or
N-acetylglucosamine can be calculated.
[0050] Methods for Producing Factor VII Preparations Having a
Predetermined Pattern of N-linked Oligosaccharides
[0051] Preparations of Factor VII, Factor VII variants, or Factor
VII-related polypeptides, each having a predetermined pattern of
N-linked oligosaccharides, may be produced using any appropriate
host cell that expresses Factor VII or Factor VII-related
polypeptides.
[0052] Host cells: In some embodiments, the host cells are human
cells expressing an endogenous Factor VII gene. In these cells, the
endogenous gene may be intact or may have been modified in situ, or
a sequence outside the Factor VII gene may have been modified in
situ to alter the expression of the endogenous Factor VII gene. Any
human cell capable of expressing an endogenous Factor VII gene may
be used.
[0053] In other embodiments, heterologous host cells are programmed
to express human Factor VII from a recombinant gene. The host cells
may be vertebrate, insect, or fungal cells. Preferably, the cells
are mammalian cells capable of the entire spectrum of mammalian
N-linked glycosylation; O-linked glycosylation; and
.gamma.-carboxylation. See, e.g., U.S. Pat. No. 4,784,950.
Preferred mammalian cell lines include the CHO (ATCC CCL 61), COS-1
(ATCC CRL 1650), baby hamster kidney (BHK) and HEK293 (ATCC CRL
1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A
preferred BHK cell line is the tk.sup.- ts13 BHK cell line
(Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110,
1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell
line is available from the American Type Culture Collection, 12301
Parklawn Dr., Rockville, Md. 20852, under ATCC accession number CRL
10314. A tk.sup.- ts13 BHK cell line is also available from the
ATCC under accession number CRL 1632. In addition, a number of
other cell lines may be used, including Rat Hep I (Rat hepatoma;
ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK
(ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1)
and DUKX cells (CHO cell line) (Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA 77:4216-4220, 1980). (DUKX cells also referred to as
CXB11 cells), and DG44 (CHO cell line) (Cell, 33:405, 1983, and
Somatic Cell and Molecular Genetics 12:555, 1986). Also useful are
3T3 cells, Namalwa cells, myelomas and fusions of myelomas with
other cells. In a particularly preferred embodiment, the host cells
are BHK 21 cells that have been adapted to grow in the absence of
serum and have been programmed to express Factor VII. In some
embodiments, the cells may be mutant or recombinant cells that
express a qualitatively or quantitatively different spectrum of
glycosylation enzymes (such as, e.g., glycosyl transferases and/or
glycosidases) than the cell type from which they were derived. The
cells may also be programmed to express other heterologous peptides
or proteins, including, e.g., truncated forms of Factor VII. In one
embodiment, the host cells are CHO cells that have been programmed
to co-express both the Factor VII polypeptide of interest (i.e.,
Factor VII or a Factor-VII-related polypeptide) and another
heterologous peptide or polypeptide such as, e.g., a modifying
enzyme or a Factor VII fragment.
[0054] Methods: The present invention encompasses methods for
producing a preparation comprising any of the glycoform patterns
described above as (i)-(vi) and, in further embodiments, methods
for optimizing the glycoform distribution of Factor VII and Factor
VII-related polypeptides. These methods are carried out by the
steps of:
[0055] (a) culturing a cell expressing Factor VII or Factor
VII-related polypeptides under a first set of predetermined culture
conditions;
[0056] (b) recovering Factor VII or FactorVII-related polypeptides
from the culture to obtain a preparation comprising the
polypeptides; and
[0057] (c) analyzing the structure of the oligosaccharides linked
to the polypeptides to determine a glycoform pattern.
[0058] The methods may further comprise:
[0059] (d1) altering the culture conditions of step (a) to achieve
a second set of predetermined culture conditions;
[0060] (e1) repeating steps (b)-(d2) until a desired glycoform
pattern is achieved.
[0061] Alternatively, the methods may further comprise
[0062] (d2) treating the preparation chemically and/or
enzymatically to alter the oligosaccharide structure; and
[0063] (e2) repeating steps (b)-(d2) until a desired glycoform
pattern is achieved.
[0064] These methods may further comprise the step of subjecting
preparations having predetermined glycoform patterns to at least
one test of bioactivity (including, e.g., clotting, Factor X
proteolysis, or TF binding) or other functionality (such as, e.g.,
pharmacokinetic profile or stability), and correlating particular
glycoform patterns with particular bioactivity or functionality
profiles in order to identify a desired glycoform pattern.
[0065] The variables in the culture conditions that may be altered
in step (d1) include, without limitation: the cell of origin, such
as, e.g., a cell derived from a different species than originally
used; or a mutant or recombinant cell having alterations in one or
more glycosyltransferases or glycosidases or other components of
the glycosylation apparatus (see, Grabenhorst et al.,
Glycoconjugate J. 16:81, 1999; Bragonzi et al., Biochem. Biophys.
Acta 1474:273, 2000; Weikert, Nature Biotechnol. 17:1116, 1999);
the level of expression of the polypeptide; the metabolic
conditions such as, e.g., glucose or glutamine concentration; the
absence or presence of serum; the concentration of vitamin K;
protein hydrolysates, hormones, trace metals, salts as well as
process parameters like temperature, dissolved oxygen level and
pH.
[0066] The enzymatic treatments that may be used in step (d2) to
modify the oligosaccharide pattern of a preparation include,
without limitation, treatment with one or more of sialidase
(neuraminidase), galactosidase, fucosidase; galactosyl transferase,
fucosyl transferase, and/or sialyltransferase, in a sequence and
under conditions that achieve a desired modification in the
distribution of oligosaccharide chains having particular terminal
structures. Glycosyl transferases are commercially available from
Calbiochem (La Jolla, Calif.) and glycosidases are commercially
available from Glyko, Inc., (Novato, Calif.).
[0067] In one series of embodiments, host cells expressing Factor
VII or a related polypeptide are subjected to specific culture
conditions in which they secrete glycosylated Factor VII
polypeptides having the desired pattern of oligosaccharide
structures described above as any of (i)-(vi). Such culture
conditions include, without limitation, a reduction in, or complete
absence of, serum. Preferably, the host cells are adapted to grow
in the absence of serum and are cultured in the absence of serum
both in the growth phase and in the production phase. Such
adaptation procedures are described, e.g., in Scharfenberg, et al.,
Animal Cell Technology Developments towards the 21.sup.st Century,
E. C. Beuvery et al. (Eds.), Kluwer Academic Publishers, pp.
619-623, 1995 (BHK and CHO cells); Cruz, Biotechnol. Tech.
11:117-120, 1997 (insect cells); Keen, Cytotechnol. 17:203-211,
1995 (myeloma cells); Berg et al., Biotechniques 14:972-978, 1993
(human kidney 293 cells). In a preferred embodiment, the growth
medium that is added to the cells contains no protein or other
component that was isolated from an animal tissue or an animal cell
culture. See, e.g., Example 1 below. Typically, in addition to
conventional components, a medium suitable for producing Factor VII
contains Vitamin K at a concentration between 0.1-50 mg/liter,
which is required for .gamma.-carboxylation of glutamine residues
in Factor VII.
[0068] In another series of embodiments, the glycoforms of the
invention are produced by subjecting a preparation of Factor VII or
Factor VII-related polypeptides to enzymatic and/or chemical
modification of the N-linked oligosaccharides contained
therein.
[0069] Factor VII Preparations
[0070] As used herein, a "Factor VII preparation" refers to a
plurality of Factor VII polypeptides, Factor VIIa polypeptides, or
Factor VII-related polypeptides, including variants and chemically
modified forms, that have been separated from the cell in which
they were synthesized.
[0071] Separation of polypeptides from their cell of origin may be
achieved by any method known in the art, including, without
limitation, removal of cell culture medium containing the desired
product from an adherent cell culture; centrifugation or filtration
to remove non-adherent cells; and the like.
[0072] Optionally, Factor VII polypeptides may be further purified.
Purification may be achieved using any method known in the art,
including, without limitation, affinity chromatography, such as,
e.g., on an anti-Factor VII antibody column (see, e.g., Wakabayashi
et al., J. Biol. Chem. 261:11097, 1986; and Thim et al., Biochem.
27:7785, 1988); hydrophobic interaction chromatography;
ion-exchange chromatography; size exclusion chromatography;
electrophoretic procedures (e.g., preparative isoelectric focusing
(IEF), differential solubility (e.g., ammonium sulfate
precipitation), or extraction and the like. See, generally, Scopes,
Protein Purification, Springer-Verlag, New York, 1982; and Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989. Following purification, the preparation preferably
contains less than about 10% by weight, more preferably less than
about 5% and most preferably less than about 1%, of non-Factor VII
proteins derived from the host cell.
[0073] Factor VII and Factor VII-related polypeptides may be
activated by proteolytic cleavage, using Factor XIIa or other
proteases having trypsin-like specificity, such as, e.g., Factor
IXa, kallikrein, Factor Xa, and thrombin. See, e.g., Osterud et
al., Biochem. 11:2853 (1972); Thomas, U.S. Pat. No. 4,456,591; and
Hedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively,
Factor VII may be activated by passing it through an ion-exchange
chromatography column, such as Mono Q.RTM. (Pharmacia) or the like.
The resulting activated Factor VII may then be formulated and
administered as described below.
[0074] Functional Properties of Factor VII Preparations
[0075] The preparations of Factor VII polypeptides and Factor
VII-related polypeptides having predetermined oligosaccharide
patterns according to the invention exhibit improved functional
properties relative to reference preparations. The improved
functional properties may include, without limitation, a) physical
properties such as, e.g., storage stability; b) pharmacokinetic
properties such as, e.g., bioavailability and half-life; and c)
immunogenicity in humans.
[0076] A reference preparation refers to a preparation comprising a
polypeptide that is identical to that contained in the preparation
of the invention to which it is being compared (such as, e.g.,
wild-type Factor VII or a particular variant or chemically modified
form) except for exhibiting a different pattern of
asparagine-linked glycosylation. For example, reference
preparations typically comprise one or more of the following
glycoform patterns: (i) less than about 93% (such as, e.g. less
than about 92% or less than about 90%) of the oligosaccharide
chains contain at least one sialic acid residue; (ii) at least
about 6% (such as, e.g., at least about 7.5% or at least about 10%)
of the oligosaccharide chains lack any sialic acid (i.e., are
neutral); (iii) at least about 10% (such as, e.g., at least about
12.5% or at least about 15%) of the oligosaccharide chains contain
at least one terminal galactose residue; (iv) at least about 15%
(such as, e.g., at least about 20% or at least about 25%) of the
oligosaccharide chains contain at least one terminal
N-acetylgalactosamine residue; (v) at least about 25% (such as,
e.g., at least about 30% or at least about 35%) of the
oligosaccharide chains contain at least one uncapped antenna (i.e.,
contain at least one terminal galactose or N-acetylgalactosamine
residue); or (vi) essentially undetectable levels (such as, e.g.,
less than about 0.2%) of antennary fucose residues.
[0077] Storage stability of a Factor VII preparation may be
assessed by measuring (a) the time required for 20% of the
bioactivity of a preparation to decay when stored as a dry powder
at 25.degree. C and/or (b) the time required for a doubling in the
proportion of Factor VIIa aggregates in the preparation.
[0078] In some embodiments, the preparations of the invention
exhibit an increase of at least about 30%, preferably at least
about 60% and more preferably at least about 100%, in the time
required for 20% of the bioactivity to decay relative to the time
required for the same phenomenon in a reference preparation, when
both preparations are stored as dry powders at 25.degree. C.
Bioactivity measurements may be performed using any of a clotting
assay, proteolysis assay, TF-binding assay, or TF-independent
thrombin generation assay.
[0079] In some embodiments, the preparations of the invention
exhibit an increase of at least about 30%, preferably at least
about 60%, and more preferably at least about 100%, in the time
required for doubling of aggregates relative to a reference
preparation, when both preparations are stored as dry powders at
25.degree. C. The content of aggregates is determined by gel
permeation HPLC on a Protein Pak 300 SW column (7.5.times.300 mm)
(Waters, 80013) as follows. The column is equilibrated with Eluent
A (0.2 M ammonium sulfate, 5% isopropanol, pH adjusted to 2.5 with
phosphoric acid, and thereafter pH is adjusted to 7.0 with
triethylamine), after which 25 .mu.g of sample is applied to the
column. Elution is with Eluent A at a flow rate of 0.5 ml/min for
30 min, and detection is achieved by measuring absorbance at 215
nm. The content of aggregates is calculated as the peak area of the
Factor VII aggregates/total area of Factor VII peaks (monomer and
aggregates).
[0080] "Bioavailability" refers to the proportion of an
administered dose of a Factor VII or Factor VII-related preparation
that can be detected in plasma at predetermined times after
administration. Typically, bioavailability is measured in test
animals by administering a dose of between about 25-250 .mu.g/kg of
the preparation; obtaining plasma samples at predetermined times
after administration; and determining the content of Factor VII or
Factor VII-related polypeptides in the samples using one or more of
a clotting assay (or any bioassay), an immunoassay, or an
equivalent. The data are typically displayed graphically as [Factor
VII] v. time and the bioavailability is expressed as the area under
the curve (AUC). Relative bioavailability of a test preparation
refers to the ratio between the AUC of the test preparation and
that of the reference preparation.
[0081] In some embodiments, the preparations of the present
invention exhibit a relative bioavailability of at least about
110%, preferably at least about 120%, more preferably at least
about 130% and most preferably at least about 140% of the
bioavailability of a reference preparation. The bioavailability may
be measured in any mammalian species, preferably dogs, and the
predetermined times used for calculating AUC may encompass
different increments from 10 min-8 h.
[0082] "Half-life" refers to the time required for the plasma
concentration of Factor VII polypeptides of Factor VII-related
polypeptides to decrease from a particular value to half of that
value. Half-life may be determined using the same procedure as for
bioavailability. In some embodiments, the preparations of the
present invention exhibit an increase in half-life of at least
about 0.25 h, preferably at least about 0.5 h, more preferably at
least about 1 h, and most preferably at least about 2 h, relative
to the half-life of a reference preparation.
[0083] "Immunogenicity" of a preparation refers to the ability of
the preparation, when administered to a human, to elicit a
deleterious immune response, whether humoral, cellular, or both.
Factor VIIa polypeptides and Factor VIIa-related polypeptides are
not known to elicit detectable immune responses in humans.
Nonetheless, in any human sub-population, there may exist
individuals who exhibit sensitivity to particular administered
proteins. Immunogenicity may be measured by quantifying the
presence of anti-Factor VII antibodies and/or Factor VII-responsive
T-cells in a sensitive individual, using conventional methods known
in the art. In some embodiments, the preparations of the present
invention exhibit a decrease in immunogenicity in a sensitive
individual of at least about 10%, preferably at least about 25%,
more preferably at least about 40% and most preferably at least
about 50%, relative to the immunogenicity for that individual of a
reference preparation.
[0084] Pharmaceutical Compositions and Methods of Use
[0085] The preparations of the present invention may be used to
treat any Factor VII-responsive syndrome, such as, e.g., bleeding
disorders, including, without limitation, those caused by clotting
factor deficiencies (e.g., haemophilia A and B or deficiency of
coagulation factors XI or VII); by thrombocytopenia or von
Willebrand's disease, or by clotting factor inhibitors, or
excessive bleeding from any cause. The preparations may also be
administered to patients in association with surgery or other
trauma or to patients receiving anticoagulant therapy.
[0086] Preparations comprising Factor VII-related polypeptides
according to the invention, which have substantially reduced
bioactivity relative to wild-type Factor VII, may be used as
anticoagulants, such as, e.g., in patients undergoing angioplasty
or other surgical procedures that may increase the risk of
thrombosis or occlusion of blood vessels as occurs, e.g., in
restenosis. Other medical indications for which anticoagulants are
prescribed include, without limitation, deep vein thrombosis,
pulmonary embolism, stroke, disseminated intravascular coagulation
(DIC), fibrin deposition in lungs and kidneys associated with
gram-negative endotoxemia, myocardial infarction; Acute Respiratory
Distress Syndrome (ARDS), Systemic Inflammatory Response Syndrome
(SIRS), Hemolytic Uremic Syndrome (HUS), MOF, and TTP.
[0087] Pharmaceutical compositions comprising the Factor VII and
Factor VII-related preparations according to the present are
primarily intended for parenteral administration for prophylactic
and/or therapeutic treatment. Preferably, the pharmaceutical
compositions are administered parenterally, i.e., intravenously,
subcutaneously, or intramuscularly. They may be administered by
continuous or pulsatile infusion.
[0088] Pharmaceutical compositions or formulations comprise a
preparation according to the invention in combination with,
preferably dissolved in, a pharmaceutically acceptable carrier,
preferably an aqueous carrier or diluent. A variety of aqueous
carriers may be used, such as water, buffered water, 0.4% saline,
0.3% glycine and the like. The preparations of the invention can
also be formulated into liposome preparations for delivery or
targeting to the sites of injury. Liposome preparations are
generally described in, e.g., U.S. Pat. Nos. 4,837,028, 4,501,728,
and 4,975,282. The compositions may be sterilised by conventional,
well-known sterilisation techniques. The resulting aqueous
solutions may be packaged for use or filtered under aseptic
conditions and lyophilised, the lyophilised preparation being
combined with a sterile aqueous solution prior to
administration.
[0089] The compositions may contain pharmaceutically acceptable
auxiliary substances or adjuvants, including, without limitation,
pH adjusting and buffering agents and/or tonicity adjusting agents,
such as, for example, sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, etc.
[0090] The concentration of Factor VII or Factor VII-related
polypeptides in these formulations can vary widely, i.e., from less
than about 0.5% by weight, usually at or at least about 1% by
weight to as much as 15 or 20% by weight and will be selected
primarily by fluid volumes, viscosities, etc., in accordance with
the particular mode of administration selected.
[0091] Thus, a typical pharmaceutical composition for intravenous
infusion could be made up to contain 250 ml of sterile Ringer's
solution and 10 mg of the preparation. Actual methods for preparing
parenterally administrable compositions will be known or apparent
to those skilled in the art and are described in more detail in,
for example, Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing Company, Easton, Pa. (1990).
[0092] The compositions containing the preparations of the present
invention can be administered for prophylactic and/or therapeutic
treatments. In therapeutic applications, compositions are
administered to a subject already suffering from a disease, as
described above, in an amount sufficient to cure, alleviate or
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as "therapeutically
effective amount". Effective amounts for each purpose will depend
on the severity of the disease or injury as well as the weight and
general state of the subject. In general, however, the effective
amount will range from about 0.05 mg up to about 500 mg of the
preparation per day for a 70 kg subject, with dosages of from about
1.0 mg to about 200 mg of the preparation per day being more
commonly used. It will be understood that determining an
appropriate dosage may be achieved using routine experimentation,
by constructing a matrix of values and testing different points in
the matrix.
[0093] Local delivery of the preparations of the present invention,
such as, for example, topical application, may be carried out,
e.g., by means of a spray, perfusion, double balloon catheters,
stent, incorporated into vascular grafts or stents, hydrogels used
to coat balloon catheters, or other well established methods. In
any event, the pharmaceutical compositions should provide a
quantity of the preparation sufficient to effectively treat the
subject.
[0094] The pharmaceutical compositions of the invention may further
comprise other bioactive agents, such as, e.g., non-Factor
VII-related coagulants or anticoagulants.
[0095] The following examples are intended as non-limiting
illustrations of the present invention.
EXAMPLE 1
[0096] Production and Analysis of a Factor VII Preparation
Exhibiting an Altered Glycoform Pattern
[0097] The following experiment was performed to produce a Factor
VII preparation having an altered glycoform pattern.
[0098] I. Production: A BHK cell line transformed with a Factor
VII-encoding plasmid was adapted to growth in suspension culture in
the absence of serum. The cells were propagated sequentially in
spinner cultures and as the cell number increased, the volume was
gradually increased by addition of new medium.
[0099] Finally, 6 l of seed culture were inoculated into a
100-liter production bioreactor containing macroporous Cytopore 1
carriers (Pharmacia), after which the suspension cells became
immobilized in the carriers. The culture was maintained at
36.degree. C. at a pH of 6.7-6.9 and a DO of 50%. The volume in the
production bioreactor was gradually increased by addition of new
medium as the cell number increased. When the cell density reached
approximately 2.times.10.sup.6 cells/ml, the production phase was
initiated and a medium change was performed every 24 hours:
Agitation was stopped to allow for sedimentation of the
cell-containing carriers, and 80% of the culture supernatant was
then harvested and replaced with new medium. The harvested culture
supernatant was filtered to remove non-trapped cells and cell
debris and was then transferred for further processing.
[0100] During the production phase the cells reached
3-6.times.10.sup.6 cells/ml and a titer of 2-7 mg Factor
VII/liter.
[0101] II. Analysis of the glycoform pattern of recombinant Factor
VII
[0102] The oligosaccharide patterns of the following preparations
were compared: (a) recombinant Factor VII preparations produced as
described in part I (n=7); and two reference preparations: (b)
recombinant Factor VII preparations produced in BHK cells in the
presence of calf serum (n=10); and (c) a Factor VII preparation
purified from human plasma.
[0103] The N-linked oligosaccharides were released from the
polypeptides by chemical cleavage (hydrazinolysis, on a
GlycoPrep1000 unit, Oxford GlycoSciences) or by enzymatic cleavage
(N-glycosidase F from, eg., Boehringer Mannheim). The released
oligosaccharides were labeled with 2-aminobenzamide (using a signal
labelling kit, K-404, Oxford GlycoSciences or Glyko). The labeled
oligosa ccharides were analysed using anion-exchange HPLC on a
CarboPac PA100 column (4.times.250 mm, Dionex, P/N 43055) with a
Guard column (4.times.50 mm, Dionex, P/N 43054). The column was
equilibrated with 150 mM sodium hydroxide and eluted with a
gradient of 0-150 mM sodium acetate, 150 mM sodium hydroxide.
Oligosaccharides were detected using fluorescence, with excitation
at 330 nm and emission at 420 nm.
[0104] The relative contents of the various Factor VII
oligosaccharide structures (Klausen et al., 1998) were calculated
as the relative peak areas for the carbohydrate peaks in the
anion-exchange HPLC analysis. Based on the structural elements of
each oligosaccharide, it was assigned to one of the following: (i)
chains containing at least one sialic acid; (ii) chains lacking any
sialic acid (i.e., neutral); (iii) chains containing at least one
terminal galactose residue; (iv) chains containing at least one
terminal N-acetylgalactosamine residue; and (v) chains containing
at least one uncapped antenna (i.e., at least one terminal
galactose or N-acetylgalactosamine residue). Finally, the sum of
the relative contents of the oligosaccharide chains assigned to
each group was calculated as a percentage of the total
oligosaccharide chains. The standard deviation of this
determination was calculated to be 0.08% (intraday variation); 0.7%
(day-to-day variation); and 0.5% (1-100 .mu.g interval).
[0105] The resulting glycoform patterns are illustrated in the
following table:
1 (i) (ii) (iii) (iv) (v) a 93.1-98.7 1.3-6.9 5.9-16.4 5.9-8.7
11.7-23.9 b 88.3-92.5 7.5-12.9 9.4-16.8 19.0-28.6 30.1-39.0 c 99.5%
<0.5% 2-3% 0% 2-3%
[0106] The recombinant Factor VII preparations produced according
to this Example (i.e., in the absence of serum) exhibit a glycoform
pattern that differs from both the glycoform pattern of recombinant
Factor VII produced in the presence of serum and native Factor VII
isolated from human plasma. The oligosaccharides of recombinant
Factor VII produced in the absence of serum are sialylated to a
higher extent than those produced in the presence of serum and
contain less neutral chains and less chains that terminate in
either galactose or N-acetylgalatosamine.
[0107] III. Bioavailability:
[0108] The following experiment was performed to compare the
bioavailability of two Factor VII preparations produced as above (I
and II) with that of two reference Factor VII preparations (i.e.,
produced in the presence of serum) (A and B).
[0109] Groups of 8 rats were administered either a test preparation
or a reference preparation at a dose of 25 .mu.g/kg (.apprxeq.100
.mu.g/rat) in a glycylglycine buffer (pH 7.4) containing sodium
chloride (7.87 mg/ml), calcium chloride dihydrate (1.48 mg/ml),
mannitol (2.5 mg/ml) and polysorbate 80. Blood samples were
withdrawn at 10 min and 30 min following the initial
administration. Plasma was obtained from the samples and Factor VII
was quantified by ELISA. Bioavailability of each sample is
expressed as the dose-adjusted area under the plasma concentration
curve for Factor VII based on the 10 and 30-min samples
(AUC.sub.10-30/dose). The relative bioavailability is expressed as
the ratio between the mean AUC.sub.10-30/dose of the test and
reference samples X 100. The 90% confidence limits for the relative
bioavailability were calculated from the 90% confidence limits for
differences between preparations.
[0110] The results are summarized in the Table below. (The %
sialylation of each preparation, which was measured as described
above, is indicated in parentheses).
2 relative 90% conf. 90% conf. test reference bioavailability lower
upper I A 128.6 116.1 141.1 (97.5%) (93%) I B 154.9 141.2 168.5
(97.5%) (86%) II A 117.3 104.8 129.8 (96.7%) 93% II B 141.2 127.5
154.8 (96.7%) (86%)
[0111] The results indicate that even relatively small differences
in the proportion of oligosaccharide chains having at least one
sialic acid residue, such as, e.g., between 93% and 96 or 97%, can
have a marked impact on bioavailability (increase of 20-30%). A 10%
increase in the % sialylation, moreover, causes a 40-50% increase
in bioavailability.
EXAMPLE 2
[0112] Analysis of Factor VII Preparations Exhibiting an Altered
Glycoform Pattern
[0113] Factor VII was produced as described in Example 1 above,
with the exception that the Factor VII was harvested from 500-I
cultures. Glycoform analysis was performed as described in Example
1. Three independent preparations (A, B, and C) were analyzed and
compared with a reference preparation (D).
[0114] Bioavailability was measured in a dog model as follows: The
experiment was performed as a four leg cross-over study in 12
Beagle dogs divided in four groups. All animals received each of
the three test preparations A, B, and C and the reference
preparation D at a dose of .apprxeq.90 .mu.g/kg in a glycylglycine
buffer (pH 5.5) containing sodium chloride (2.92 mg/ml), calcium
chloride dihydrate (1.47 mg/ml), mannitol (30 mg/ml) and
polysorbate 80. Blood samples were withdrawn at 10, 30, and 60
minutes and 2, 3, 4, 6 and 8 hours following the initial
administration. Plasma was obtained from the samples and Factor VII
was quantified by ELISA.
[0115] Bioavailability of each sample is expressed as the
dose-adjusted area under the plasma concentration curve for Factor
VII (AUC/dose). The relative bioavailability is expressed as the
ratio between the mean AUC/dose of the test and reference
preparation X 100 and 90% confidence limits for the relative
bioavailability were calculated.
[0116] The results are summarized in the Table below. The %
sialylation of each preparation, which was measured as described in
Example 1 above, is indicated in parentheses.
3 Relative 90% conf. limit 90% conf. limit Test Reference
bioavailability lower upper A D 144 135 153 (98.7%) (88.2%) B D 127
119 136 (95.9%) (88.2%) C D 112 105 120 (93.1%) (88.2%)
[0117] The results indicate that small differences in the
proportion of oligosaccharide chains having at least one sialic
acid residue have a marked impact on bioavailability of Factor VII.
A 10% increase in the % sialylation causes a 30-50% increase in
bioavailability with a close to linear relationship for the three
test preparations and the reference preparation.
EXAMPLE 3
[0118] Factor VII Preparations Exhibiting an Altered Glycoform
Pattern
[0119] The following experiment was performed to produce a Factor
VII preparation having an altered glycoform pattern.
[0120] I. Construction of cell line and Factor VII production:
[0121] A plasmid vector pLN174 for expression of human FVII has
been described (Persson and Nielsen. 1996. FEBS Lett. 385:
241-243). Briefly, it carries the cDNA nucleotide sequence encoding
human FVII including the propeptide under the control of a mouse
metallothionein promoter for transcription of the inserted cDNA,
and mouse dihydrofolate reductase cDNA under the control of an SV40
early promoter for use as a selectable marker.
[0122] For construction of a plasmid vector encoding a
gamma-carboxylation recognition sequence, a cloning vector
(pBluescript II KS+, Stratagene) containing cDNA encoding FVII
including its propeptide was used (pLN171). (Persson et al. 1997.
J. Biol. Chem. 272: 19919-19924). A nucleotide sequence encoding a
stop codon was inserted into the cDNA encoding FVII after the
propeptide of FVII by inverse PCR-mediated mutagenesis using this
cloning vector. The template plasmid was denatured by treatment
with NaOH followed by PCR with Pwo (Boehringer-Mannheim) and Taq
(Perkin-Elmer) polymerases with the following primers:
4 a) 5'-AGC GTT TTA GCG CCG GCG CCG GTG CAG GAC-3' (SEQ ID NO.19)
b) 5'-CGC CGG CGC TAA AAC GCT TTC CTG GAG GAG CTG CGG CC-3' (SEQ ID
NO.20)
[0123] The resulting mix was digested with DpnI to digest residual
template DNA and Escherichia coli were transformed with the PCR
product. Clones were screened for the presence of the mutation by
sequencing. The cDNA from a correct clone was transferred as a
BamHI-EcoRI fragment to the expression plasmid pcDNA3 (Invitrogen).
The resulting plasmid was termed pLN329. CHO K1 cells (ATCC CCI61)
were transfected with equal amounts of pLN174 and pLN329 with the
Fugene6 method (Boehriner-Mannheim). Transfectants were selected by
the addition of methotrexate to 1 .mu.M and G-418 to 0.45 mg/ml.
The pool of transfectants were cloned by limiting dilution and FVII
expression from the clones was measured.
[0124] A high producing clone was further subcloned and a clone E11
with a specific FVII expression of 2.4 pg/cell/day in
Dulbecco-modified Eagle's medium with 10% fetal calf serum was
selected. The clone was adapted to serum free suspension culture in
a commercially available CHO medium (JRH Bioscience) free of animal
derived components.
[0125] The adapted cells were propagated sequentially in spinner
cultures and as the cell number increased, the volume was gradually
increased by addition of new medium. After 25 days, 6 l of spinner
culture were inoculated into a 50-liter bioreactor. The cells were
propagated in the bioreactor and as the cell number increased, the
volume was gradually increased by addition of new medium.
[0126] Finally, 50 l of seed culture were inoculated into a
500-liter production bioreactor containing macroporous Cytopore 1
carriers (Pharmacia), after which the suspension cells became
immobilized in the carriers. The culture was maintained at
36.degree. C. at a pH of 7.0-7.1 and a Dissolved Oxygen Tension
(DOT) of 50% of saturation. The volume in the bioreactor was
gradually increased by addition of new medium as the cell number
increased. When the cell density reached approximately
10-12.times.105 cells/ml, the production phase was initiated and a
medium change was performed every 24 hours: agitation was stopped
to allow for sedimentation of the cell-containing carriers, and 80%
of the culture supernatant was then harvested and replaced with new
medium. The harvested culture supernatant was filtered to remove
non-trapped cells (i.e. cells that were not immobilized in
carriers) and cell debris and was then transferred for further
processing.
[0127] During the production phase the cells reached 2-3.times.107
cells/ml and a titer of 8 mg Factor VII/liter.
[0128] II. Glycoform Analysis:
[0129] A. The oligosaccharide pattern of a Factor VII preparation
produced as described above (a) was compared with those of (b)
recombinant Factor VII preparations produced in BHK cells in the
presence of calf serum and (c) a Factor VII preparation purified
from human plasma. The methods used were essentially as described
in Example 1.
[0130] The results are shown in the Table below. The
oligosaccharide assignments are as follows: (i) chains containing
at least one sialic acid; (ii) chains lacking any sialic acid
(i.e., neutral); (iii) chains containing at least one terminal
galactose residue; (iv) chains containing at least one terminal
N-acetylgalactosamine residue; and (v) chains containing at least
one uncapped antenna (i.e., at least one terminal galactose or
N-acetylgalactosamine residue).
5 (i) (ii) (iii) (iv) (v) a 95.2 4.8 22.9 0.1 23.0 b 88.3-92.5
7.5-12.9 9.4-16.8 19.0-28.6 30.1-39.0 c 99.5% <0.5% 2-3% 0%
2-3%
[0131] B. The oligosaccharide patterns of five independent Factor
VII preparations produced as described in this Example (a) were
compared with those of (b) recombinant Factor VII preparations
produced in BHK cells in the presence of calf serum and (c) a
Factor VII preparation purified from human plasma, using the
analytical methods described in Example 1.
[0132] Based on the structural elements of each oligosaccharide, it
was assigned to one of the following: (i) chains containing at
least one sialic acid; (ii) chains lacking any sialic acid (i.e.,
neutral); (iii) chains containing at least one fucose linked to the
antenna. Finally, the sum of the relative contents of the
oligosaccharide chains assigned to each group was calculated as a
percentage of the total oligosaccharide chains. The standard
deviation of this determination was calculated to be 0.08%
(intraday variation); 0.7% (day-to-day variation); and 0.5% (1-100
.mu.g interval).
[0133] The resulting glycoform patterns are illustrated in the
following Table:
6 (i) (ii) (iii) a 89.0-97.9% 2.1-11.0% 6.3-21.3% b 88.3-92.5%
7.5-12.9% 0% c 99.5% <0.5% 0%
[0134] The recombinant Factor VII preparations produced according
to Example 1 (i.e., produced in the absence of serum by the CHO
cell line) exhibit a glycoform pattern that differs from both the
glycoform pattern of recombinant Factor VII produced in the
presence of serum and native Factor VII isolated from human plasma.
The oligosaccharides of recombinant Factor VII produced in the
absence of serum by the CHO 282.4 cell line include structures with
fucose linked to the antenna, which are absent from both of the
reference preparations. Two of the structures have been purified
and characterized by matrix assisted laser desorption ionisation
mass spectrometry, by treatment with linkage specific fucosidase
enzymes and by anion-exchange HPLC as described above. The two
structures have been shown to contain the sialyl Lewis x structure,
i.e., fucose linked .alpha.1.fwdarw.3 to an antennary
N-acetylglucosamine in a sialylated oligosaccharide.
[0135] III. Bioactivity:
[0136] Five Factor VII preparations produced as described in this
Example were analyzed for (a) thrombin generation and (b) binding
to tissue factor (TF) and compared with recombinant Factor VII
produced in BHK cells in the presence of serum (reference). The
following Table correlates the glycoform patterns (% of
oligosaccharide chains containing sialic acid and the % containing
fucosylated antenna) and the two bioactivities.
7 Thrombin Oligosaccharide generation Factor VII Pattern (% of TF
binding Preparation % Sialyl % Fucosyl reference) Kd (nM) 1 98 6
125 2.8 2 94 13 123 2.0 3 93 14 126 1.8 4 88 16 145 3.3 5 86 21 158
2.8 reference 86-93 0 100 2.2-6.6
[0137] The results indicate that Factor VII preparations having
fucosylated antennae exhibit higher TF-independent Factor VII
activity (as exhibited, e.g. by thrombin generation) than Factor
VII preparations lacking fucosylated antennae.
EXAMPLE 4
[0138] In Vitro Hydrolysis Assay
[0139] The following method can be used to assay Factor VIIa
bioactivity. The assay is carried out in a microtiter plate
(MaxiSorp, Nunc, Denmark). The chromogenic substrate
D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), at a
final concentration of 1 mM, is added to Factor VIIa (final
concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0.1 M
NaCl, 5 mM CaCl.sub.2 and 1 mg/ml bovine serum albumin. The
absorbance at 405 nm is measured continuously in a SpectraMax.TM.
340 plate reader (Molecular Devices, USA). The absorbance developed
during a 20-minute incubation, after subtraction of the absorbance
in a blank well containing no enzyme, is used to calculate the
ratio between the activities of a test and a reference Factor
VIIa.
EXAMPLE 5
[0140] In Vitro Proteolysis Assay
[0141] The following method can be used to assay Factor VIIa
bioactivity. The assay is carried out in a microtiter plate
(MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) and Factor X (0.8
microM) in 100 .mu.l 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5
mM CaCl.sub.2 and 1 mg/ml bovine serum albumin, are incubated for
15 min. Factor X cleavage is then stopped by the addition of
50.mu.l 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and
1 mg/ml bovine serum albumin. The amount of Factor Xa generated is
measured by addition of the chromogenic substrate
Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final
concentration 0.5 mM. The absorbance at 405 nm is measured
continuously in a SpectraMax.TM. 340 plate reader (Molecular
Devices, USA). The absorbance developed during 10 minutes, after
subtraction of the absorbance in a blank well containing no FVIIa,
is used to calculate the ratio between the proteolytic activities
of a test and a reference Factor VIIa.
[0142] All patents, patent applications, and literature references
referred to herein are hereby incorporated by reference in their
entirety.
[0143] Many variations of the present invention will suggest
themselves to those skilled in the art in light of the above
detailed description. Such obvious variations are within the full
intended scope of the appended claims.
* * * * *