U.S. patent application number 11/579401 was filed with the patent office on 2008-12-11 for o-linked glycoforms of polypeptides and method to manufacture them.
This patent application is currently assigned to NOVO NORDISK HEALTHCARE A/G. Invention is credited to Niels Kristian Klausen, Daniel Rasmussen.
Application Number | 20080305518 11/579401 |
Document ID | / |
Family ID | 34968774 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080305518 |
Kind Code |
A1 |
Klausen; Niels Kristian ; et
al. |
December 11, 2008 |
O-Linked Glycoforms Of Polypeptides And Method To Manufacture
Them
Abstract
The present invention relates to compositions comprising
glycoproteins having altered patterns of O-linked glycosylation, in
particular Factor VII, Factor IX, and methods for making these.
Inventors: |
Klausen; Niels Kristian;
(Gentofte, DK) ; Rasmussen; Daniel; (Copenhagen O,
DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
NOVO NORDISK HEALTHCARE A/G
Zurich
CH
|
Family ID: |
34968774 |
Appl. No.: |
11/579401 |
Filed: |
May 3, 2005 |
PCT Filed: |
May 3, 2005 |
PCT NO: |
PCT/EP05/52024 |
371 Date: |
July 18, 2008 |
Current U.S.
Class: |
435/68.1 ;
530/384; 530/395 |
Current CPC
Class: |
C07K 14/745 20130101;
A61P 7/04 20180101; A61P 7/02 20180101 |
Class at
Publication: |
435/68.1 ;
530/395; 530/384 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2004 |
DK |
PA 2004 00712 |
Jun 4, 2004 |
DK |
PA 2004 00882 |
Claims
1. A preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is
linked to a sugar chain by a Glc-O-Ser/Thr covalent bond, X1 and X2
each represent any amino acid residue and the preparation has a
substantially uniform serine/threonine-linked glycosylation
pattern.
2. The preparation according to claim 1, wherein the glycosylation
pattern is at least 80% uniform.
3. The preparation according to claim 1, wherein the
serine/threonine-linked sugar chain is Xyl-Xyl-Glc-.
4. The preparation according to claim 1, wherein the
serine/threonine-linked sugar chain is Xyl-Glc-.
5. The preparation according to claim 1, wherein the
serine/threonine-linked sugar chain is Glc-.
6. The preparation according to claim 1, wherein the glycoprotein
is selected from the group consisting of Factor VII polypeptides,
Factor VII-related polypeptides, Factor IX polypeptides, Factor
IX-related polypeptides, Factor X polypeptides, Factor X-related
polypeptides, Factor XII polypeptides, and protein Z
polypeptides.
7. The preparation according to claim 6, wherein the glycoprotein
is human Factor VII.
8. A preparation according to claim 6, wherein the glycoprotein is
a variant of Factor VII and wherein the ratio between the activity
of the Factor VII-variant and the activity of native human factor
VIIa (wild-type FVIIa) is at least about 1.25 when tested in the
"In Vitro Hydrolysis Assay" or in the "In vitro Proteolysis
Assay".
9. The preparation according to claim 6, wherein the glycoprotein
is human Factor IX or a human Factor IX sequence variant.
10. A method for making a preparation according to claim 5
comprising: (a) obtaining a preparation of a precursor glycoprotein
containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the
serine/threonine is linked to a sugar chain by Glc-O-Ser/Thr
covalent bond and X1 and X2 each represent any amino acid residue;
and (b) contacting the preparation obtained in step (a) with an
.alpha.-xylosidase under conditions appropriate for removing xylose
residues from the precursor glycoprotein, thereby producing the
glycoprotein.
11. The method according to claim 10, further including the step of
isolating the glycoprotein prepared in step (b).
12. The method according to claim 10, wherein the glycosylation is
a serine glycosylation.
13. The method according to claim 10 10, further including the step
of analysing the structure of the sugar chain linked to the
glycoprotein to determine a glycoform pattern, and, optionally,
repeating step (b) until the desired glycoform pattern is
achieved.
14. A method for making a preparation according to in claim 6
comprising: (a) obtaining a preparation of a polypeptide containing
a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein X1 and X2 each represent
any amino acid residue; and (b) contacting the preparation obtained
in step (a) with a O-glucosyltransferase and an activated glucose
donor under conditions appropriate for transferring a glucose
residue from the glucose donor moiety to the serine/threonine
thereby producing the glycoprotein.
15. The method according to claim 14, further including the step of
isolating the glycoprotein prepared in step (b).
16. The method according to claim 14, wherein the glycosylation is
a serine glycosylation.
17. The method according to claim 14, further including the step of
analyzing the structure of the sugar chain linked to glycoproteins
in the preparation to determine if the glycoproteins have a desired
glycoform pattern, and, optionally, repeating step (b) until the
desired glycoform pattern is achieved.
18. A method for making a preparation according to claim 4
comprising: (a) obtaining a preparation of a precursor glycoprotein
containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the
serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr
covalent bond and X1 and X2 each represent any amino acid residue;
and (b) contacting the preparation obtained in step (a) with (i)
UDP-D-xylose: .beta.-D-glucoside .alpha.-1,3-D-xylosyltransferase
and (ii) an activated xylosyl donor under conditions appropriate
for transferring a xylose residue from an xylose donor moiety to an
acceptor moiety, thereby producing the glycoprotein.
19. The method according to claim 18, further including the step of
isolating the glycoprotein prepared in step (b).
20. The method according to claim 18, wherein the glycosylation is
a serine glycosylation.
21. The method according to claim 18, further including the step of
analyzing the structure of the sugar chain linked to glycoproteins
in the preparation to determine if the glycoproteins have a desired
glycoform pattern, and, optionally, repeating step (b) until the
desired glycoform pattern is achieved.
22. The method according to claim 18, further including the step of
removing terminal xylose-residues by subjecting the preparation
obtained in step (a) to the method described in claim 10 prior to
step (b).
23. A method for making a preparation according to claim 3
comprising: (a) obtaining a preparation of a precursor glycoprotein
containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the
serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr
covalent bond and X1 and X2 each represent any amino acid residue;
(b) contacting the preparation obtained in step (a) with
UDP-D-xylose: .beta.-D-glucoside .alpha.-1,3-D-xylosyltransferase
and an activated xylosyl donor under conditions appropriate for
transferring a xylose residue from a xylose donor moiety to an
acceptor moiety; and (c) contacting the preparation obtained in
step (b) with UDP-D-xylose: .alpha.-D-xyloside
.alpha.-1,3-xylosyltransferase and an activated xylosyl donor under
conditions appropriate for transferring a xylose residue from a
xylose donor moiety to an acceptor moiety, thereby producing the
glycoprotein.
24. The method according to claim 23, further including the step of
isolating the preparation obtained in step (b) prior to subjecting
the preparation to step (c).
25. The method according to claim 23, further including the step of
isolating the glycoprotein prepared in step (c).
26. The method according to claim 23, wherein the glycosylation is
a serine glycosylation.
27. The method according to claim 23, further including the step of
analyzing the structure of the sugar chain linked to the
glycoproteins in the preparation to determine if the glycoproteins
have a desired glycoform pattern, and, optionally, repeating step
(b) and/or step (c) until the desired glycoform pattern is
achieved.
28. The method according to claim 23, further including the step of
removing terminal xylose-residues by subjecting the preparation
obtained in step (a) to the method described in claim 10 prior to
step (b).
29. The preparation according to claim 2, wherein the glycosylation
pattern is at least 90% uniform.
30. The preparation according to claim 29, wherein the
glycosylation pattern is at least 98% uniform.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions comprising
glycoproteins having altered patterns of O-linked glycosylation, in
particular Factor VII, Factor IX and other blood clotting
factors.
BACKGROUND OF THE INVENTION
[0002] The biological activity of many glycoproteins is highly
dependent upon the presence or absence of particular
oligosaccharide structures attached to the glycoprotein. The
glycosylation pattern of a therapeutic glycoprotein can affect
numerous aspects of the therapeutic efficacy, such as, e.g,
solubility, resistance to proteolytic attack, thermal inactivation,
immunogenicity, half-life, bioactivity, bioavailability, and
stability.
[0003] Glycosylation is a complex post-transitional modification
that is cell dependent. Following translation, proteins are
transported into the endoplasmic reticulum (ER), glycosylated and
sent to the Golgi for further processing and subsequent targeting
and/or secretion. During glycosylation, either N-linked or O-linked
glycoproteins are formed.
[0004] Serum proteins involved in coagulation or fibrinolysis,
including, e.g., Factor VII and Factor IX 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.
[0005] 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 posttranslational
modifications, including, e.g., asparagine-linked (N-linked)
glycosylation; serine- or threonine-linked (O-linked)
glycosylation; and T-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 identical
polypeptides are having different covalently linked oligosaccharide
structures.
[0006] 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
glycoprotein preparations, particularly preparations comprising
recombinant Factor IX or recombinant human Factor VII or modified
Factor VII or Factor VII-related polypeptides that contain
predetermined glycoform patterns.
SUMMARY OF THE INVENTION
[0007] The present invention relates to preparations comprising
polypeptides that exhibit predetermined serine or threonine-linked
glycoform patterns. The preparations are at least about 80%
homogenous in respect of the attached glycans or oligosaccharide
chains, preferably at least about 90%, at least about 95%, or at
least about 98% homologous.
[0008] As used herein, a glycoform pattern refers to the
distribution within the preparation of oligosaccharide chains
having varying structures that are covalently linked to a serine or
threonine residue located in an EGF-like domain in the amino acid
backbone of the polypeptide.
[0009] In one aspect, the invention provides a preparation of a
glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif and
wherein said serine/threonine forms part of a Glc-O-Ser/Thr
covalent bond, said preparation containing a substantially uniform
serine/threonine-linked glycosylation pattern.
[0010] In one embodiment of the invention, the glycosylation
pattern is at least 80% uniform, preferably at least 85%, at least
90%, at least 95%, or at least 98% uniform.
[0011] In one embodiment, the serine/threonine-linked glycans are
Xyl-Xyl-Glc-; in another, the glycans are Xyl-Glc-; in yet another,
the glycans are Glc-.
[0012] In different embodiments the glycoproteins are selected from
the group of: Factor VII polypeptides, Factor VII-related
polypeptides, Factor IX polypeptides, Factor X polypeptides, Factor
XII polypeptides, and protein Z polypeptides. In a preferred
embodiment, the glycoprotein is selected from the group of: Human
Factor VII, Factor VII sequence variants, human Factor IX, and
Factor IX sequence variants. In one embodiment, the glycoprotein is
a Factor VII variant wherein the ratio between the activity of the
Factor VII-variant and the activity of native human factor VIIa
(wild-type FVIIa) is at least about 1.25 when tested in the "In
Vitro Hydrolysis Assay" as described in the pre-sent description,
preferably at least about 2.0, or at least about 4.0.
[0013] In another aspect the invention provides methods for making
preparations of glycoproteins containing Cys-X1-Ser/Thr-X2-Pro-Cys
motifs and wherein said serine/threonine forms part of a
Glc-O-Ser/Thr covalent bond, said preparations containing a
substantially uniform serine/threonine-linked glycosylation
pattern. The methods are useful for remodelling or altering the
glycosylation pattern present on a glycoprotein upon its initial
expression.
[0014] More particular, the present invention provide a general
enzymatic methodology for the modification of glycans (in
particular O-linked glycans) of glycoproteins, in order to improve
or enhance their pharmaceutically properties. One method involves
treatment of the glycoprotein with xylosidases in order to remove
any terminal xylose residues; other methods includes attachment of
xylose residues to the exposed glucose or xylose residues on the
glycoprotein by treatment with xylosyltransferases; a third method
includes attachment of glucose residues to serine and/or threonine
amino acid residues in the polypeptide backbone thereby creating a
glycosylated polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the serine 52 glycosylation of wt-Factor
VII.
[0016] FIG. 2 shows an O-glycosylation mapping of Factor VII.
[0017] FIG. 3 shows a reaction scheme for the making of a
preparation of glycoproteins exhibiting a predetermined
serine/threonine-linked glycosylation.
[0018] FIG. 4 shows a chromatogram from first HIC cycle showing
fractions "A" and "B".
[0019] FIG. 5 shows a chromatogram obtained by reloading fraction
"A" onto the HIC column; Glc-O-Ser52-FVII was identified in the
peak fraction, fraction 10.
[0020] FIG. 6 shows a chromatogram obtained by reloading fraction
"B" onto the HIC column; Xyl-Xyl-Glc-O-Ser52-FVII was identified in
the peak fraction, fraction 15.
[0021] FIG. 7A shows a tryptic peptide map of the peak fraction,
fraction 10; the arrow indicates the Glc-O-Ser52
O-glycopeptide.
[0022] FIG. 7B shows a tryptic peptide map of the peak fraction,
fraction 15; the arrow indicates the Xyl-Xyl-Glc-O-Ser52
O-glycopeptide.
[0023] FIG. 8A shows a total mass analysis of the peak fraction,
fraction 10; the arrow indicates the Glc-O-Ser52-rFVIIa
O-glycoform.
[0024] FIG. 8B shows a total mass analysis of the peak fraction,
fraction 15; the arrow indicates the Xyl-Xyl-Glc-O-rFVIIa
O-glycoform.
DETAILED DESCRIPTION
[0025] The following abbreviations are used herein:
[0026] Glc=glucosyl
[0027] Xyl=xylosyl
[0028] Ser=serine (one letter code: S)
[0029] Thr=threonine (one letter code: T)
[0030] Pro=proline (one letter code: P)
[0031] Cys=cysteine (one letter code: C)
[0032] FVII=Factor VII
[0033] FVIIa=activated (two-chain) Factor VII
[0034] FIX=Factor IX
[0035] FIXa=activated (two-chain) Factor IX
[0036] As used herein, a "glycoform pattern" (or "glycosylation
pattern") refers to the distribution within the preparation of
oligosaccharide chains having varying structures that are
covalently linked to a serine or threonine residue located in the
amino acid backbone of the polypeptide.
[0037] "Homogeneity" refers to the structural consistency across a
population of polypeptides with conjugated glycans. Thus, a
glycoprotein preparation is said to be about 100% homologous if all
contained glycoprotein molecules contain identical glycans attached
to the relevant glycosylation site. For example, a preparation of
Factor VII polypeptides is said to be at least 90% homologous if at
least 90% of the Factor VII polypeptide molecules contain the
glycan of interest attached to serine 52 (e.g.,
Xyl-Xyl-Glc-O-Ser52).
[0038] "Substantially uniform glycoform" or "substantially uniform
glycosylation" or "substantially uniform glycosylation pattern",
when referring to a glycopeptide species, refers to the percentage
of acceptor moieties, i.e., serine or threonine residues, that are
glycosylated by the glycan of interest. For example, in the case of
Factor VII, a substantially uniform glycosylation patterns exists
if substantially all (as defined below) of the serine residues in
position 52 are glycosylated with the glycan of interest. It is
understood by one skilled in the art that the starting material may
contain glycosylated serine and/or threonine residues that are
glycosylated with a species having the same structure as the glycan
of interest. Thus, the calculated percent glycosylation includes
serine/threonine residues that are glycosylated with the glycan of
interest according to the invention, as well as those
serine/threonine residues already glycosylated with the glycan of
interest in the starting material.
[0039] The term "substantially" is intended to mean that at least
about 80%, such as at least about 90%, at least about 95%, or at
least about 98% of the serine/threonine residues in the
glycoprotein is glycosylated with a predetermined, specific glycan
or glycan of interest. The glycosylation pattern is typically
determined by one or more methods known to those skilled in the
art, such as, e.g., tryptic digestion followed by high performance
liquid chromatography (HPLC), liquid-chromatography-mass
spectrometry (LCMS), matrix assisted laser desorption mass time of
flight spectrometry (MALDITOF), capillary electrophoresis, and the
like.
[0040] The term "acceptor moiety" is intended to encompass the
group or moiety to which a desired oligo- or mono-saccharide group
is transferred such as, without limitation, the serine/threonine
residue located within a Cys-X1-Ser/Thr-X2-Pro-Cys motif, a
Glc-residue covalently linked to such a serine/threonine residue,
or a Xyl-residue covalently linked to a Glc-residue or a
Xyl-residue in a Glc-O-Ser/Thr or Xyl-Glc-O-Ser/Thr moiety,
respectively.
[0041] The term "saccharide donor moiety" is intended to encompass
an activated saccharide donor molecule (e.g., a desired oligo- or
mono-saccharide structure such as, for example, a
xylosyl-xylosyl-donor, xylosyl-donor, or glycosyl-donor) having a
leaving group (e.g., xylose-UDP or glucose-UDP) suitable for the
donor moiety acting as a substrate for the relevant catalysing
enzyme (e.g. glycosyltransferase, xylosidase or
xylosyltransferase).
[0042] Oligosaccharides are considered to have a reducing and a
non-reducing end, whether or not the saccharide at the reducing end
is in fact a reducing sugar. In accordance with accepted
nomenclature, oligosaccharides are depicted herein with the
non-reducing end on the left and the reducing end on the right
(e.g., Xyl-Xyl-Glc-O-Ser)
EGF Domain-Containing Polypeptides
[0043] The term "EGF domain-containing polypeptides" is intended to
encompass peptides, oligopeptides and polypeptides containing one
or more epidermal growth factor (EGF)-like domain(s). EGF domains
or repeats are small motifs with about 40 amino acids defined by 6
conserved cysteines forming three disulfide bonds. EGF
domain-containing polypeptides all contain a consensus sequence for
O-glucose modification: Cys1-X1-Ser/Thr-X2-Pro-Cys2 (i.e., a
Cys1-X1-Ser-X2-Pro-Cys2 or a Cys1-X1-Thr-X2-Pro-Cys2 consensus
sequence) where Cys1 and Cys2 are the first and second conserved
cysteines of the EGF repeat and X1 and X2 independently is any
amino acid.
[0044] The term "glycoprotein" is intended to encompass EGF
domain-containing polypeptides containing one or more glycans
attached to one or more serine/threonine amino acid residues of the
EGF-domain located in the back bone amino acid sequence of the
polypeptide.
[0045] As used herein, the term "glycan" or, interchangeable,
"sugar chain", "oligosaccharide chain" or "oligosaccharide moiety"
refers to the entire oligosaccharide structure that is covalently
linked to a single serine/threonine residue. The glycan may
comprise one or more saccharide units; examples of glycans include,
e.g., Glc-, Xyl-Glc-, and Xyl-Xyl-Glc-.
[0046] The term "O-glycosylation site" is intended to indicate the
glycosylation site at serine/threonine (i.e., Ser or Thr) located
within the motif Cys1-X1-Ser/Thr-X2-Pro-Cys2 where Cys1 and Cys2
are the first and second conserved cysteines of the EGF repeat and
X1 and X2 independently is any amino acid. These include the
glycosylation site at position Ser-52 (552) of human wt-FVII and
the corresponding residues in homologous polypeptides such as,
without limitation, FVII sequence variants and FIX polypeptides.
The term "corresponding residues" is intended to indicate the Ser
or Thr amino acid residue corresponding to the Ser52 residue of
wild-type Factor VII (see FIG. 1) when the sequences are aligned.
Amino acid sequence homology/identity is conveniently determined
from aligned sequences, using a suitable computer program for
sequence alignment, such as, e.g., the ClustalW program, version
1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22:
4673-4680). For example, the wt-factor VII Ser52-residue
corresponds to the Ser53-residue of wt-Factor IX. It is further to
be understood that polypeptide variants may be created containing
non-naturally occurring Cys-X1-Ser/Thr-X2-Pro-Cys motifs and
thereby containing non-naturally occurring O-glycosylation sites
that can be glycosylated in accordance with the present invention.
In one embodiment of the invention, the O-glycosylation site is a
serine-glycosylation site and the motif is Cys1-X1-Ser-X2-Pro-Cys2.
In another embodiment, the O-glycosylation site is a
threonine-glycosylation site and the motif is
Cys1-X1-Thr-X2-Pro-Cys2.
[0047] The term "terminal glucose" is intended to encompass glucose
residues linked as the terminal sugar residue in a glycan, or
oligosaccharide chain, i.e., the terminal sugar of each antenna is
glucose. The term "terminal xylose" is intended to encompass xylose
residues linked as the terminal sugar residue in a glycan, or
oligosaccharide chain.
Enzymes
[0048] Protein O-glycosyltransferase may be prepared as described,
e.g., in Shao et al. (Glycobiology 12(11): 763-770 (2002)).
[0049] The alpha-xylosidase enzymes may be prepared, e.g., as
described by Monroe et al. (Plant Physiology and Biochemistry
41:877-885 (2003)).
[0050] The enzyme, UDP-D-xylose: .beta.-D-glucoside
.alpha.-1,3-D-xylosyltransferase can be prepared from HepG2 cells
as described by Omichi et al. (Eur. J. Biochem. 245:143-146
(1997)).
[0051] The enzyme, UDP-D-xylose: .alpha.-D-xyloside
.alpha.1,3-xylosyltransferase can be prepared from HepG2 cells as
described by Minamida et al. ((J. Biochem. (Tokyo) 120: 1002-1006
(1996)).
[0052] UDP-beta-D-glucose is commercially available from, e.g.,
Sigma (Sigma U4625)
[0053] UDP-D-xylose is commercially available from, e.g., Sigma
(Sigma U5875)
Glycoproteins
[0054] The motif: Cys-X1-Ser/Thr-X2-Pro-Cys appears to be primarily
found in epidermal growth factor (EGF) domains of multi-modular
proteins such as coagulation and fibrinolytic factors. The motif is
a consensus sequence for O-glucose modification whereby a
serine-glucose (Glc-O-Ser) or threonine-glucose (Glc-O-Thr) bond is
formed. Coagulation factors VII, IX, X and XII as well as plasma
Protein Z, Fibrillin and thrombospondin have all been shown to
contain the Cys-X1-Ser/Thr-X2-Pro-Cys consensus sequence. Of these,
Factors VII and IX and Protein Z have been described to contain the
consensus sequence Cys-X1-Ser-X2-Pro-Cys.
[0055] The thrombospondins are a family of extracellular proteins
that participate in cell-to-cell and cell-to-matrix communication.
The proteins are secreted from platelets. They regulate cellular
phenotype during tissue genesis and repair.
[0056] Protein Z is a vitamin k-dependent plasma protein whose
structure is similar to that of Factors VII, IX and X. In contrast
to these proteins, however, Protein Z is not the zymogen of a
serine protease because it lacks the His and Ser residues of the
catalytic triad. Like Proteins C and S, Protein Z participates in
limiting the coagulation response, believably by assisting in
inhibition of activated Factor X (FXa).
[0057] Factor X (Stuart Prower Factor) is a vitamin K-dependent
serine protease which participates in the blood clotting process by
participating in activation of prothrombin into thrombin.
[0058] Factor XII (Hageman factor) is a blood coagulation factor
activated by contact with the sub-endothelial surface of an injured
vessel. Along with prekallikrein, it serves as the contact factor
that initiates the intrinsic pathway of blood coagulation.
Kallikrein activates factor XII to XIIa.
[0059] Factor IX (Christmas factor) is a vitamin K-dependent serine
protease which participates in the blood clotting process by
participating in activation of FX into FXa.
[0060] Factor VII (proconvertin) is a vitamin K-dependent serine
protease which participates in the blood clotting process by
participating in activation of prothrombin into thrombin. FVII is
activated into FVIIa by contact with exposed tissue factor (TF) at
sites of injury of the vessel wall.
Factor VII Polypeptides and Factor VII-Related Polypeptides
[0061] As used herein, the terms "Factor VII polypeptide" or "FVII
polypeptide" means any protein comprising the amino acid sequence
1-406 of wild-type human Factor VIIa (i.e., a polypeptide having
the amino acid sequence disclosed in U.S. Pat. No. 4,784,950),
variants thereof as well as Factor VII-related polypeptides, Factor
VII derivatives and Factor VII conjugates. This includes FVII
variants, Factor VII-related polypeptides, Factor VII derivatives
and Factor VII conjugates exhibiting substantially the same or
improved biological activity relative to wild-type human Factor
VIIa.
[0062] 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. Such variants of Factor VII may exhibit different properties
relative to human Factor VII, including stability, phospholipid
binding, altered specific activity, and the like.
[0063] As used herein, "wild type human FVIIa" is a polypeptide
having the amino acid sequence disclosed in U.S. Pat. No.
4,784,950.
[0064] As used herein, "Factor VII-related polypeptides"
encompasses polypeptides, including variants, in which the Factor
VIIa biological activity has been substantially modified, such as
reduced, relative to the activity of wild-type Factor VIIa. These
polypeptides include, without limitation, Factor VII or Factor VIIa
into which specific amino acid sequence alterations have been
introduced that modify or disrupt the bioactivity of the
polypeptide.
[0065] The term "Factor VII derivative" as used herein, is intended
to designate a FVII polypeptide exhibiting substantially the same
or improved biological activity relative to wild-type Factor VII,
in which one or more of the amino acids of the parent peptide have
been genetically and/or chemically and/or enzymatically modified,
e.g. by alkylation, glycosylation, PEGylation, acylation, ester
formation or amide formation or the like. This includes but is not
limited to PEGylated human Factor VIIa, cysteine-PEGylated human
Factor VIIa and variants thereof. Non-limiting examples of Factor
VII derivatives includes GlycoPegylated FVII derivatives as
disclosed in WO 03/31464 and US Patent applications US 20040043446,
US 20040063911, US 20040142856, US 20040137557, and US 20040132640
(Neose Technologies, Inc.); FVII conjugates as disclosed in WO
01/04287, US patent application 20030165996, WO 01/58935, WO
03/93465 (Maxygen ApS) and WO 02/02764, US patent application
20030211094 (University of Minnesota).
[0066] The term "improved biological activity" refers to FVII
polypeptides with i) substantially the same or increased
proteolytic activity compared to recombinant wild type human Factor
VIIa or ii) to FVII polypeptides with substantially the same or
increased TF binding activity compared to recombinant wild type
human Factor VIIa or iii) to FVII polypeptides with substantially
the same or increased half life in blood plasma compared to
recombinant wild type human Factor VIIa. The term "PEGylated human
Factor VIIa" means human Factor VIIa, having a PEG molecule
conjugated to a human Factor VIIa polypeptide. It is to be
understood, that the PEG molecule may be attached to any part of
the Factor VIIa polypeptide including any amino acid residue or
carbohydrate moiety of the Factor VIIa polypeptide. The term
"cysteine-PEGylated human Factor VIIa" means Factor VIIa having a
PEG molecule conjugated to a sulfhydryl group of a cysteine
introduced in human Factor VIIa.
[0067] Non-limiting examples of Factor VII variants having
substantially the same or increased proteolytic activity compared
to recombinant wild type human Factor VIIa 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); oxidized forms of Factor VIIa (Kornfelt et al.,
Arch. Biochem. Biophys. 363:43-54, 1999); FVII variants as
disclosed in PCT/DK02/00189 (corresponding to WO 02/077218); and
FVII variants exhibiting increased proteolytic stability as
disclosed in WO 02/38162 (Scripps Research Institute); FVII
variants having a modified Gla-domain and exhibiting an enhanced
membrane binding as disclosed in WO 99/20767, U.S. Pat. No.
6,017,882 and U.S. Pat. No. 6,747,003, US patent application
20030100506 (University of Minnesota) and WO 00/66753, US patent
applications US 20010018414, US 2004220106, and US 200131005, U.S.
Pat. No. 6,762,286 and U.S. Pat. No. 6,693,075 (University of
Minnesota); and FVII variants as disclosed in WO 01/58935, U.S.
Pat. No. 6,806,063, US patent application 20030096338 (Maxygen
ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO
04/083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS), as well as
in WO 04/108763 (Canadian Blood Services).
[0068] Non-limiting examples of FVII variants having increased
biological activity compared to wild-type FVIIa include FVII
variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218,
PCT/DK02/00635 (corresponding to WO 03/027147), Danish patent
application PA 2002 01423 (corresponding to WO 04/029090), Danish
patent application PA 2001 01627 (corresponding to WO 03/027147);
WO 02/38162 (Scripps Research Institute); and FVIIa variants with
enhanced activity as disclosed in JP 2001061479
(Chemo-Sero-Therapeutic Res Inst.). Examples of variants of factor
VII include, without limitation, L305V-FVII,
L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,
V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,
V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,
V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,
K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,
V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII,
L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII,
L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII,
L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII,
L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,
L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII,
L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII,
L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII,
L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII,
L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,
L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII,
S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,
S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII,
S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII,
K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII,
K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,
K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII,
K316Q/V158T-FVII, S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII,
S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII,
S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII,
S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII,
S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII,
S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII,
S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII,
S314E/L305V/V158D/E296V/M298Q-FVII,
S314E/L305V/V158T/E296V/M298Q-FVII,
S314E/L305V/V158T/K337A/M298Q-FVII,
S314E/L305V/V158T/E296V/K337A-FVII,
S314E/L305V/V158D/K337A/M298Q-FVII,
S314E/L305V/V158D/E296V/K337A-FVII,
S314E/L305V/V158D/E296V/M298Q/K337A-FVII,
S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,
K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII,
K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII,
K316H/L305V/K337A/V158T-FVII, K316H/L305V/K337A/M298Q-FVII,
K316H/L305V/K337A/E296V-FVII, K316H/L305V/K337A/V158D-FVII,
K316H/L305V/V158D/M298Q-FVII, K316H/L305V/V158D/E296V-FVII,
K316H/L305V/V158T/M298Q-FVII, K316H/L305V/V158T/E296V-FVII,
K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII,
K316H/L305V/V158T/E296V/M298Q-FVII,
K316H/L305V/V158T/K337A/M298Q-FVII,
K316H/L305V/V158T/E296V/K337A-FVII,
K316H/L305V/V158D/K337A/M298Q-FVII,
K316H/L305V/V158D/E296V/K337A-FVII,
K316H/L305V/V158D/E296V/M298Q/K337A-FVII,
K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,
K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII,
K316Q/L305V/M298Q-FVII, K316Q/L305V/V158T-FVII,
K316Q/L305V/K337A/V158T-FVII, K316Q/L305V/K337A/M298Q-FVII,
K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/K337A/V158D-FVII,
K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII,
K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII,
K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII,
K316Q/L305V/V158T/E296V/M298Q-FVII,
K316Q/L305V/V158T/K337A/M298Q-FVII,
K316Q/L305V/V158T/E296V/K337A-FVII,
K316Q/L305V/V158D/K337A/M298Q-FVII,
K316Q/L305V/V158D/E296V/K337A-FVII,
K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,
K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,
F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII,
F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII,
F374Y/L305V/K337A-FVII, F374Y/L305V/V158D-FVII,
F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,
F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII,
F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVII,
F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,
F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII,
F374Y/V158D/M298Q-FVII, F374Y/V158D/E296V-FVII,
F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,
F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII,
F374Y/S314E/M298Q-FVII, F374Y/E296V/M298Q-FVII,
F374Y/L305V/K337A/V158D-FVII, F374Y/L305V/K337A/E296V-FVII,
F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVII,
F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V-FVII,
F374Y/L305V/V158D/M298Q-FVII, F374Y/L305V/V158D/S314E-FVII,
F374Y/L305V/E296V/M298Q-FVII, F374Y/L305V/E296V/V158T-FVII,
F374Y/L305V/E296V/S314E-FVII, F374Y/L305V/M298Q/V158T-FVII,
F374Y/L305V/M298Q/S314E-FVII, F374Y/L305V/V158T/S314E-FVII,
F374Y/K337A/S314E/V158T-FVII, F374Y/K337A/S314E/M298Q-FVII,
F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII,
F374Y/K337A/V158T/M298Q-FVII, F374Y/K337A/V158T/E296V-FVII,
F374Y/K337A/M298Q/E296V-FVII, F374Y/K337A/M298Q/V158D-FVII,
F374Y/K337A/E296V/V158D-FVII, F374Y/V158D/S314E/M298Q-FVII,
F374Y/V158D/S314E/E296V-FVII, F374Y/V158D/M298Q/E296V-FVII,
F374Y/V158T/S314E/E296V-FVII, F374Y/V158T/S314E/M298Q-FVII,
F374Y/V158T/M298Q/E296V-FVII, F374Y/E296V/S314E/M298Q-FVII,
F374Y/L305V/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/K337A/S314E-FVII,
F374Y/E296V/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A-FVII,
F374Y/L305V/E296V/M298Q/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A-FVII,
F374Y/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158D/K337A/S314E-FVII,
F374Y/V158D/M298Q/K337A/S314E-FVII,
F374Y/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q-FVII,
F374Y/L305V/V158D/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A-FVII,
F374Y/L305V/V158D/M298Q/S314E-FVII,
F374Y/L305V/V158D/E296V/S314E-FVII,
F374Y/V158T/E296V/M298Q/K337A-FVII,
F374Y/V158T/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158T/K337A/S314E-FVII,
F374Y/V158T/M298Q/K337A/S314E-FVII,
F374Y/V158T/E296V/K337A/S314E-FVII,
F374Y/L305V/V158T/E296V/M298Q-FVII,
F374Y/L305V/V158T/M298Q/K337A-FVII,
F374Y/L305V/V158T/E296V/K337A-FVII,
F374Y/L305V/V158T/M298Q/S314E-FVII,
F374Y/L305V/V158T/E296V/S314E-FVII,
F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,
F374Y/L305V/E296V/K337A/V158T/S314E-FVII,
F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,
S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, T106N-FVII,
K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII,
R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; and FVII having
substitutions, additions or deletions in the amino acid sequence
from 233Thr to 240Asn; FVII having substitutions, additions or
deletions in the amino acid sequence from 304Arg to 329Cys; and
FVII having substitutions, additions or deletions in the amino acid
sequence from 153Ile to 223Arg.
[0069] 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%, such as, e.g., at
least about 50%, at least about 75%, at least about 90%, at least
about 120, at least about 130, or at least about 150% 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
below. 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.
[0070] 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, "General Methods"
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, "General Methods" below); and (v) measuring
generation of thrombin in a TF-independent in vitro system (see,
"General Methods" below).
Factor IX Polypeptides and Factor IX-Related Polypeptides
[0071] The present invention encompasses factor IX polypeptides,
such as, e.g., those having the amino acid sequence disclosed in,
e.g., Jaye et al., Nucleic Acids Res. 11: 2325-2335, 1983.
(wild-type human factor IX).
[0072] In practicing the present invention, any factor IX
polypeptide may be used that is effective in preventing or treating
bleeding. This includes factor IX polypeptides derived from blood
or plasma, or produced by recombinant means.
[0073] As used herein, "factor IX polypeptide" encompasses, without
limitation, factor IX, as well as factor IX-related polypeptides.
The term "factor IX" is intended to encompass, without limitation,
polypeptides having the amino acid sequence as described in Jaye et
al., Nucleic Acids Res. 1983 (see above) (wild-type human factor
IX), as well as wild-type Factor IX derived from other species,
such as, e.g., bovine, porcine, canine, murine, and salmon Factor
IX. It further encompasses natural allelic variations of Factor IX
that may exist and occur from one individual to another. Also,
degree and location of glycosylation or other post-translation
modifications may vary depending on the chosen host cells and the
nature of the host cellular environment. The term "Factor IX" is
also intended to encompass Factor IX 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 IXa.
[0074] "Factor IX-related polypeptides" include, without
limitation, factor IX polypeptides that have either been chemically
modified relative to human factor IX and/or contain one or more
amino acid sequence alterations relative to human factor IX (i.e.,
factor IX variants), and/or contain truncated amino acid sequences
relative to human factor IX (i.e., factor IX fragments). Such
factor IX-related polypeptides may exhibit different properties
relative to human factor IX, including stability, phospholipid
binding, altered specific activity, and the like.
[0075] The term "factor IX-related polypeptides" are intended to
encompass such 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
IXa-related polypeptides" or "activated factor IX-related
polypeptides".
[0076] As used herein, "factor IX-related polypeptides"
encompasses, without limitation, polypeptides exhibiting
substantially the same or improved biological activity relative to
wild-type human factor IX, as well as polypeptides, in which the
factor IX biological activity has been substantially modified or
reduced relative to the activity of wild-type human factor IX.
These polypeptides include, without limitation, factor IX or factor
IXa that has been chemically modified and factor IX variants into
which specific amino acid sequence alterations have been introduced
that modify or disrupt the bioactivity of the polypeptide.
[0077] It further encompasses polypeptides with a slightly modified
amino acid sequence, for instance, polypeptides having a modified
N-terminal end including N-terminal amino acid deletions or
additions, and/or polypeptides that have been chemically modified
relative to human factor IX.
[0078] Factor IX-related polypeptides, including variants of factor
IX, whether exhibiting substantially the same or better bioactivity
than wild-type factor IX, or, alternatively, exhibiting
substantially modified or reduced bioactivity relative to wild-type
factor IX, include, without limitation, polypeptides having an
amino acid sequence that differs from the sequence of wild-type
factor IX by insertion, deletion, or substitution of one or more
amino acids.
[0079] Factor IX-related polypeptides, including variants,
encompass those that exhibit at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 100%, at least about 110%, at least about
120%, and at least about 130%, of the specific activity of
wild-type factor IX that has been produced in the same cell type,
when tested in the factor IX activity assay as described in the
present specification.
[0080] Factor IX-related polypeptides, including variants, having
substantially the same or improved biological activity relative to
wild-type factor IX encompass those that exhibit at least about
25%, preferably at least about 50%, more preferably at least about
75%, more preferably at least about 100%, more preferably at least
about 110%, more preferably at least about 120%, and most
preferably at least about 130% of the specific biological activity
of wild-type human factor IX that has been produced in the same
cell type when tested in one or more of the specific factor IX
activity assays as described. For purposes of the invention, factor
IX biological activity may be quantified as described later in the
present description (see "General Methods").
[0081] Factor IX-related polypeptides, including variants, having
substantially reduced biological activity relative to wild-type
factor IX 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 IX that has been produced in the same cell type when tested
in one or more of the specific factor IX activity assays as
described above.
[0082] Non-limiting examples of factor IX polypeptides include
plasma-derived human factor IX as described, e.g., in Chandra et
al., Biochem. Biophys. Acta 1973, 328:456; Andersson et al.,
Thromb. Res. 1975, 7:451; Suomela et al., Eur. J. Biochem. 1976,
71:145.
[0083] Suitable assays for testing for factor IX activity, and
thereby providing means for selecting suitable factor IX variants
for use in the present invention, can be performed as simple in
vitro tests as described, for example, in Wagenvoord et al.,
Haemostasis 1990; 20(5):276-88. Factor IX biological activity may
also be quantified by measuring the ability of a preparation to
correct the clotting time of factor IX-deficient plasma, e.g., as
described in Nilsson et al., 1959. (Nilsson I M, Blombaeck M,
Thilen A, von Francken I., Carriers of haemophilia A--A laboratory
study, Acta Med Scan 1959; 165:357). In this assay, biological
activity is expressed as units/ml plasma (1 unit corresponds to the
amount of FIX present in normal pooled plasma.
[0084] In some embodiments of the invention, the factor IX are
factor IX-related polypeptides wherein the ratio between the
activity of said factor IX polypeptide and the activity of native
human factor IX (wild-type factor IX) is at least about 1.25 when
tested in the "chromogenic assay" (see below); in other
embodiments, the ratio is at least about 2.0; in further
embodiments, the ratio is at least about 4.0.
O-Linked Glycosylation
[0085] In practicing the present invention, the pattern of
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. Biochem. 318:34 (1994); Harvey et al., Organic Mass
Spectrometry 29:752 (1994).
[0086] The relative content of O-glycoforms can be determined, for
example, by tryptic peptide mapping. In short, the glycoprotein is
digested with trypsin and the polypeptides containing the
O-glycosylation site are separated according to the glycan
structure by RP-HPLC chromatography, mass spectrometry or another
suitable analytical separation technique. If necessary in order to
obtaining a suitable separation, the glycoprotein can prior to the
digestion with trypsin be reduced and alkylated and the polypeptide
chain containing the O-glycosylation site is purified by, e.g.
RP-HPLC chromatography. Then the purified polypeptide is subjected
to tryptic digestion followed by analysis as described above.
Methods for Producing Glycoprotein Preparations Having a
Predetermined Pattern of O-Linked Oligosaccharides
[0087] The origin of the acceptor glycoprotein is not a critical
aspect of the invention. Typically, the glycoprotein will be
expressed in a cultured prokaryote cell or eukaryote cell such as a
mammalian, yeast insect, fungal or plant cell. The protein,
however, may also be isolated from a natural source such as plasma,
serum or blood. The glycoprotein can either be a full length
protein or a fragment.
[0088] The invention provides compositions that include
glycoprotein species that have a substantially uniform
glycosylation pattern. The methods are useful for remodelling or
altering the glycosylation pattern present on a glycoprotein upon
its initial expression. Thus, the methods of the invention provide
a practical means for large-scale preparation of glycoforms having
pre-selected or pre-determined uniform derivatization patterns. The
methods are particularly well suited for modification of
therapeutic peptides, including but not limited to, glycoproteins
that are incompletely glycosylated during production in cell
culture cells or transgenic animals. However, the preparations and
compositions of the invention may also be prepared by purification
of natural sources, such as plasma, serum or blood, or cell culture
fluids and isolating the desired glycoforms therefrom.
[0089] The polypeptides to be re-modelled in accordance with the
invention are typically prepared by cell culture processes.
Suitable host cells include, without limitation, human cells
expressing an endogenous gene such as, e.g., a Factor VII, IX, X,
or XII gene or a protein Z gene. In these cells, the endogenous
gene may be intact or may have been modified in situ, or a sequence
outside the endogenous gene may have been modified in situ to alter
the expression of the endogenous glycoprotein gene. Any human cell
capable of expressing an endogenous glycoprotein gene may be used.
Other, included host cells are heterologous host cells programmed
to express a glycoprotein such as, e.g., human Factor VII or IX or
X or XII 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. Nos. 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. Suitable host cells include
BHK 21 cells that have been adapted to grow in the absence of serum
and have been programmed to express Factor VII. 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. The host cells may also be 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.
[0090] Methods: The present invention encompasses methods for
producing a preparation comprising a predetermined
serine/threonine-linked glycoform pattern as described above and,
in further embodiments, methods for optimizing the glycoform
distribution of a glycoprotein (see FIG. 3). The individual process
steps described can be applied in different combinations in order
to obtain the desired glycoform pattern. Non-limiting examples are
given below.
[0091] In one aspect, these methods are carried out by the steps
of:
[0092] (a) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond from a cell in which it
is prepared; e.g., from an engineered cell (cell culture) or by
isolating the glycoprotein from a natural source;
[0093] (b) contacting the glycoprotein preparation with an
activated donor of the desired mono- or oligosaccharide moiety and
an enzyme suitable for transferring the desired mono- or
oligo-saccharide group under conditions appropriate for
transferring the mono- or oligo-saccharide group from the donor
moiety to the acceptor moiety, thereby producing the glycopeptide
having an altered glycosylation pattern.
[0094] In another aspect, these methods are carried out by the
steps of:
[0095] (aa) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond from a cell in which it
is prepared; e.g., from an engineered cell (cell culture) or by
isolating the glycoprotein from a natural source;
[0096] (bb) contacting the glycoprotein preparation with an enzyme
suitable for removing the terminal mono- or oligo-saccharide group
under conditions appropriate for removing said mono- or
oligo-saccharide group, thereby producing the glycopeptide having
an altered glycosylation pattern.
[0097] In one embodiment, the methods comprise a combination of
steps (b) and (bb). In one embodiment the methods further comprise
a step of isolating the glycoprotein having an altered
glycosylation pattern.
In one embodiment, the methods comprise a further step of:
[0098] Analyzing the structure of the oligosaccharides linked to
the polypeptides to determine a glycoform pattern, and, optionally,
repeating steps (b) and/or (bb) until a desired glycoform pattern
is achieved.
[0099] 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.
[0100] In one embodiment, the desired glycoform pattern is a
substantially uniform glucose-O-serine/threonine glycosylation: In
this embodiment, wherein the initially obtained glycoprotein
contains terminal xylose the method (METHOD B) comprises the steps
of:
[0101] (a) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond from a cell in which it
is prepared; e.g., from an engineered cell (cell culture) or by
isolating the glycoprotein from a natural source;
[0102] (b) contacting the preparation obtained in step (a) with a
xylosidase under conditions appropriate for removing xylose
residues from the glycoprotein, thereby producing the glycoprotein
having an altered glycosylation pattern.
[0103] In one embodiment, the method further includes the step of
isolating the glycoprotein prepared in step b having a
Glc-O-Ser/Thr glycosylation.
[0104] In one embodiment, the method further includes the step of
analysing the structure of the oligosaccharides linked to the
polypeptides to determine a glycoform pattern, and, optionally,
repeating step (b) until the desired glycoform pattern is
achieved.
[0105] In another embodiment for making a desired glycoforms
pattern in the form of a substantially uniform
glucose-O-serine/threonine glycosylation, the method (METHOD C)
comprises the steps of:
[0106] (a) obtaining a preparation of a polypeptide containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif, e.g., from an engineered cell
(cell culture) or by isolating the glycoprotein from a natural
source;
[0107] (b) contacting the preparation obtained in step (a) with a
O-glucosyltransferase and an activated glucose donor under
conditions appropriate for transferring a glucose residue from the
glucose donor moiety to the serine/threonine acceptor moiety,
thereby producing the polypeptide having an altered glycosylation
pattern.
[0108] In one embodiment, the method further includes the step of
isolating the glycoprotein prepared in step b having a
Glc-O-Ser/Thr glycosylation.
[0109] In one embodiment, the method further includes the step of
analysing the structure of the oligosaccharides linked to the
polypeptides to determine a glycoform pattern, and, optionally,
repeating step (b) until the desired glycoform pattern is
achieved.
[0110] In one embodiment, the desired glycoform pattern is a
substantially uniform xylose-glucose-O-serine/threonine
glycosylation: In this embodiment, the method (METHOD A1) comprises
the steps of:
[0111] (a) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond; e.g., from an
engineered cell (cell culture) or by isolating the glycoprotein
from a natural source;
[0112] (b) contacting the preparation obtained in step (a) with
UDP-D-xylose: .beta.-D-glucoside .alpha.-1,3-D-xylosyltransferase
and an activated xylosyl donor under conditions appropriate for
transferring a xylose residue from the xylose donor moiety to the
acceptor moiety, thereby producing the glycopeptide having an
altered glycosylation pattern.
[0113] In one embodiment, the method further includes the step of
isolating the glycoprotein prepared in step b having a
Xyl-Glc-O-Ser/Thr glycosylation.
[0114] In one embodiment, the method further includes the step of
analysing the structure of the oligosaccharides linked to the
polypeptides to determine a glycoform pattern, and, optionally,
repeating step (b) until the desired glycoform pattern is
achieved.
[0115] In one embodiment, the method further includes the step of
removing terminal xylose-residues by subjecting the preparation
obtained in step (a) to METHOD B prior to step (b).
[0116] In one embodiment, the desired glycoform pattern is a
substantially uniform xylose-xylose-glucose-O-serine/threonine
glycosylation: In this embodiment, the method (METHOD A2) comprises
the steps of:
[0117] (a) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond; e.g., from an
engineered cell (cell culture) or by isolating the glycoprotein
from a natural source;
[0118] (b) contacting the preparation obtained in step (a) with
UDP-D-xylose: .beta.-D-glucoside .alpha.-1,3-D-xylosyltransferase
and an activated xylosyl donor under conditions appropriate for
transferring a xylose residue from the xylose donor moiety to the
acceptor moiety, thereby producing the glycopeptide having an
altered glycosylation pattern.
[0119] (c) contacting the preparation obtained in step (b) with
UDP-D-xylose: .alpha.-D-xyloside .alpha.-1,3-xylosyltransferase and
an activated xylosyl donor under conditions appropriate for
transferring a xylose residue from the xylose donor moiety to the
acceptor moiety, thereby producing the glycopeptide having an
altered glycosylation pattern.
[0120] In one embodiment, the method further includes the step of
isolating the preparation obtained in step (b) prior to subjecting
the preparation to step (c).
[0121] In one embodiment, the method further includes the step of
isolating the glycoprotein prepared in step (c) having a
Xyl-Xyl-Glc-O-Ser/Thr glycosylation.
[0122] In one embodiment, the method further includes the step of
analysing the structure of the oligosaccharides linked to the
polypeptides to determine a glycoform pattern, and, optionally,
repeating step (b) and/or step (c) until the desired glycoform
pattern is achieved.
[0123] In one embodiment, the method further includes the step of
removing terminal xylose-residues by subjecting the preparation
obtained in step (a) to METHOD B prior to step (b).
[0124] In different embodiments, the glycoprotein exhibits
substantially uniform Xyl-Xyl-Glc-O-Ser glycosylation,
Xyl-Glc-O-Ser glycosylation, and Glc-O-Ser glycosylation; Ser being
the serine of the contained Cys-X1-Ser-X2-Pro-Cys motif (X1 and X2
independently being any amino acid residue). In other, different
embodiments, the glycoprotein exhibits substantially uniform
Xyl-Xyl-Glc-O-Thr glycosylation, Xyl-Glc-O-Thr glycosylation, and
Glc-O-Thr glycosylation; Thr being the threonine of the contained
Cys-X1-Thr-X2-Pro-Cys motif (X1 and X2 independently being any
amino acid residue).
[0125] In different embodiments, the polypeptides are selected from
the list of: Factor VII polypeptides, Factor VII-related
polypeptides, Factor IX polypeptides, Factor IX-related
polypeptides, Factor X polypeptides, and Factor X-related
polypeptides.
[0126] In preferred embodiments, the glycoprotein preparation is
selected from the list of:
Factor VII polypeptides exhibiting substantially uniform
Xyl-Xyl-Glc-O-Ser52 glycosylation, Factor VII polypeptides
exhibiting substantially uniform Xyl-Glc-O-Ser52 glycosylation
Factor VII polypeptides exhibiting substantially uniform
Glc-O-Ser52 glycosylation Factor VII-related polypeptides
exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser52 glycosylation
Factor VII-related polypeptides exhibiting substantially uniform
Xyl-Glc-O-Ser52 glycosylation Factor VII-related polypeptides
exhibiting substantially uniform Glc-O-Ser52 glycosylation Factor
VII variants exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser52
glycosylation Factor VII variants exhibiting substantially uniform
Xyl-Glc-O-Ser52 glycosylation Factor VII variants exhibiting
substantially uniform Glc-O-Ser52 glycosylation Factor IX
polypeptides exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser53
glycosylation Factor IX polypeptides exhibiting substantially
uniform Xyl-Glc-O-Ser53 glycosylation Factor IX polypeptides
exhibiting substantially uniform Glc-O-Ser53 glycosylation Factor
IX-related polypeptides exhibiting substantially uniform
Xyl-Xyl-Glc-O-Ser53 glycosylation Factor IX-related polypeptides
exhibiting substantially uniform Xyl-Glc-O-Ser53 glycosylation
Factor IX-related polypeptides exhibiting substantially uniform
Glc-O-Ser53 glycosylation Factor IX variants exhibiting
substantially uniform Xyl-Xyl-Glc-O-Ser53 glycosylation Factor IX
variants exhibiting substantially uniform Xyl-Glc-O-Ser53
glycosylation Factor IX variants exhibiting substantially uniform
Glc-O-Ser53 glycosylation
[0127] It is to be understood that oligosaccharides such as
Xyl-Xyl- may also be transferred to the acceptor Glc-O-Ser/Thr
moiety by using a suitable transferring enzyme and an activated
Xyl-Xyl- donor.
[0128] Chromatographic method: The present invention also
encompasses hydrophobic interaction chromatographic methods for
producing a preparation comprising a predetermined
serine/threonine-linked glycoform pattern as described above, and
for purifying a O-glycosylated polypeptide having a desired
glycoform pattern from a composition comprising said polypeptide
and polypeptides having unwanted glycoform patterns.
[0129] In one aspect, the method comprises the following steps:
[0130] (a) obtaining a preparation of a glycoprotein containing a
Cys-X1-Ser/Thr-X2-Pro-Cys motif and wherein said serine/threonine
forms part of a Glc-O-Ser/Thr covalent bond from a cell in which it
is prepared; e.g., from an engineered cell (cell culture) or by
isolating the glycoprotein from a natural source;
[0131] (b) binding the glycoprotein to an hydrophobic interaction
material using a solution comprising water, optionally a salt
component, and optionally a buffer,
[0132] (c) optionally washing the hydrophobic interaction material
using a solution comprising water, optionally a salt component, and
optionally a buffer so as to elute contaminants from the
hydrophobic interaction material;
[0133] (d) washing the hydrophobic interaction material using a
solution comprising an organic modifier, water, optionally a salt
component, and optionally a buffer, at a linear or step gradient or
isocratically in salt component so as to separate glycoproteins
having a desired glycoform patter from glycoproteins not having the
desired glycoform from the hydrophobic interaction material;
[0134] (e) collecting the fraction containing the glycoproteins
having the desired glycoform pattern.
[0135] In one embodiment, the above-described methods further
includes the step of repeating steps (a) to e) by subjecting the
preparation obtained in step (e) to steps (a) to (e). This further
step may be repeated more than once if deemed necessary.
[0136] It is to be understood that the preparations according to
the invention may also be prepared by a process comprising a
combination of purification steps whereby glycoprotein species
having the desired glycosylation are captured from the cell culture
liquid or natural source of origin and the above-described
enzymatic methods.
[0137] The above-described 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.
[0138] Further enzymatic treatments may be used in connection with
the above methods to modify the oligosaccharide pattern of N- or
O-linked glycans of a preparation; such treatments 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.).
Glycoprotein Preparations
[0139] As used herein, a "glycoprotein preparation" refers to a
plurality of glycoforms that have been separated from the cell in
which they were synthesized. The glycoprotein preparation include
inactivated forms, activated forms, functionally related
polypeptides such as, e.g., variants and chemically modified forms,
that have been separated from the cell in which they were
synthesized.
[0140] For example, 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 or isolated from a
natural source. Likewise, a "Factor IX preparation" refers to a
plurality of Factor IX polypeptides, Factor IXa polypeptides, or
Factor IX-related polypeptides, including variants or chemically
modified forms, that have been separated from the cell in which
they were synthesized or isolated from a natural source (e.g.,
plasma, serum, blood).
[0141] 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.
[0142] Optionally, the 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 or anti-Factor IX 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-related proteins derived from the host cell.
[0143] Factor VII and Factor VII-related polypeptides, Factor IX
and Factor IX-related polypeptides, or Factor X and Factor
X-related polypeptides, respectively, 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, IX or
X, respectively, may be activated by passing it through an
ion-exchange chromatography column, such as Mono Q.RTM. (Pharmacia)
or the like. The resulting activated polypeptide, e.g., Factor VII,
may then be formulated and administered as described below.
Functional Properties of Glycoprotein Preparations
[0144] The preparations of glycoproteins having predetermined
oligosaccharide patterns according to the invention (including
Factor VII polypeptides, Factor VII-related polypeptides, Factor IX
polypeptides and Factor IX-related polypeptides) 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; c) immunogenicity in humans, and d) biological activity,
such as, e.g., clotting activity.
[0145] 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 wild-type Factor IX or a particular variant
or chemically modified form) except for exhibiting a different
pattern of serine/threonine-linked glycosylation.
[0146] Storage stability of a glycoprotein (e.g., 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 (e.g., Factor VIIa) aggregates of
said glycoprotein in the preparation.
[0147] 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.
[0148] 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 contents of aggregates may be determined
according to methods known to the skilled person, such as, e.g.,
gel permeation HPLC methods. For example, the content of Factor VII
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).
[0149] "Bioavailability" refers to the proportion of an
administered dose of a (e.g., Factor VII or Factor VII-related)
glycoprotein 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 (e.g., Factor VII or Factor VII-related)
glycosylated 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 polypeptide [e.g.,
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.
[0150] 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.
[0151] "Half-life" refers to the time required for the plasma
concentration of (e.g., Factor VII polypeptides of Factor
VII-related polypeptides) the glycoprotein 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.
[0152] "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.
Pharmaceutical compositions and Methods of Use
[0153] The preparations of the present invention may be used to
treat any syndrome responsive to the relevant glycoprotein. Factor
VII-, FIX and FX-responsive syndromes, respectively, include
syndromes 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.
[0154] 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
pre-scribed 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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).
[0160] 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.
[0161] 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,
stents, 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.
[0162] The pharmaceutical compositions of the invention may further
comprise other bioactive agents, such as, e.g., non-Factor
VII-related coagulants or anticoagulants.
EXPERIMENTALS
General Methods
[0163] .alpha.-xylosidase Assay
[0164] The .alpha.-xylosidase assays are conducted in an
appropriate buffer, e.g. 50 mM sodium acetate, pH 4.5, containing a
suitable substrate, e.g. the O-glycopeptides that can be obtained
from the O-glycopeptide map of the relevant glycoprotein (e.g.,
rFVIIa). The reaction is stopped after an appropriate time that can
be determined experimentally, by e.g. addition of trifluoroacetic
acid, and the assay mixtures are analysed by HPLC.
.alpha.-Xylosyltransferase Assay
[0165] The .alpha.-xylosyltransferase assays are conducted in an
appropriate buffer, e.g. 10 mM Hepes, pH 7.2, 0.1% Triton X-100,
0.5 mM UDP-Xylose (Sigma U5875), containing a suitable substrate,
e.g. the O-glycopeptides that can be obtained from the
O-glycopeptide map of the relevant glycoprotein (e.g., rFVIIa) or
the pyridyl-aminated oligosaccharides prepared as described in
Minamida et al. (Minamida et. al., Detection of UDP-D-xylose:
.alpha.-D-xyloside .alpha.1-3xylosyltransferase activity in human
hepatoma cell line HepG2. J. Biochem. 120 1002-1006, 1996). The
reaction is stopped after an appropriate time, that can be
determined experimentally, by e.g. addition of trifluoroacetic
acid, and the assay mixtures are analysed by HPLC.
[0166] The .alpha.-xylosidase and .alpha.-xylosyltransferase assays
are optimized for time and, optionally for temperature and pH.
O-Glucosyltransferase Assay.
[0167] The O-glucosyltransferase assays are conducted, e.g., as
described by Shao et al. (Glycobiology 12(11) 763-770 2002).
Factor VII Assays
[0168] A suitable assay for testing for factor VIIa activity and
thereby selecting suitable factor VIIa variants can be performed as
a simple preliminary in vitro test. The assay is also suitable for
selecting suitable factor VIIa variants.
In Vitro Hydrolysis Assay
[0169] Native (wild-type) factor VIIa and factor VIIa variant (both
hereafter referred to as "factor VIIa") may be assayed for specific
activities. They may also be assayed in parallel to directly
compare their specific activities. 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), final concentration 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 variant and wild-type factor
VIIa:
Ratio=(A.sub.405 nm factor VIIa variant)/(A.sub.405 nm factor VIIa
wild-type).
[0170] Based thereon, factor VIIa variants with an activity
comparable to or higher than native factor VIIa may be identified,
such as, for example, variants where the ratio between the activity
of the variant and the activity of native factor VII (wild-type
FVII) is around, versus above 1.0.
[0171] The activity of factor VIIa or factor VIIa variants may also
be measured using a physiological substrate such as factor X,
suitably at a concentration of 100-1000 nM, where the factor Xa
generated is measured after the addition of a suitable chromogenic
substrate (eg. S-2765). In addition, the activity assay may be run
at physiological temperature.
In Vitro Proteolysis Assay
[0172] Native (wild-type) Factor VIIa and Factor VIIa variant (both
hereafter referred to as "Factor VIIa") are assayed in parallel to
directly compare their specific activities. The assay is carried
out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa
(10 nM) and Factor X (0.8 microM) in 100 microL 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 microL 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 variant and wild-type Factor VIIa:
Ratio=(A405 nm Factor VIIa variant)/(A405 nm Factor VIIa
wild-type).
[0173] Based thereon, factor VIIa variants with an activity
comparable to or higher than native factor VIIa may be identified,
such as, for example, variants where the ratio between the activity
of the variant and the activity of native factor VII (wild-type
FVII) is around 1, versus above 1.0.
Thrombin Generation Assay:
[0174] The ability of factor VII or factor VII-related polypeptides
(e.g., variants) to generate thrombin can be measured in an assay
comprising all relevant coagulation factors and inhibitors at
physiological concentrations and activated platelets (as described
on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547
which is hereby incorporated as reference).
[0175] Clot Assays.
1st Generation Assay
[0176] The activity of the Factor VII polypeptides may also be
measured using a one-stage clot assay essentially as described in
WO 92/15686 or U.S. Pat. No. 5,997,864. Briefly, the sample to be
tested is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 .mu.L is
incubated with 100 .mu.L of Factor VII deficient plasma and 200
.mu.L of thromboplastin C containing 10 mM Ca2+. Clotting times are
measured and compared to a standard curve using a reference
standard or a pool of citrated normal human plasma in serial
dilution.
2nd Generation Assay:
[0177] Essentially the same, except that recombinant human tissue
factor is used instead for thromboplastin C.
Factor IX Assay
Test for Factor IX Activity:
[0178] Suitable assays for testing for factor IX activity, and
thereby providing means for selecting suitable factor IX variants
for use in the present invention, can be performed as simple in
vitro tests as described, for example, in Wagenvoord et al.,
Haemostasis 1990; 20(5):276-88
[0179] Factor IX biological activity may also be quantified by
measuring the ability of a preparation to correct the clotting time
of factor IX-deficient plasma, e.g., as described in Nilsson et
al., 1959. (Nilsson I M, Blombaeck M, Thilen A, von Francken I.,
Carriers of haemophilia A--A laboratory study, Acta Med Scan 1959;
165:357). In this assay, biological activity is expressed as
units/ml plasma (1 unit corresponds to the amount of FIX present in
normal pooled plasma.
EXAMPLES
[0180] The following examples are intended as non-limiting
illustrations of the present invention.
Example 1
Preparation of .alpha.-Xylosidase by Extraction and
Purification
[0181] The enzyme, .alpha.-xylosidase, can be prepared from various
sources, e.g. from plant material as described by Monroe et al.
(Plant Physiology and Biochemistry 41:877-885 (2003)). For example,
plant tissues from e.g. Arabidopsis thaliana are ground in a mortar
and pestle with quartz sand in two volumes of Buffer A (40 mM
Hepes, pH 7.0, 1 M NaCl), and the filtered extract is centrifuged
at 15000.times.g for 15 min. Ammonium sulfate is added to for
example 80% saturation. Precipated proteins are collected by
centrifugation at 15000.times.g for 15 min and redissolved in
Buffer A. The .alpha.-xylosidase is purified by chromatography, for
example on a Concanavalin A-Sepharose column, on an anion-exchange
column and/or on other chromatographic columns known for the
skilled person. Fractions are collected during elution and the
fractions containing the .alpha.-xylosidase enzyme are identified
by use of the .alpha.-xylosidase assay.
Example 2
Preparation of .alpha.-Xylosidase by Cloning and Expression in E.
coli and Purification
[0182] Genes encoding .alpha.-xylosidases, which can hydrolyse
alpha xylosidic bonds, have been cloned and characterized
previously and genes showing significant homology to characterized
.alpha.-xylosidases have been annotated in the genomes from several
prokaryotic and eukaryotic organisms. The gene sequences are
available in databases such as SWISS-PROT or NCBI and can be
amplified by PCR from genomic DNA from the respective organisms.
Several candidates were chosen for cloning and expression in E.
coli after searching protein databases for the presence of
.alpha.-xylosidase proteins. The following candidate genes were
selected on the basis of already existing annotation in databases
(A), previous published characterization(P) or based on homology
analysis to known axylosidases(H): gene tm0308 (Thermotoga
maritima: A); gene bt3085 (2139 bp) and gene bt3659(2475 bp)
(Bacteroides thetaiotaomicron: A); gene bf0551 (2238 bp) and gene
bf1247 (2538 bp) (Bacteroides fragilis: A); gene bl02681(2310
bp)(Bacillus licheniformis: H); gene bh1905 (2328 bp)(Bacillus
halodurans: H), gene xylS (2196 bp)(Sulfolobus solfataricus: P);
gene yicI (2319 bp) (Escherichia coli: P)
Strategy for Cloning and Expression of .alpha.-Xylosidases in E.
coli
[0183] The SignalP software (Bendtsen, J. D. et al. J. Mol. Biol.,
340:783-795, 2004) is used to evaluate whether a signal peptide is
potentially present in the N-terminal of the candidate enzymes.
BF0551, BF1247, BT3085, BT3659 are presumably secreted as indicated
by a strong prediction of a signal peptidase I cleavage site. A
methionine codon encoding a start-methionine is included in front
of the first amino acid following the predicted cleavage site.
[0184] Purified genomic DNA from Bacteroides thetaiotaomicron (ATCC
29148D), Bacteroides fragilis (ATCC 25285D), Bacillus haludurans
(ATCC 21591D&BAA-125D), Sulfolobus solfataricus (ATCC 35092D),
Thermotoga maritima (ATCC 43589D) is obtained from American Type
Culture Collection. In case of E. coli (strain K-12 derivative) and
Bacillus licheniformis (ATCC 28450), genomic DNA is prepared from
bacterial cells cultivated overnight in LB medium using the DNeasy
tissue kit (Qiagen) according to the manufactures instructions.
[0185] Forward and reverse primers for PCR amplification are
designed with an extension in the 5'-ends comprising the
restriction enzyme cleavage sites NdeI (or XbaI) and XmaI,
respectively. PCR is performed using the following conditions: 1)
95.degree. C. for 3 min: denaturation, 2) 94.degree. C. for 30 sec:
denaturation, 3) 55.degree. C. or 60.degree. C. for 30 sec:
annealing, 4) 72.degree. C. for 2 min: elongation. Step 2-4 is
repeated for 15 cycles. PCR products are separated on 1% ethidium
bromide agarose gels and bands showing the correct predicted sizes
are excised from the gels and purified using the GFX DNA
purification kit (Amersham Pharmacia). Purified PCR products are
cloned into the pCR2.1TOPO vector according to the instructions of
the manufacturer (Invitrogen). Clones showing the correct
restriction enzyme cleavage profile are sequenced to evaluate the
DNA sequence. The insert representing the .alpha.-xylosidase genes
are released from the pCR2.1TOPO vector using the relevant
restriction enzymes. A pET11a E. coli expression vector (Novagen)
containing a NdeI (and XbaI) and a XmaI site is cleaved with
relevant restriction enzymes and the vector part is purified as
described for the PCR products. Vector and inserts are ligated
together using the Rapid Ligation Kit (Roche) according to the
manufacturer's instructions.
[0186] Ligation products are transformed into E. coli TOP10
(Invitrogen) cells by means of chemical transformation or heat
shock methods known to the skilled persons. Cells are plated on
LB/ampecillin(Amp)-medium culture plates overnight. Single colonies
are selected from plates and grown overnight in LB/Amp medium.
Purified pET plasmids from each colony are screened for the
presence of correct inserts using restriction cleavage enzymes and
evaluation of sizes of released inserts.
[0187] E. Coli Rosetta DE3 (Novagen) is transformed with pET
plasmids containing the axylosidase genes and plated on
chloramphinicol(Cam)/Amp LB plates. Cells from overnight plates are
resuspended in liquid Cam/Amp LB medium and diluted to
OD.sub.600=0.1. Cells in liquid medium are propagated until
OD.sub.600=0.4-0.8. Cells are then equilibrated to a temperature of
18.degree. C. for 30 min. and protein induction is induced with 0.5
mM IPTG o/n at 18.degree. C. Cells are harvested and pellets are
re-suspended in a buffer (e.g., 25 mM Tris HCl pH 7 or 10 mM
potassium phosphate buffer pH 7) to a cell density corresponding to
OD.sub.600=.about.10. Cells are sonicated on ice for 3-7 times
15-30 sec with interruptions of 30 sec on ice. Cell debris is
removed by centrifugation and supernatants are assayed for
activity.
Assay for .alpha.-Xylosidase Activity
[0188] Supernatants resulting from sonication are evaluated on
p-nitrophenyl .alpha.-D xylopyranoside (Sigma) for presence of
.alpha.-xylosidase activity. Crude enzyme is incubated with 5 mM
p-nitrophenyl .alpha.-D xylopyranoside at 37.degree. C. for 1-2
hours in a buffer (e.g., 10 mM potassium buffer pH 7 or a 25 mM
Tris HCl pH 7 buffer). Crude enzymes are also assayed on a fragment
of human FVII comprising the Xyl-Xyl-Glc-O-Ser52 glycosylation
(peptide fragment consisting of amino acid residues 39-62 of FVII)
to evaluate whether the enzyme can cleave the alpha-1,3 xylosidic
bonds. The incubation with peptide is performed for 3 hours or
overnight at 37.degree. C. Peptide samples incubated with or
without axylosidase are then evaluated by MALDI MS directly after
incubation to evaluate whether the enzyme can remove zero, one or
two xylose sugars from the glycopeptide.
Purification of .alpha.-Xylosidases
[0189] A partial purification of the expressed .alpha.-xylosidase
is performed prior to incubation with rFVII. Supernatants (from
approximately 20-50 ml cell culture) obtained after cell disruption
in a suitable buffer (eg. a 10 mM phosphate buffer pH 7). In case
of enzymes coming from thermophiles (eg. tm0308, BH1905, XylS),
supernatants are also heated at 50-70.degree. C. for 30 min, cooled
on ice for 10 min and precipitate is removed by centrifugation for
15 min at 15.000 G in order to remove thermo-labile E. coli
contaminants.
[0190] The supernatants are sterile filtrated and applied to a 1 ml
DEAE FF column (Amersham Pharmacia). The purification is performed
with the AKTA explorer (Amersham Pharmacia) FPLC with the following
buffers: Buffer A: 25 mM sodium phosphate pH 7, Buffer B: 25 mM
sodium phosphate pH 7 and 1 M NaCl. After the application is
loaded, unbound sample is washed out with buffer A for 5 CV. A
gradient from 0-100% buffer B is used for 20 CV during which the
target protein is eluted in fractions. After purification,
fractions comprising the main peak in the resulting chromatogram
are assayed by incubation on p-nitrophenyl .alpha.-D xylopyranoside
or by SDS PAGE. The fractions containing the axylosidase activity
are diluted in a 20 mM Tris HCl pH 7, 2 mM CaCl.sub.2 buffer and
concentrated on Vivaspin 20 50.000 MWCO columns (Vivascience) by
centrifugation at 2900 rpm.
[0191] O-Glycoforms of rFVIIa with Exclusively Glucose at Serine
52
[0192] The O-glycoforms of rFVIIa with exclusively glucose at
serine 52 are obtained by incubation of rFVIIa in an appropriate
buffer, e.g. glycylglycine or 20 mM Tris HCl pH 7.0, 2 mM
CaCl.sub.2, with purified .alpha.-xylosidase for an appropriate
time, that can be determined experimentally. Mass spectra
visualizing the deglycosylation are obtained by analysing rFVII
.alpha.-xylosidase incubations ESI-MS (Q-STAR).
[0193] The resulting glycan-remodeled rFVIIa is purified from the
.alpha.-xylosidase enzyme by for example anion-exchange
chromatography or gel filtration or suitable combinations. The
purity of the prepared O-glycoform of rFVIIa is verified by the
O-glycopeptide map of rFVIIa.
Example 3
Preparation of .alpha.-Xylosidase by Cloning and Expression of T.
maritima Putative .alpha.-Xylosidase Gene (tm0308) in E. coli and
Purification
[0194] The above strategy (see Example 2) was followed all the way
to a conclusion for tm0308. The T. maritima putative
.alpha.-xylosidase gene (tm0308) was PCR amplified and cloned into
a E. coli pET11a vector. Soluble tm0308 could be obtained after
expression in an E. coli Rosetta (DE3) expression strain and
evaluation of a crude TM0308 preparation on a p-nitrophenyl
.alpha.-D xylopyranoside, clearly indicated .alpha.-xylosidase
activity. The .alpha.-xylosidase was partly purified using DEAE FF
chromatography followed by up-concentration by ultra filtration.
The partly purified enzyme was incubated with FVII in a 25 mM Tris
pH 7, 2 mM CaCl.sub.2 buffer at different enzyme/FVII ratios for 3
hours at 50.degree. C. or overnight at 37.degree. C. Controls with
identical compositions of .alpha.-xylosidase and rFVII, to which
synthetic substrate was added, showed that the enzyme was active
under these conditions and it was possible to visualize FVII with
and without xylosidase treatment on SDS-gels and by ESI-MS.
However, no significant removal of the xylose sugars linked to
Glc-O-Ser52 could be detected in this first experiment. In
contrast, removal of xylose from a purified reduced and alkylated
FVIIa peptide comprising Xyl-Xyl-Glc-O-Ser52 was observed.
Example 4
Preparation of .alpha.-Xylosidase by Cloning and Expression in E.
coli
[0195] The following constructs have been cloned into the pET
expression vectors in accordance with the strategy described in
Example 2: Gene bl2681(2310 bp)(Bacillus licheniformis: H); gene
bl1905 (2328 bp)(Bacillus halodurans: H), gene xylS (2196
bp)(Sulfolobus solfataricus: P); gene yicI (2319 bp) (Escherichia
coli: P).
The constructs will be expressed in Rosetta, isolated, purified,
and evaluated for .alpha.-xylosidase activity in accordance with
the above-described strategy. Each .alpha.-xylosidase will be
incubated with rFVIIa in an appropriate buffer, e.g. glycylglycine
or 20 mM Tris HCl pH 7.0, 2 mM CaCl.sub.2, for an appropriate time
that can be determined experimentally and MS Spectra visualizing
the deglycosylation will be obtained by analysing rFVII
.alpha.-xylosidase incubations ESI-LC-MS (Q-STAR).
[0196] The resulting glycan-remodeled rFVIIa will be purified from
the .alpha.-xylosidase enzyme by for example anion-exchange
chromatography or gel filtration or suitable combinations thereof.
The purity of the prepared O-glycoform of rFVIIa is verified by the
O-glycopeptide map of rFVIIa.
Example 5
Preparation of Truncated .alpha.-Xylosidase by Cloning and
Expression in E. coli
[0197] The crystal structure of YicI was recently solved. Thus,
cloning of a truncated .alpha.-xylosidase enzyme representing an
active, catalytical domain of the YicI protein (or other similar
.alpha.-xylosidases) may be possible and is being planned, since a
smaller enzyme, if active, may better access the
Xyl-Xyl-Glc-O-Ser52 present in native rFVIIa. A domain comprising
the active site in the enzyme is predicted from the structure. Gene
sequence encoding this part of the YicI sequence is prepared from
the already existing YicI pET11a plasmid for an example by PCR
amplification of relevant areas of the YicI gene, The primers used
for PCR will have extensions with restriction enzyme sites that can
be used for ligation of the truncated YicI gene into the pET11a
vector. The truncated enzyme will after expression and purification
be evaluated for its potential for deglycosylation of rFVIIa as
described above.
Example 6
Preparation of rFVIIa with Exclusively Xylose-Glucose at Serine 52
or Exclusively Xylosexylose-Glucose at Serine 52 by
.alpha.-Xylosyltransferase Treatment
Preparation of .alpha.-Xylosyltransferase
[0198] The enzyme, UDP-D-xylose: .beta.-D-glucoside
.alpha.-1,3-D-xylosyltransferase, can be prepared from HepG2 cells
as described by Omichi et al. (1997). In short, HepG2 cells are
grown in a medium supplemented with 10% fetal calf serum. The
microsomal fraction is prepared by homogenisation of the cells
followed by centrifugation. The .alpha.-xylosyltransferase enzyme
is purified by chromatography, for example on an anion-exchange
column and/or on other chromatographic columns known for the
skilled person. Fractions are collected during elution and the
fractions containing the .alpha.-xylosyltransferase enzyme are
identified by use of the .alpha.-xylosyltransferase assay.
[0199] The enzyme, UDP-D-xylose: .beta.-D-xyloside
.alpha.1,3-xylosyltransferase, can be prepared from HepG2 cells as
described by Minamida et al. (1996). In short, HepG2 cells are
grown in a medium supplemented 10% fetal calf serum. The microsomal
fraction is pre-pared by homogenisation of the cells followed by
centrifugation. The .alpha.-xylosyltransferase enzyme is purified
by chromatography, for example on an anion-exchange column and/or
on other chromatographic columns known for the skilled person.
Fractions are collected during elution and the fractions containing
the .alpha.-xylosyltransferase enzyme are identified by use of the
.alpha.-xylosyltransferase assay.
.alpha.-Xylosyltransferase Assay
[0200] The .alpha.-xylosyltransferase assays are conducted in an
appropriate buffer, e.g. 10 mM Hepes, pH 7.2, 0.1% Triton X-100,
0.5 mM UDP-Xylose (Sigma U5875), containing a suitable substrate,
e.g. the O-glycopeptides that can be obtained from the
O-glycopeptide map of rFVIIa or the pyridylaminated
oligosaccharides prepared as described in Minamida et al. (Minamida
et. al., Detection of UDP-D-xylose: .alpha.-D-xyloside
.alpha.1-3xylosyltransferase activity in human hepatoma cell line
HepG2. J. Biochem. 120 1002-1006, 1996). The reaction is stopped
after an appropriate time, that can be determined experimentally,
by e.g. addition of trifluoroacetic acid, and the assay mixtures
are analysed by HPLC.
O-Glycoforms of rFVIIa with Exclusively Xylose-Glucose- at Serine
52
[0201] The O-glycoforms of rFVIIa with exclusively xylose-glucose
at serine 52 are obtained by (1) treatment of rFVIIa with
xylosidase as described above, (2) purification of the xylosidase
treated rFVIIa from the xylosidase by for example anion-exchange
chromatography, and (3) by incubation of xylosidase-treated rFVIIa
in an appropriate buffer, e.g. glycylglycine, pH 7.0, 10 mM calcium
chloride, with purified UDP-D-xylose: .beta.-D-glucoside
.alpha.-1,3-D-xylosyltransferase and UDP-D-xylose for an
appropriate time, that can be determined experimentally. The
resulting glyco-remodelled rFVIIa is purified from the
UDP-D-xylose: .beta.-D-glucoside .alpha.-1,3-D-xylosyltransferase
enzyme by for example anion-exchange chromatography. The purity of
the prepared O-glycoform of rFVIIa is verified by the
O-glycopeptide map of rFVIIa.
O-Glycoforms of rFVIIa with Exclusively Xylose-Xylose-Glucose- at
Serine 52
[0202] The O-glycoforms of with xylose-xylose-glucose at serine 52
are obtained by (1) treatment of rFVIIa with xylosidase as
described above, (2) purification of the xylosidase treated rFVIIa
from the xylosidase by for example anion-exchange chromatography,
(3) further treatment with UDP-D-xylose: .beta.-D-glucoside
.alpha.-1,3-D-xylosyltransferase and UDP-D-xylose as described
above, and (4) by incubation of the product in an appropriate
buffer, e.g. glycylglycine, pH 7.0, 10 mM calcium chloride, with
purified UDP-D-xylose: .alpha.-D-xyloside
.alpha.1,3-xylosyltransferase and UDP-D-xylose for an appropriate
time, that can be determined experimentally. The resulting
glyco-remodelled rFVIIa is purified from the UDP-D-xylose:
.alpha.-D-xyloside .alpha.1,3-xylosyltransferase enzyme by for
example anion-exchange chromatography. The purity of the prepared
O-glycoform of rFVIIa is verified by the O-glycopeptide map of
rFVIIa.
Example 7
Analysis of O-Glycoform Pattern of rFVIIa
[0203] Tryptic Peptide Mapping of the rFVIIa Light Chain
[0204] The relative content of the O-glycoforms of rFVIIa is
determined by tryptic peptide mapping of the rFVIIa light chain.
The rFVIIa is reduced and alkylated and the rFVIIa light chain is
purified on a RP-HPLC column eluted with an acetonitrile gradient
in water:trifluoroacetic acid. The purified rFVIIa light chain is
buffer-exchanged to Tris buffer, pH 7.5 and digested with trypsin.
The tryptic digest of the rFVIIa light chain is analysed on a
RP-HPLC column (for example Nucleosil C18, 5.mu., 300 .ANG.,
4.0.times.250 mm, Macherey-Nagel 720065) eluted with an
acetonitrile gradient (0%-45% acetonitrile in 100 min) in water:
trifluoroacetic acid (see FIG. 2). Flow is 1.0 ml/min and detection
is UV at 215 nm.
[0205] The peaks containing the O-glycopeptides of rFVIIa are
eluted after approx. 60-65 min where the 1st and the 3rd peak
contain O-glycopeptides with a xylose-xylose-glucose-linked to
serine 52, and the 2nd and 4th peak contain O-glycopeptides with a
glucose linked to serine 52.
[0206] Similarly, the 1st and the 2nd peak contain O-glycopeptides
with a tetrasacharide linked to serine 60, and the 3rd and the 4th
peak contain O-glycopeptides with a fucose linked to serine 60.
Tryptic Peptide Mapping of rFVIIa
[0207] The O-glycoform pattern can be analysed by tryptic peptide
mapping of rFVIIa. The rFVIIa is buffer-exchanged to Tris buffer,
pH 7.5, and digested with trypsin. The tryptic digest of the rFVIIa
is analysed on a RP-HPLC column (for example Nucleosil C18, 5.mu.,
300 .ANG., 4.0.times.250 mm, Macherey-Nagel 720065) eluted with an
acetonitrile gradient (0%-45% acetonitrile in 100 min) in water:
trifluoroacetic acid. Flow is 1.0 ml/min and detection is UV at 215
nm.
[0208] The peaks containing the O-glycopeptides of rFVIIa are
eluted after approx. 67-70 min where the 1st peak contains
O-glycopeptides with a xylose-xylose-glucose linked to serine 52,
and the 2nd peak contains O-glycopeptides with a glucose linked to
serine 52.
Total Mass Analysis of rFVIIa
[0209] The O-glycoform pattern can be analysed by total mass
analysis of rFVIIa. The rFVIIa is desalted on a Millipore ZipTip C4
column equilibrated with 0.1% formic acid and eluted with 3% formic
acid in 90% methanol. The eluted sample is analysed by the
nanospray technique on a Qstar XL mass spectrometer.
[0210] The major peak at approximately 50500 Da represents rFVIIa
O-glycoforms with a glucose linked to serine 52 and the major peak
at approximately 50800 Da represents rFVIIa O-glycoforms with a
xylose-xylose-glucose linked to serine 52.
Example 8
Purification of Glc-O-Ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII
[0211] Glc-O-ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII was purified
using two cycles of hydrophobic interaction chromatography (HIC).
The column (1.0 cm in inner diameter.times.7.0 cm length=5.5 ml
column volume (CV)) packed with Toso Haas TSK-Gel phenyl 5 PW, was
equilibrated with 5 CV 10 mM histidine, 10 mM CaCl2, 2.0 M
NH4-acetate, pH 6.0. The column was loaded with approximately 2.5
mg of FVII pr. ml resin. To the load solution 2.0 M NH4-acetate and
10 mM CaCl2 was added prior to load. The column was washed with 5
CV 10 mM histidine, 10 mM CaCl.sub.2, 2.0 M NH4-acetate, pH 6.0.
Elution was performed using a 20 CV linear gradient from 10 mM
histidine, 10 mM CaCl2, 2.0 M NH4-acetate, pH 6.0 to 10 mM
histidine, 10 mM CaCl2, pH 6.0. The purification was performed at a
flow rate of 6 CV/h and at a temperature of 5.degree. C. Fractions
were collected during elution.
[0212] The FVII eluted in two overlapping major peaks (see FIG. 4:
Chromatogram from first HIC cycle). Fractions containing the first
peak were pooled (fraction "A", FIG. 4) and further purified by a
second cycle of HIC, using the same chromatographic procedure as
for the first HIC cycle (see FIG. 5: Chromatogram obtained by
reloading fraction "A" onto the HIC column). Fractions containing
the second major peak (fraction "B", FIG. 4) were pooled as well
and further purified by a second cycle of HIC, using the same
chromatographic procedure as for the first HIC cycle (see FIG. 6:
Chromatogram obtained by reloading fraction "B" onto the HIC
column).
[0213] Purified Glc-O-Ser52-FVII was identified in the peak
fraction, fraction 10 (FIG. 5), obtained by reloading fraction "A"
onto the second HIC step. Purified Xyl-Xyl-Glc-O-Ser52-FVII was
identified in the peak fraction, fraction 15 (FIG. 6), obtained by
reloading fraction "B" onto the second HIC step. The identification
was obtained by tryptic peptide mapping of rFVIIa as described in
Example 7 (FIGS. 7A and 7B) and by total mass analysis of rFVIIa as
described in Example 7 (FIGS. 8A and 8B). Both analyses showed a
high content of Glc-O-Ser52-rFVIIa and a low content of
Xyl-Xyl-Glc-O-Ser52-rFVIIa in the peak fraction, Fraction 10, and a
low content of Glc-O-Ser52-rFVIIa and a high content of
Xyl-Xyl-Glc-O-Ser52-rFVIIa in the peak fraction, Fraction 15. A
quantitation of the content of the O-glycoforms in the two peak
fractions could not be obtained due to relatively low rFVIIa
content in the fractions (FIGS. 7A and 7B: Tryptic peptide mapping:
Other peptide fragments of rFVIIa co-eluted with or eluted close to
the O-glycopeptides, and the content of O-glycopeptides in low
amounts could therefore not be determined.) (FIGS. 8A and 8B: Total
mass analysis: Other O- and/or N-glycoforms of rFVIIa, for example
N-glycoforms of rFVIIa lacking one N-acetylneuraminic acid,
appeared in the mass spectra, and the content of O-glycoforms of
rFVIIa in low amounts could therefore not be determined).
[0214] The specific activities of the peak fractions obtained from
the HIC (Table 1) were determined by the 1st generation clotting
assay. It was found that the Glc-O-Ser52-rFVIIa O-glycoform had a
low specific activity while the Xyl-Xyl-Glc-O-Ser52-rFVIIa
O-glycoform had a high specific activity.
TABLE-US-00001 TABLE 1 Specific activities determined using the 1st
generation clotting assay for the peak fractions obtained from HIC.
The content of rFVIIa was determined by HPLC. Sample Specific
activity PS5002-014 Frak. 10 44 IU/.mu.g PS5002-015 Frak. 15 61
IU/.mu.g PS5002-014/015 starting material 53 IU/.mu.g
Example 9
Purification by Hydrophobic Interaction Chromatography
[0215] Highly purified Glc-O-Ser52-rFVIIa preparations and highly
purified Xyl-Xyl-Glc-O-Ser52-rFVIIa preparations can be obtained by
repeated purification on the hydrophobic interaction chromatography
as described above. Highly purified Glc-O-Ser52-rFVIIa and
Xyl-Xyl-Glc-O-Ser52-rFVIIa preparations with higher rFVIIa content
can be obtained by increasing the amount of starting material for
the hydrophobic interaction chromatography performed as described
above. The content of each O-glycoform of rFVIIa in the highly
purified preparations with higher rFVIIa content can be quantitated
by tryptic peptide mapping of the rFVIIa light chain as described
in Example 7. The specific activities of the highly purified
preparations can be determined by the 1st generation clotting assay
as above.
* * * * *