U.S. patent application number 11/367189 was filed with the patent office on 2006-09-14 for coagulation factor vii polypeptides.
This patent application is currently assigned to Novo Nordisk HealthCare A/G. Invention is credited to Soren E. Bjorn, Henrik Ostergaard, Egon Persson.
Application Number | 20060205036 11/367189 |
Document ID | / |
Family ID | 36971482 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060205036 |
Kind Code |
A1 |
Ostergaard; Henrik ; et
al. |
September 14, 2006 |
Coagulation Factor VII polypeptides
Abstract
The present invention relates to novel coagulation Factor VII
polypeptides, polynucleotide constructs encoding such polypeptides,
as well as vectors and host cells comprising and expressing the
polynucleotide, pharmaceutical compositions, uses and methods of
treatment.
Inventors: |
Ostergaard; Henrik;
(Roskilde, DK) ; Bjorn; Soren E.; (Lyngby, DK)
; Persson; Egon; (Ballerup, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;PATENT DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk HealthCare A/G
Zurich
CH
|
Family ID: |
36971482 |
Appl. No.: |
11/367189 |
Filed: |
March 3, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/DK04/00594 |
Sep 9, 2004 |
|
|
|
11367189 |
Mar 3, 2006 |
|
|
|
Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/325; 514/14.3; 530/383; 536/23.5 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
9/00 20180101; C12N 9/6437 20130101; C12Y 304/21021 20130101; C07K
14/745 20130101; C12N 9/647 20130101; A61P 7/04 20180101; A61K
38/00 20130101 |
Class at
Publication: |
435/069.6 ;
435/320.1; 435/325; 530/383; 514/012; 536/023.5 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C07H 21/04 20060101 C07H021/04; A61K 38/36 20060101
A61K038/36; C07K 14/745 20060101 C07K014/745 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 2003 |
DK |
PA 2003 01296 |
Claims
1. A Factor VII polypeptide with a Tissue Factor binding affinity
lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa, wherein said Factor VII
polypeptide is selected from the group consisting of: i) a Factor
VII polypeptide comprising one or more amino acid substitutions at
positions selected from the group consisting of: K18X.sub.1,
R36X.sub.2, S43X.sub.3, K62X.sub.4, Q64X.sub.5, L65X.sub.6,
I69X.sub.7, F71X.sub.8, L73X.sub.9, P74X.sub.10, E77X.sub.11,
G78X.sub.12, R79X.sub.13, K85X.sub.14, Q88X.sub.15, N93X.sub.16,
F275X.sub.17, R277X.sub.18, M306X.sub.19, T307X.sub.20,
Q308X.sub.21, D309X.sub.22, or R379X.sub.23 corresponding to amino
acids at positions of SEQ ID NO: 1, wherein X.sub.1 is E, D, A, or
F; X.sub.2 is E, D, K, A, F, or N; X.sub.3 is E, D, K, R, A, or F;
X.sub.4 is D, A, or F; X.sub.5 is E, D, K, R, A, F, or N; X.sub.6
is E, D, K, R, A, or F; X.sub.7 is C, D, E, F, G, H, K, L, M, N, P,
Q, R, S, T, V, W, or Y; X.sub.8 is A, G, H, I, K, L, M, P, R, S, T,
V, or W; X.sub.9 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,
W, or Y; X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,
V, W, or Y; X.sub.11 is A, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W, or Y; X.sub.12 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y; X.sub.13 is A, D, E, F, G, H, I, K, L, M, N, P, Q,
S, T, V, W, or Y; X.sub.14 is A, D, E, F, G, I, L, M, P, S, T, V,
W, or Y; X.sub.15 is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W, or Y; X.sub.16 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S,
T, V, W, or Y; X.sub.17 is A, D, E, G, I, K, L, M, N, P, Q, R, S,
T, V, W, or Y; X.sub.18 is A, D, E, F, G, H, I, K, L, M, P, Q, S,
T, V, W, or Y; X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T,
V, W, or Y; X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, V, W, or Y; X.sub.21 is A, D, E, F, G, H, I, K, L, M, N, P, R,
S, T, V, W, or Y; X.sub.22 is A, F, G, H, I, K, L, M, N, P, Q, R,
V, W, or Y; and X.sub.23 is A, D, E, F, G, H, I, K, L, M, P, Q, S,
T, V, W, or Y; and/or ii) a Factor VII polypeptide in which one or
more amino acids corresponding to amino acids at positions selected
from K18, R36, K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79,
K85, Q88, N93, F275, R277, M306, Q308, D309, or R379 of SEQ ID NO:
1 have been substituted with a cysteine amino acid residue, and
wherein the cysteine amino acid residue may optionally be
conjugated with a chemical group that increases the molecular
weight of the Factor VII polypeptide; and/or iii) a Factor VII
polypeptide comprising one or more N-glycosylation site N-Xaa-S/T
introduced by amino acid substitutions corresponding to amino acids
starting at positions selected from R36, Q64, I69, F71, P74, E77,
G78, Q88, N93, F275, M306, T307, or D309 of SEQ ID NO: 1, wherein
Xaa is any amino acid except P.
2. The Factor VII polypeptide according to claim 1, further
comprising one or more amino acid substitutions selected from the
group consisting of L305V, L305V/M306D/D309S, L3051, L305T, F374P,
V158T/M298Q, V158D/E296V/M298Q, K337A, M298Q, V158D/M298Q,
L305V/K337A, V158D/E296V/M298Q/L305V, V158D/E296V/M298Q/K337A,
V158D/E296V/M298Q/L305V/K337A, K157A, E296V, E296V/M298Q,
V158D/E296V, V158D/M298K, and S336G, L305V/K337A, L305V/V158D,
L305V/E296V, L305V/M298Q, L305V/V158T, L305V/K337A/V158T,
L305V/K337A/M298Q, L305V/K337A/E296V, L305V/K337A/V158D,
L305V/V158D/M298Q, L305V/V158D/E296V, L305V/V158T/M298Q,
L305V/V158T/E296V, L305V/E296V/M298Q, L305V/V158D/E296V/M298Q,
L305V/V158T/E296V/M298Q, L305V/V158T/K337A/M298Q,
L305V/V158T/E296V/K337A, L305V/V158D/K337A/M298Q,
L305V/V158D/E296V/K337A, L305V/V158D/E296V/M298Q/K337A,
L305V/V158T/E296V/M298Q/K337A, S314E/K316H, S314E/K316Q,
S314E/L305V, S314E/K337A, S314E/V158D, S314E/E296V, S314E/M298Q,
S314E/V158T, K316H/L305V, K316H/K337A, K316H/V158D, K316H/E296V,
K316H/M298Q, K316H/V158T, K316Q/L305V, K316Q/K337A, K316Q/V158D,
K316Q/E296V, K316Q/M298Q, K316Q/V158T, S314E/L305V/K337A,
S314E/L305V/V158D, S314E/L305V/E296V, S314E/L305V/M298Q,
S314E/L305V/V158T, S314E/L305V/K337A/V158T,
S314E/L305V/K337A/M298Q, S314E/L305V/K337A/E296V,
S314E/L305V/K337A/V158D, S314E/L305V/V158D/M298Q,
S314E/L305V/V158D/E296V, S314E/L305V/V158T/M298Q,
S314E/L305V/V158T/E296V, S314E/L305V/E296V/M298Q,
S314E/L305V/V158D/E296V/M298Q, S314E/L305V/V158T/E296V/M298Q,
S314E/L305V/V158T/K337A/M298Q, S314E/L305V/V158T/E296V/K337A,
S314E/L305V/V158D/K337A/M298Q, S314E/L305V/V158D/E296V/K337A,
S314E/L305V/V158D/E296V/M298Q/K337A,
S314E/L305V/V158T/E296V/M298Q/K337A, K316H/L305V/K337A,
K316H/L305V/V158D, K316H/L305V/E296V, K316H/L305V/M298Q,
K316H/L305V/V158T, K316H/L305V/K337A/V158T,
K316H/L305V/K337A/M298Q, K316H/L305V/K337A/E296V,
K316H/L305V/K337A/V158D, K316H/L305V/V158D/M298Q,
K316H/L305V/V158D/E296V, K316H/L305V/V158T/M298Q,
K316H/L305V/V158T/E296V, K316H/L305V/E296V/M298Q,
K316H/L305V/V158D/E296V/M298Q, K316H/L305V/V158T/E296V/M298Q,
K316H/L305V/V158T/K337A/M298Q, K316H/L305V/V158T/E296V/K337A,
K316H/L305V/V158D/K337A/M298Q, K316H/L305V/V158D/E296V/K337A,
K316H/L305V/V158D/E296V/M298Q/K337A,
K316H/L305V/V158T/E296V/M298Q/K337A, K316Q/L305V/K337A,
K316Q/L305V/V158D, K316Q/L305V/E296V, K316Q/L305V/M298Q,
K316Q/L305V/V158T, K316Q/L305V/K337AN/158T,
K316Q/L305V/K337A/M298Q, K316Q/L305V/K337A/E296V,
K316Q/L305V/K337A/V158D, K316Q/L305V/V158D/M298Q,
K316Q/L305V/V158D/E296V, K316Q/L305V/V158T/M298Q,
K316Q/L305V/V158T/E296V, K316Q/L305V/E296V/M298Q,
K316Q/L305V/V158D/E296V/M298Q, K316Q/L305V/V158T/E296V/M298Q,
K316Q/L305V/V158T/K337A/M298Q, K316Q/L305V/V158T/E296V/K337A,
K316Q/L305V/V158D/K337A/M298Q, K316Q/L305V/V158D/E296V/K337A,
K316Q/L305V/V158D/E296V/M298Q/K337A,
K316Q/L305V/V158T/E296V/M298Q/K337A, F374Y/K337A, F374Y/V158D,
F374Y/E296V, F374Y/M298Q, F374Y/V158T, F374Y/S314E, F374Y/L305V,
F374Y/L305V/K337A, F374Y/L305V/V158D, F374Y/L305V/E296V,
F374Y/L305V/M298Q, F374Y/L305V/V158T, F374Y/L305V/S314E,
F374Y/K337A/S314E, F374Y/K337A/V158T, F374Y/K337A/M298Q,
F374Y/K337A/E296V, F374Y/K337A/V158D, F374Y/V158D/S314E,
F374Y/V158D/M298Q, F374Y/V158D/E296V, F374Y/V158T/S314E,
F374Y/V158T/M298Q, F374Y/V158T/E296V, F374Y/E296V/S314E,
F374Y/S314E/M298Q, F374Y/E296V/M298Q, F374Y/L305V/K337A/V158D,
F374Y/L305V/K337A/E296V, F374Y/L305V/K337A/M298Q,
F374Y/L305V/K337A/V158T, F374Y/L305V/K337A/S314E,
F374Y/L305V/V158D/E296V, F374Y/L305V/V158D/M298Q,
F374Y/L305V/V158D/S314E, F374Y/L305V/E296V/M298Q,
F374Y/L305V/E296V/V158T, F374Y/L305V/E296V/S314E,
F374Y/L305V/M298Q/V158T, F374Y/L305V/M298Q/S314E,
F374Y/L305V/V158T/S314E, F374Y/K337A/S314E/V158T,
F374Y/K337A/S314E/M298Q, F374Y/K337A/S314E/E296V,
F374Y/K337A/S314E/V158D, F374Y/K337A/V158T/M298Q,
F374Y/K337A/V158T/E296V, F374Y/K337A/M298Q/E296V,
F374Y/K337A/M298Q/V158D, F374Y/K337A/E296V/V158D,
F374Y/V158D/S314E/M298Q, F374Y/V158D/S314E/E296V,
F374Y/V158D/M298Q/E296V, F374Y/V158T/S314E/E296V,
F374Y/V158T/S314E/M298Q, F374Y/V158T/M298Q/E296V,
F374Y/E296V/S314E/M298Q, F374Y/L305V/M298Q/K337A/S314E,
F374Y/L305V/E296V/K337A/S314E, F374Y/E296V/M298Q/K337A/S314E,
F374Y/L305V/E296V/M298Q/K337A, F374Y/L305V/E296V/M298Q/S314E,
F374Y/V158D/E296V/M298Q/K337A, F374Y/V158D/E296V/M298Q/S314E,
F374Y/L305V/V158D/K337A/S314E, F374Y/V158D/M298Q/K337A/S314E,
F374Y/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q,
F374Y/L305V/V158D/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A,
F374Y/L305V/V158D/M298Q/S314E, F374Y/L305V/V158D/E296V/S314E,
F374Y/V158T/E296V/M298Q/K337A, F374Y/V158T/E296V/M298Q/S314E,
F374Y/L305V/V158T/K337A/S314E, F374Y/V158T/M298Q/K337A/S314E,
F374Y/V158T/E296V/K337A/S314E, F374Y/L305V/V158T/E296V/M298Q,
F374Y/L305V/V158T/M298Q/K337A, F374Y/L305V/V158T/E296V/K337A,
F374Y/L305V/V158T/M298Q/S314E, F374Y/L305V/V158T/E296V/S314E,
F374Y/E296V/M298Q/K337A/V158T/S314E,
F374Y/V158D/E296V/M298Q/K337A/S314E,
F374Y/L305V/V158D/E296V/M298Q/S314E,
F374Y/L305V/E296V/M298Q/V158T/S314E,
F374Y/L305V/E296V/M298Q/K337A/V158T,
F374Y/L305V/E296V/K337A/V158T/S314E,
F374Y/L305V/M298Q/K337A/V158T/S314E,
F374Y/L305V/V158D/E296V/M298Q/K337A,
F374Y/L305V/V158D/E296V/K337A/S314E,
F374Y/L305V/V158D/M298Q/K337A/S314E,
F374Y/L305V/E296V/M298Q/K337A/V158T/S314E,
F374Y/L305V/V158D/E296V/M298Q/K337A/S314E, S52A, S60A; R152E,
S344A, P11Q/K33E, T106N, K143N/N145T, V253N, R290N/A292T, G291N,
R315N/V317T; K143N/N145T/R315N/V317T; one or more substitutions,
additions or deletions in the amino acid sequence from 233Thr to
240Asn; and one or more substitutions, additions or deletions in
the amino acid sequence from 304Arg to 329Cys.
3. The Factor VII polypeptide according to claim 1, wherein the
dissociation constant K.sub.d of said Factor VII polypeptide is
higher than 5 nM,
4. The Factor VII polypeptide according to claim 3, wherein the
dissociation constant K.sub.d of said Factor VII polypeptide is
higher than 20 nM.
5. The Factor VII polypeptide according to claim 4, wherein the
dissociation constant K.sub.d of said Factor VII polypeptide is
dissociation constant K.sub.d of said Factor VII polypeptide is
higher than 100 nM.
6. The Factor VII polypeptide according to claim 5, wherein the
dissociation constant K.sub.d of said Factor VII polypeptide is
dissociation constant K.sub.d of said Factor VII polypeptide is
higher than 1 .mu.M.
7. A composition comprising a Factor VII polypeptide according to
claim 1.
8. A pharmaceutical composition comprising a Factor VII polypeptide
according to claim 1 and a pharmaceutically acceptable carrier or
diluent.
9. A polynucleotide construct encoding a Factor VII polypeptide
according to claim 1.
10. The polynucleotide construct according to claim 9, wherein said
construct is a vector.
11. A eukaryotic host cell comprising the polynucleotide construct
according to claim 9.
12. The eukaryotic host cell according to claim 11, wherein said
cell is of mammalian origin.
13. The eucaryotic host cell according to claim 12, wherein said
cell is selected from the group consisting of CHO cells, BHK cells
or HEK cells.
14. A method for producing the Factor VII polypeptide, the method
comprising (i) cultivating a eukaryotic host cell according to
claim 11 in an appropriate growth medium under conditions allowing
protein synthesis from said polynucleotide construct and (ii)
recovering said Factor VII polypeptide from the culture medium.
15. A method for the treatment of bleeding episodes or bleeding
disorders in a subject or for the enhancement of the normal
haemostatic system, the method comprising administering to a
subject in need thereof a therapeutically or prophylactically
effective amount of a Factor VII polypeptide as defined in claim 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel human coagulation
Factor VII polypeptides, as well as polynucleotide constructs
encoding such polypeptides, vectors and host cells comprising and
expressing the polynucleotide, pharmaceutical compositions
comprising Factor VII polypeptides, uses and methods of
treatment.
BACKGROUND OF THE INVENTION
[0002] Blood coagulation is a process consisting of a complex
interaction of various blood components (or factors) that
eventually gives rise to a fibrin clot. Generally, the blood
components, which participate in what has been referred to as the
coagulation "cascade", are enzymatically inactive proteins
(proenzymes or zymogens) that are converted to proteolytic enzymes
by the action of an activator (which itself is an activated
clotting factor). Coagulation factors that have undergone such a
conversion are generally referred to as "active factors", and are
designated by the addition of the letter "a" to the name of the
coagulation factor (e.g. Factor VIIa).
[0003] Initiation of the haemostatic process is mediated by the
formation of a complex between tissue factor, exposed as a result
of injury to the vessel wall, and Factor VIIa. This complex then
converts Factors IX and X to their active forms. Factor Xa converts
limited amounts of prothrombin to thrombin on the tissue
factor-bearing cell. Thrombin activates platelets and Factors V and
VIII into Factors Va and VIIIa, both cofactors in the further
process leading to the full thrombin burst. This process includes
generation of Factor Xa by Factor IXa (in complex with factor
VIIIa) and occurs on the surface of activated platelets. Thrombin
finally converts fibrinogen to fibrin resulting in formation of a
fibrin clot.
[0004] Factor VII is a trace plasma glycoprotein that circulates in
blood as a single-chain zymogen. The zymogen is catalytically
inactive. Single-chain Factor VII may be converted to two-chain
Factor VIIa by Factor Xa, Factor XIIa, Factor IXa, Factor VIIa or
thrombin in vitro. Factor Xa is believed to be the major
physiological activator of Factor VII. The conversion of zymogen
Factor VII into the activated two-chain molecule occurs by cleavage
of an internal Arg.sub.152-Ile.sub.153 peptide bond.
[0005] It is often desirable to stimulate the coagulation cascade
in a subject. Factor VIIa has been used to control bleeding
disorders that have several causes such as clotting factor
deficiencies (e.g. haemophilia A and B or deficiency of coagulation
Factors XI or VII) or clotting factor inhibitors. Factor VIIa has
also been used to control excessive bleeding occurring in subjects
with a normally functioning blood clotting cascade (no clotting
factor deficiencies or inhibitors against any of the coagulation
factors). Such bleeding may, for example, be caused by a defective
platelet function, thrombocytopenia or von Willebrand's disease.
Bleeding is also a major problem in connection with surgery and
other forms of tissue damage.
[0006] European Patent No. 200,421 (ZymoGenetics) relates to the
nucleotide sequence encoding human Factor VII and the recombinant
expression of Factor VII in mammalian cells.
[0007] Dickinson et al. (Proc. Nat. Acad. Sci. USA 93, 14379-14384,
1996) discloses Factor VII polypeptides wherein certain amino acids
have been individually replaced by Ala. Iwanaga et al. (Thromb.
Haemost. (supplement august 1999), 466, abstract 1474) relates to
Factor VIIa variants wherein residues 316-320 are deleted or
residues 311-322 are replaced with the corresponding residues from
trypsin.
[0008] There is still a need in the art for improved Factor VII
polypeptides having procoagulant activity. In particular, there is
a need for Factor VII polypeptides with increased TF independent
activity.
SUMMARY OF THE INVENTION
[0009] The present invention relates to Factor VII polypeptides
with increased TF independent activity, such as novel coagulation
Factor VII polypeptides with the same or increased activity
compared to wild type Factor VIIa and with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa.
[0010] In a first aspect, the present invention relates to a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa with the proviso that the Factor VII polypeptide
is not I69A-FVII.
[0011] In a second aspect, the invention relates to a composition
comprising a Factor VII polypeptide with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa with the proviso that the
Factor VII polypeptide is not I69A-FVII.
[0012] In a third aspect, the invention relates to a pharmaceutical
composition comprising a Factor VII polypeptide with a Tissue
Factor binding affinity lower than recombinant wild type human
Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa; and
optionally, a pharmaceutically acceptable carrier.
[0013] In a further aspect, the invention relates to a
polynucleotide construct encoding a Factor VII polypeptide with a
Tissue Factor binding affinity lower than recombinant wild type
human Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa with
the proviso that the Factor VII polypeptide is not I69A-FVII.
[0014] In a further aspect, the invention relates to a eukaryotic
host cell comprising a polynucleotide construct encoding a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa with the proviso that the Factor VII polypeptide
is not I69A-FVII.
[0015] In a further aspect, the invention relates to a method for
producing a Factor VII polypeptide with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa with the proviso that the
Factor VII polypeptide is not I69A-FVII, the method comprising
cultivating a eukaryotic host cell comprising a polynucleotide
construct encoding the Factor VII polypeptide in an appropriate
growth medium under conditions allowing protein synthesis from said
polynucleotide construct and recovering said Factor VII polypeptide
from the culture medium.
[0016] In a further aspect, the invention relates to a use of a
Factor VII polypeptide with a Tissue Factor binding affinity lower
than recombinant wild type human Factor VIIa and substantially the
same activity or increased activity compared to recombinant wild
type human Factor VIIa; for the preparation of a medicament for the
treatment of bleeding episodes or for the enhancement of the normal
haemostatic system.
[0017] In a further aspect, the invention relates to a method for
the treatment of bleeding episodes or bleeding disorders in a
subject or for the enhancement of the normal haemostatic system,
the method comprising administering to a subject in need thereof a
therapeutically or prophylactically effective amount of a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa.
[0018] In a further aspect, the invention relates to a Factor VII
polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa, wherein said Factor VII polypeptide is selected
from: [0019] i) a Factor VII polypeptide comprising one or more
amino acid substitution selected from K18X.sub.1, R36X.sub.2,
S43X.sub.3, K62X.sub.4, Q64X.sub.5, L65X.sub.6, I69X.sub.7,
F71X.sub.8, L73X.sub.9, P74X.sub.10, E77X.sub.11, G78X.sub.12,
R79X.sub.13, K85X.sub.14, Q88X.sub.15, N93X.sub.16, F275X.sub.17,
R277X.sub.18, M306X.sub.19, T307X.sub.20, Q308X.sub.21,
D309X.sub.22, or R379X.sub.23 corresponding to amino acids at
positions of SEQ ID NO: 1, which different amino acid decreases the
Tissue Factor binding affinity, wherein [0020] X.sub.1 is E, D, A,
or F; [0021] X.sub.2 is E, D, K, A, F, or N; [0022] X.sub.3 is E,
D, K, R, A, or F; [0023] X.sub.4 is D, A, or F; [0024] X.sub.5 is
E, D, K, R, A, F, or N; [0025] X.sub.6 is E, D, K, R, A, or F;
[0026] X.sub.7 is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,
W, or Y; [0027] X.sub.8 is A, G, H, I, K, L, M, P, R, S, T, V, or
W; [0028] X.sub.9 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,
V, W, or Y; [0029] X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W, or Y; [0030] X.sub.11 is A, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W, or Y; [0031] X.sub.12 is A, D, E, F, H, I, K,
L, M, N, P, Q, R, S, T, V, W, or Y; [0032] X.sub.13 is A, D, E, F,
G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; [0033] X.sub.14 is A,
D, E, F, G, I, L, M, P, S, T, V, W, or Y; [0034] X.sub.15 is A, D,
E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; [0035] X.sub.16
is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; [0036]
X.sub.17 is A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
[0037] X.sub.18 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y; [0038] X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T, V,
W, or Y; [0039] X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, V, W, or Y; [0040] X.sub.21 is A, D, E, F, G, H, I, K, L, M,
N, P, R, S, T, V, W, or Y; [0041] X.sub.22 is A, F, G, H, I, K, L,
M, N, P, Q, R, V, W, or Y; and [0042] X.sub.23 is A, D, E, F, G, H,
I, K, L, M, P, Q, S, T, V, W, or Y; [0043] and/or [0044] ii) a
Factor VII polypeptide, wherein one or more amino acid
corresponding to amino acids at positions selected from K18, R36,
K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85, Q88, N93,
F275, R277, M306, Q308, D309, or R379 of SEQ ID NO: 1 have been
substituted with a cysteine amino acid residue, which cysteine
amino acid residue decreases the Tissue Factor binding affinity,
and which cysteine amino acid residue is optionally conjugated with
a chemical group, which chemical group increases the molecular
weight of said Factor VII polypeptide; [0045] and/or [0046] iii) a
Factor VII polypeptide comprising one or more N-glycosylation site
N-Xaa-S/T introduced by amino acid substitutions corresponding to
amino acids starting at positions selected from R36, Q64, I69, F71,
P74, E77, G78, Q88, N93, F275, M306, T307, or D309 of SEQ ID NO: 1,
wherein Xaa is any amino acid except P, which introduced
N-glycosylation site decreases the Tissue Factor binding
affinity.
[0047] In a further aspect, the invention relates to a composition
comprising a Factor VII polypeptide with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa, wherein said Factor VII
polypeptide is selected from: [0048] i) a Factor VII polypeptide
comprising one or more amino acid substitution selected from
K18X.sub.1, R36X.sub.2, S43X.sub.3, K62X.sub.4, Q64X.sub.5,
L65X.sub.6, I69X.sub.7, F71X.sub.8, L73X.sub.9, P74X.sub.10,
E77X.sub.11, G78X.sub.12, R79X.sub.13, K85X.sub.14, Q88X.sub.15,
N93X.sub.16, F275X.sub.17, R277X.sub.18, M306X.sub.19,
T307X.sub.20, Q308X.sub.21, D309X.sub.22, or R379X.sub.23
corresponding to amino acids at positions of SEQ ID NO: 1, which
different amino acid decreases the Tissue Factor binding affinity,
wherein [0049] X.sub.1 is E, D, A, or F; [0050] X.sub.2 is E, D, K,
A, F, or N; [0051] X.sub.3 is E, D, K, R, A, or F; [0052] X.sub.4
is D, A, or F; [0053] X.sub.5 is E, D, K, R, A, F, or N; [0054]
X.sub.6 is E, D, K, R, A, or F; [0055] X.sub.7 is C, D, E, F, G, H,
K, L, M, N, P, Q, R, S, T, V, W, or Y; [0056] X.sub.8 is A, G, H,
I, K, L, M, P, R, S, T, V, or W; [0057] X.sub.9 is A, D, E, F, G,
H, I, K, M, N, P, Q, R, S, T, V, W, or Y; [0058] X.sub.10 is A, D,
E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; [0059] X.sub.11
is A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; [0060]
X.sub.12 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or
Y; [0061] X.sub.13 is A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T,
V, W, or Y; [0062] X.sub.14 is A, D, E, F, G, I, L, M, P, S, T, V,
W, or Y; [0063] X.sub.15 is A, D, E, F, G, H, I, K, L, M, N, P, R,
S, T, V, W, or Y; [0064] X.sub.16 is A, D, E, F, G, H, I, K, L, M,
P, Q, R, S, T, V, W, or Y; [0065] X.sub.17 is A, D, E, G, I, K, L,
M, N, P, Q, R, S, T, V, W, or Y; [0066] X.sub.18 is A, D, E, F, G,
H, I, K, L, M, P, Q, S, T, V, W, or Y; [0067] X.sub.19 is A, E, F,
G, H, I, K, L, P, Q, R, S, T, V, W, or Y; [0068] X.sub.20 is A, D,
E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; [0069] X.sub.21
is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; [0070]
X.sub.22 is A, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and
[0071] X.sub.23 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y; [0072] and/or [0073] ii) a Factor VII polypeptide, wherein
one or more amino acid corresponding to amino acids at positions
selected from K18, R36, K62, Q64, L65, I69, F71, L73, P74, E77,
G78, R79, K85, Q88, N93, F275, R277, M306, Q308, D309, or R379 of
SEQ ID NO: 1 have been substituted with a cysteine amino acid
residue, which cysteine amino acid residue decreases the Tissue
Factor binding affinity, and which cysteine amino acid residue is
optionally conjugated with a chemical group, which chemical group
increases the molecular weight of said Factor VII polypeptide;
[0074] and/or [0075] iii) a Factor VII polypeptide comprising one
or more N-glycosylation site N-Xaa-S/T introduced by amino acid
substitutions corresponding to amino acids starting at positions
selected from R36, Q64, I69, F71, P74, E77, G78, Q88, N93, F275,
M306, T307, or D309 of SEQ ID NO: 1, wherein Xaa is any amino acid
except P, which introduced N-glycosylation site decreases the
Tissue Factor binding affinity.
[0076] In a further aspect, the invention relates to a
pharmaceutical composition comprising a Factor VII polypeptide with
a Tissue Factor binding affinity lower than recombinant wild type
human Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa,
wherein said Factor VII polypeptide is selected from: [0077] i) a
Factor VII polypeptide comprising one or more amino acid
substitution selected from K18X.sub.1, R36X.sub.2, S43X.sub.3,
K62X.sub.4, Q64X.sub.5, L65X.sub.6, I69X.sub.7, F71X.sub.8,
L73X.sub.9, P74X.sub.10, E77X.sub.11, G78X.sub.12, R79X.sub.13,
K85X.sub.14, Q88X.sub.15, N93X.sub.16, F275X.sub.17, R277X.sub.18,
M306X.sub.19, T307X.sub.20, Q308X.sub.21, D309X.sub.22, or
R379X.sub.23 corresponding to amino acids at positions of SEQ ID
NO: 1, which different amino acid decreases the Tissue Factor
binding affinity, wherein [0078] X.sub.1 is E, D, A, or F; [0079]
X.sub.2 is E, D, K, A, F, or N; [0080] X.sub.3 is E, D, K, R, A, or
F; [0081] X.sub.4 is D, A, or F; [0082] X.sub.5 is E, D, K, R, A,
F, or N; [0083] X.sub.6 is E, D, K, R, A, or F; [0084] X.sub.7 is
C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; [0085]
X.sub.8 is A, G, H, I, K, L, M, P, R, S, T, V, or W; [0086] X.sub.9
is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; [0087]
X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or
Y; [0088] X.sub.11 is A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, or Y; [0089] X.sub.12 is A, D, E, F, H, I, K, L, M, N, P, Q, R,
S, T, V, W, or Y; [0090] X.sub.13 is A, D, E, F, G, H, I, K, L, M,
N, P, Q, S, T, V, W, or Y; [0091] X.sub.14 is A, D, E, F, G, I, L,
M, P, S, T, V, W, or Y; [0092] X.sub.15 is A, D, E, F, G, H, I, K,
L, M, N, P, R, S, T, V, W, or Y; [0093] X.sub.16 is A, D, E, F, G,
H, I, K, L, M, P, Q, R, S, T, V, W, or Y; [0094] X.sub.17 is A, D,
E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; [0095] X.sub.18 is
A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, or Y; [0096]
X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, or Y;
[0097] X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V,
W, or Y; [0098] X.sub.21 is A, D, E, F, G, H, I, K, L, M, N, P, R,
S, T, V, W, or Y; [0099] X.sub.22 is A, F, G, H, I, K, L, M, N, P,
Q, R, V, W, or Y; and [0100] X.sub.23 is A, D, E, F, G, H, I, K, L,
M, P, Q, S, T, V, W, or Y; [0101] and/or [0102] ii) a Factor VII
polypeptide, wherein one or more amino acid corresponding to amino
acids at positions selected from K18, R36, K62, Q64, L65, I69, F71,
L73, P74, E77, G78, R79, K85, Q88, N93, F275, R277, M306, Q308,
D309, or R379 of SEQ ID NO: 1 have been substituted with a cysteine
amino acid residue, which cysteine amino acid residue decreases the
Tissue Factor binding affinity, and which cysteine amino acid
residue is optionally conjugated with a chemical group, which
chemical group increases the molecular weight of said Factor VII
polypeptide; [0103] and/or [0104] iii) a Factor VII polypeptide
comprising one or more N-glycosylation site N-Xaa-S/T introduced by
amino acid substitutions corresponding to amino acids starting at
positions selected from R36, Q64, I69, F71, P74, E77, G78, Q88,
N93, F275, M306, T307, or D309 of SEQ ID NO: 1, wherein Xaa is any
amino acid except P, which introduced N-glycosylation site
decreases the Tissue Factor binding affinity; and optionally, a
pharmaceutically acceptable carrier.
[0105] In a further aspect, the invention relates to a use of a
Factor VII polypeptide with a Tissue Factor binding affinity lower
than recombinant wild type human Factor VIIa and substantially the
same activity or increased activity compared to recombinant wild
type human Factor VIIa, wherein said Factor VII polypeptide is
selected from: [0106] i) a Factor VII polypeptide comprising one or
more amino acid substitution selected from K18X.sub.1, R36X.sub.2,
S43X.sub.3, K62X.sub.4, Q64X.sub.5, L65X.sub.6, I69X.sub.7,
F71X.sub.8, L73X.sub.9, P74X.sub.10, E77X.sub.11, G78X.sub.12,
R79X.sub.13, K85X.sub.14, Q88X.sub.15, N93X.sub.16, F275X.sub.17,
R277X.sub.18, M306X.sub.19, T307X.sub.20, Q308X.sub.21,
D309X.sub.22, or R379X.sub.23 corresponding to amino acids at
positions of SEQ ID NO: 1, which different amino acid decreases the
Tissue Factor binding affinity, wherein [0107] X.sub.1 is E, D, A,
or F; [0108] X.sub.2 is E, D, K, A, F, or N; [0109] X.sub.3 is E,
D, K, R, A, or F; [0110] X.sub.4 is D, A, or F; [0111] X.sub.5 is
E, D, K, R, A, F, or N; [0112] X.sub.6 is E, D, K, R, A, or F;
[0113] X.sub.7 is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,
W, or Y; [0114] X.sub.8 is A, G, H, I, K, L, M, P, R, S, T, V, or
W; [0115] X.sub.9 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,
V, W, or Y; [0116] X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W, or Y; [0117] X.sub.11 is A, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W, or Y; [0118] X.sub.12 is A, D, E, F, H, I, K,
L, M, N, P, Q, R, S, T, V, W, or Y; [0119] X.sub.13 is A, D, E, F,
G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; [0120] X.sub.14 is A,
D, E, F, G, I, L, M, P, S, T, V, W, or Y; [0121] X.sub.15 is A, D,
E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; [0122] X.sub.16
is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; [0123]
X.sub.17 is A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
[0124] X.sub.18 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y; [0125] X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T, V,
W, or Y; [0126] X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, V, W, or Y; [0127] X.sub.21 is A, D, E, F, G, H, I, K, L, M,
N, P, R, S, T, V, W, or Y; [0128] X.sub.22 is A, F, G, H, I, K, L,
M, N, P, Q, R, V, W, or Y; and [0129] X.sub.23 is A, D, E, F, G, H,
I, K, L, M, P, Q, S, T, V, W, or Y; [0130] and/or [0131] ii) a
Factor VII polypeptide, wherein one or more amino acid
corresponding to amino acids at positions selected from K18, R36,
K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85, Q88, N93,
F275, R277, M306, Q308, D309, or R379 of SEQ ID NO: 1 have been
substituted with a cysteine amino acid residue, which cysteine
amino acid residue decreases the Tissue Factor binding affinity,
and which cysteine amino acid residue is optionally conjugated with
a chemical group, which chemical group increases the molecular
weight of said Factor VII polypeptide; [0132] and/or [0133] iii) a
Factor VII polypeptide comprising one or more N-glycosylation site
N-Xaa-S/T introduced by amino acid substitutions corresponding to
amino acids starting at positions selected from R36, Q64, I69, F71,
P74, E77, G78, Q88, N93, F275, M306, T307, or D309 of SEQ ID NO: 1,
wherein Xaa is any amino acid except P, which introduced
N-glycosylation site decreases the Tissue Factor binding affinity;
for the preparation of a medicament for the treatment of bleeding
episodes or for the enhancement of the normal haemostatic
system.
[0134] In a further aspect, the invention relates to a method for
the treatment of bleeding episodes or bleeding disorders in a
subject or for the enhancement of the normal haemostatic system,
the method comprising administering to a subject in need thereof a
therapeutically or prophylactically effective amount of a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa, wherein said Factor VII polypeptide is selected
from: [0135] i) a Factor VII polypeptide comprising one or more
amino acid substitution selected from K18X.sub.1, R36X.sub.2,
S43X.sub.3, K62X.sub.4, Q64X.sub.5, L65X.sub.6, I69X.sub.7,
F71X.sub.8, L73X.sub.9, P74X.sub.10, E77X.sub.11, G78X.sub.12,
R79X.sub.13, K85X.sub.14, Q88X.sub.15, N93X.sub.16, F275X.sub.17,
R277X.sub.18, M306X.sub.19, T307X.sub.20, Q308X.sub.21,
D309X.sub.22, or R379X.sub.23 corresponding to amino acids at
positions of SEQ ID NO: 1, which different amino acid decreases the
Tissue Factor binding affinity, wherein [0136] X.sub.1 is E, D, A,
or F; [0137] X.sub.2 is E, D, K, A, F, or N; [0138] X.sub.3 is E,
D, K, R, A, or F; [0139] X.sub.4 is D, A, or F; [0140] X.sub.5 is
E, D, K, R, A, F, or N; [0141] X.sub.6 is E, D, K, R, A, or F;
[0142] X.sub.7 is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,
W, or Y; [0143] X.sub.8 is A, G, H, I, K, L, M, P, R, S, T, V, or
W; [0144] X.sub.9 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,
V, W, or Y; [0145] X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W, or Y; [0146] X.sub.11 is A, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W, or Y; [0147] X.sub.12 is A, D, E, F, H, I, K,
L, M, N, P, Q, R, S, T, V, W, or Y; [0148] X.sub.13 is A, D, E, F,
G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; [0149] X.sub.14 is A,
D, E, F, G, I, L, M, P, S, T, V, W, or Y; [0150] X.sub.15 is A, D,
E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; [0151] X.sub.16
is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; [0152]
X.sub.17 is A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
[0153] X.sub.18 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y; [0154] X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T, V,
W, or Y; [0155] X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, V, W, or Y; [0156] X.sub.21 is A, D, E, F, G, H, I, K, L, M,
N, P, R, S, T, V, W, or Y; [0157] X.sub.22 is A, F, G, H, I, K, L,
M, N, P, Q, R, V, W, or Y; and [0158] X.sub.23 is A, D, E, F, G, H,
I, K, L, M, P, Q, S, T, V, W, or Y; [0159] and/or [0160] ii) a
Factor VII polypeptide, wherein one or more amino acid
corresponding to amino acids at positions selected from K18, R36,
K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85, Q88, N93,
F275, R277, M306, Q308, D309, or R379 of SEQ ID NO: 1 have been
substituted with a cysteine amino acid residue, which cysteine
amino acid residue decreases the Tissue Factor binding affinity,
and which cysteine amino acid residue is optionally conjugated with
a chemical group, which chemical group increases the molecular
weight of said Factor VII polypeptide; [0161] and/or [0162] iii) a
Factor VII polypeptide comprising one or more N-glycosylation site
N-Xaa-S/T introduced by amino acid substitutions corresponding to
amino acids starting at positions selected from R36, Q64, I69, F71,
P74, E77, G78, Q88, N93, F275, M306, T307, or D309 of SEQ ID NO: 1,
wherein Xaa is any amino acid except P, which introduced
N-glycosylation site decreases the Tissue Factor binding
affinity.
[0163] In a further aspect the present invention relates to a
composition comprising wild type human FVIIa and a Factor VII
polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa.
[0164] In a further aspect the present invention relates to a
method for the treatment of bleeding episodes in a subject or for
the enhancement of the normal haemostatic system, the method
comprising administering to a subject in need thereof a
therapeutically or prophylactically effective amount of:
[0165] a) composition comprising wild type human FVIIa and a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa; or
[0166] b) a first composition comprising wild type human FVIIa and
a second composition comprising a Factor VII polypeptide with a
Tissue Factor binding affinity lower than recombinant wild type
human Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa.
[0167] In a further aspect the present invention relates to a
process for preparing a composition comprising wild type human
FVIIa and a Factor VII polypeptide with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa, wherein the process
comprises the step of: mixing wild type human FVIIa with a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa in an aqueous medium.
[0168] In a further aspect the present invention relates to a use
of a composition comprising wild type human FVIIa and a Factor VII
polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa, for the preparation of a medicament for the
treatment of bleeding episodes in a subject or for the enhancement
of the normal haemostatic system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0169] FIG. 1 The structure of correctly processed human
coagulation Factor VII, amino acids 1 to 406, with gamma
carboxylated Glu-residues (.gamma.) and glycosylation (*). The
arrow at amino acid residue 152 shows the site where single-chain
Factor VII is cleaved to be converted to activated two-chain Factor
VII (FVIIa).
[0170] FIG. 2 The full amino acid sequence of native (wild type)
human coagulation Factor VII (SEQ ID NO:1).
DETAILED DESCRIPTION OF THE INVENTION
[0171] The present invention relates to novel coagulation Factor
VII polypeptides with substantially the same or increased activity
compared to wild type Factor VIIa and with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa.
Increasing evidence suggests that therapeutic treatment with
recombinant wild type human coagulation Factor VII works by a TF
independent mechanism involving binding of FVII directly to
activated thrombocytes and a generation of activated Factor Xa and
eventually fibrin locally at the place of tissue damage. The Factor
VII polypeptides of the present invention provide an alternative to
traditional TF binding Factor VII polypeptides. Factor VII
polypeptides that do not bind to TF may be an advantage in the
therapeutic treatment of bleedings, when the exposure of TF is
high, e.g. plaque rupture or sepsis. As used herein, Factor VII
activity refers to its ability to promote blood clotting, which may
be assessed, e.g., by measuring hydrolytic or proteolytic activity
or by measuring thrombin formation.
[0172] As used herein, the terms "Factor VII polypeptide" or "FVII
polypeptide" mean wild-type human Factor VII/VIIa (i.e., a
polypeptide having the amino acid sequence disclosed in U.S. Pat.
No. 4,784,950), as well as variants of Factor VII exhibiting
substantially the same or improved biological activity relative to
wild-type Factor VII, Factor VII-related polypeptides as well as
Factor VII derivatives and Factor VII conjugates. Unless otherwise
indicated, 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.
[0173] As used herein, "Factor VII-related polypeptides"
encompasses polypeptides, including variants, in which the Factor
VIIa biological activity has been substantially modified or reduced
relative to the activity of wild-type Factor VIIa. These
polypeptides include, without limitation, Factor VII or Factor VIIa
into which specific amino acid sequence alterations have been
introduced that modify or disrupt the bioactivity of the
polypeptide.
[0174] The term "Factor VII derivative" as used herein, is intended
to designate wild-type Factor VII, variants of Factor VII
exhibiting substantially the same or improved biological activity
relative to wild-type Factor VII and Factor VII-related
polypeptides, in which one or more of the amino acids of the parent
peptide have been 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.
[0175] The optional conjugation of a cysteine amino acid residue
with the chemical group includes but are not limited to covalent
attachment of polyethylene glycol (PEG), monomethoxy-polyethylene
glycol, dextran, poly-(N-vinyl pyrrolidone)polyethylene glycol,
propylene glycol homopolymers, a polypropylene oxide/ethylene oxide
co-polymer, polypropylene glycol, polyoxyethylated polyols (e.g.,
glycerol) and polyvinyl alcohol, colominic acids or other
carbohydrate based polymers, polymers of amino acids, and biotin
derivatives. In one embodiment of the present invention, the
chemical group is a biocompatible, non-toxic, non-immunogenic and
water-soluble polymer. Preferably the chemical group is
water-soluble in all proportions.
[0176] Methods for attaching PEG groups to cysteine residues are
described in Roberts, M. J. et al, Advanced Drug Delivery Reviews
54 (2002) 459-476.
[0177] An "N-glycosylation site" has the sequence N-Xaa-S/T,
wherein Xaa is any amino acid residue except proline, N is
asparagine and S/T is either serine or threonine, such as serine or
threonine, such as threonine.
[0178] Specific examples of activated PEG polymers particularly
preferred for coupling to cysteine residues, include the following
linear PEGs: vinylsulfone-PEG (VS-PEG), such as vinylsulfone-mPEG
(VS-mPEG); maleimide-PEG (MAL-PEG), such as maleimide-mPEG
(MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), such as
orthopyridyl-disulfide-mPEG (OPSS-MPEG). Typically, such PEG or
mPEG polymers will have a size of about 2 kDa, such as about 5 kDa,
such as about 10 kD, such as about 12 kDa or such as about 20
kDa.
[0179] The skilled person will be aware that the activation method
and/or conjugation chemistry to be used depends on the functional
groups of the polymer (e.g. being amine, hydroxyl, carboxyl,
aldehyde, sulfydryl, succinimidyl, maleimide, vinysulfone or
haloacetate).
[0180] For conjugated with a chemical group to a cysteine residue
(e.g. PEGylation) the FVII polypeptide is usually treated with a
reducing agent, such as dithiothreitol (DDT),
.beta.-mercaptoethanol, or glutathione
(.gamma.-glutamylcysteinylglycine) prior to PEGylation, such as
described in Higashi, S. Matsumoto, N., Iwanaga, S. (1997) J. Biol.
Chem., 272(41), 25724-25730. The reducing agent is subsequently
removed by any conventional method, such as by desalting.
Conjugation of PEG to a cysteine residue typically takes place in a
suitable buffer at pH 6-9 at temperatures varying from 4.degree. C.
to 25.degree. C. for periods up to 16 hours.
[0181] In one embodiment of the present invention, an introduced
cysteine residue is conjugated by mixed disulfide bond-formation
with a chemical group selected from the list consisting of
glutathione (gamma-glutamylcysteinylglycine),
gamma-glutamylcysteine and cysteine present in the host cell or in
the culture medium during production of the FVII polypeptide.
[0182] In one embodiment of the invention the chemical group is
selected from the group consisting of: dendrimer, polyalkylene
oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG),
polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA),
poly-carboxylate, poly-vinylpyrolidone, polyethylene-co-maleic acid
anhydride, polystyrene-co-maleic acid anhydride, dextran,
carboxymethyl-dextran; serum protein binding-ligands, such as
compounds which bind to albumin, such as fatty acids, C5-C24 fatty
acid, aliphatic diacid (e.g. C5-C24), a structure (e.g. sialic acid
derivatives or mimetics) which inhibits the glycans from binding to
receptors (e.g. asialoglycoprotein receptor and mannose receptor),
a small organic molecule containing moieties that under
physiological conditions alters charge properties, such as
carboxylic acids or amines, or neutral sub-stituents that prevent
glycan specific recognition such as smaller alkyl substituents
(e.g., C1-C5 alkyl), a low molecular organic charged radical (e.g.
C1-C25), which may contain one or more carboxylic acids, amines
sulfonic, phosphonic acids, or combination thereof; a low molecular
neutral hydrophilic molecule (e.g. C1-C25), such as cyclodextrin,
or a polyethylene chain which may optionally branched;
polyethyleneglycol with a avarage molecular weight of 2-40 KDa; a
well defined precission polymer such as a dendrimer with an excact
molecular mass ranging from 700 to 20.000 Da, such as between
700-10.000 Da; and a substantially non-imunogenic polypeptide such
as albumin or an antibody or part of an antibody optionally
containing a Fc-domain.
[0183] 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.
[0184] 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; (iii) measuring its
physical binding to TF using an instrument based on surface plasmon
resonance (Persson, FEBS Letts. 413:359-363, 1997) and (iv)
measuring hydrolysis of a synthetic substrate.
[0185] The terms "variant" or "variants", as used herein, is
intended to designate Factor VII having the sequence of SEQ ID
NO:1, wherein one or more amino acids of the parent protein have
been substituted by another amino acid and/or wherein one or more
amino acids of the parent protein have been deleted and/or wherein
one or more amino acids have been inserted in protein and/or
wherein one or more amino acids have been added to the parent
protein. Such addition can take place either at the N-terminal end
or at the C-terminal end of the parent protein or both. The
"variant" or "variants" within this definition still have FVII
activity in its activated form. In one embodiment a variant is 70%
identical with the sequence of SEQ ID NO:1. In one embodiment a
variant is 80% identical with the sequence of SEQ ID NO:1. In
another embodiment a variant is 90% identical with the sequence of
SEQ ID NO:1. In a further embodiment a variant is 95% identical
with the sequence of SEQ ID NO:1.
[0186] Non-limiting examples of Factor VII variants having
substantially the same biological activity as wild-type Factor VII
include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem.
Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased
proteolytic stability as disclosed in U.S. Pat. No. 5,580,560;
Factor VIIa that has been proteolytically cleaved between residues
290 and 291 or between residues 315 and 316 (Mollerup et al.,
Biotechnol. Bioeng. 48:501-505, 1995); oxidized forms of Factor
VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999);
FVII variants as disclosed in PCT/DK02/00189; 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 (University of Minnesota) and WO 00/66753
(University of Minnesota); and FVII variants as disclosed in WO
01/58935 (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO 04/029091
(Maxygen ApS).
[0187] 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, Danish patent application PA 2002 01423, Danish
patent application PA 2001 01627; WO 02/38162 (Scripps Research
Institute); and FVIIa variants with enhanced activity as disclosed
in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.). Non-limiting
examples of Factor VII variants having substantially reduced or
modified biological activity relative to wild-type Factor VII
include R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990),
S344A-FVIIa (Kazama et al., J. Biol. Chem. 270:66-72, 1995),
FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15:515-520,
1998), and Factor VIIa lacking the Gla domain, (Nicolaisen et al.,
FEBS Letts. 317:245-249, 1993). Examples of variants of factor VII,
factor VII or factor VII-related polypeptides include, without
limitation, wild-type Factor VII, L305V-FVII,
L305V/M306D/D309S-FVII, L3051-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/M158T-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/M298D-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/L305
.mu.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, Factor VIIa
lacking the Gla domain; and P11Q/K33E-FVII, 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.
[0188] The terminology for amino acid substitutions used is as
follows. The first letter represents the amino acid naturally
present at a position of human wild type FVII. The following number
represents the position in human wild type FVII. The second letter
represent the different amino acid substituting for (replacing) the
natural amino acid. An example is M298Q, where a methionine at
position 298 of human wild type FVII is replaced by a glutamine. In
another example, V158T/M298Q, the valine in position 158 of human
wild type FVII is replaced by a threonine and the methionine in
position 298 of human wild type FVII is replaced by a Glutamine in
the same Factor VII polypeptide.
[0189] In a further embodiment of the invention, the factor VII
polypeptide is a polypeptide, wherein the ratio between the
activity of the Factor VII polypeptide and the activity of the wild
type human Factor VIIa is at least about 1.25. In one embodiment
the ratio between the activity of the Factor VII polypeptide and
the activity of the wild type human Factor VIIa is at least about
2.0. In a further embodiment the ratio between the activity of the
Factor VII polypeptide and the activity of the wild type human
Factor VIIa is at least about 4.0.
[0190] In a further embodiment of the invention, the factor VII
polypeptide is a polypeptide, wherein the ratio between the
activity of the Factor VII polypeptide and the activity of the wild
type human Factor VIIa is at least about 1.25 when tested in a
Factor VIIa activity assay. In one embodiment the ratio between the
activity of the Factor VII polypeptide and the activity of the wild
type human Factor VIIa is at least about 2.0 when tested in a
Factor VIIa activity assay. In a further embodiment the ratio
between the activity of the Factor VII polypeptide and the activity
of the wild type human Factor VIIa is at least about 4.0 when
tested in a Factor VIIa activity assay. The Factor VIIa activity
may be measured by the assays described under "assays".
[0191] In a further embodiment of the invention, the factor VII
polypeptide is a polypeptide, wherein the ratio between the
activity of the Factor VII polypeptide and the activity of the wild
type human Factor VIIa is at least about 1.25 when tested in the
"In Vitro Hydrolysis Assay". In one embodiment the ratio between
the activity of the Factor VII polypeptide and the activity of the
wild type human Factor VIIa is at least about 2.0 when tested in
the "In Vitro Hydrolysis Assay". In a further embodiment the ratio
between the activity of the Factor VII polypeptide and the activity
of the wild type human Factor VIIa is at least about 4.0 when
tested in the "In Vitro Hydrolysis Assay".
[0192] In a further embodiment of the invention, the factor VII
polypeptide is a polypeptide, wherein the ratio between the
activity of the Factor VII polypeptide and the activity of the wild
type human Factor VIIa is at least about 1.25 when tested in the
"In Vitro Proteolysis Assay". In one embodiment the ratio between
the activity of the Factor VII polypeptide and the activity of the
wild type human Factor VIIa is at least about 2.0 when tested in
the "In Vitro Proteolysis Assay". In a further embodiment the ratio
between the activity of the Factor VII polypeptide and the activity
of the wild type human Factor VIIa is at least about 4.0 when
tested in the "In Vitro Proteolysis Assay". In a further embodiment
the ratio between the activity of the Factor VII polypeptide and
the activity of the wild type human Factor VIIa is at least about
8.0 when tested in the "In Vitro Proteolysis Assay".
[0193] The present invention is also suitable for Factor VII/VIIa
variants with increased activity compared to wild type. Factor
VII/VIIa variants with increased activity may be found by testing
in suitable assays described in the following. These assays can be
performed as a simple preliminary in vitro test. Thus, the section
"assays" discloses a simple test (entitled "In Vitro Hydrolysis
Assay") for the activity of Factor VIIa variants of the invention.
Based thereon, Factor VIIa variants which are of particular
interest are such variants where the ratio between the activity of
the variant and the activity of wild type Factor VII is above 1.0,
e.g. at least about 1.25, preferably at least about 2.0, such as at
least about 3.0 or, even more preferred, at least about 4.0 when
tested in the "In Vitro Hydrolysis Assay".
[0194] The activity of the variants can also be measured using a
physiological substrate such as factor X ("In Vitro Proteolysis
Assay") (see under "assays"), suitably at a concentration of
100-1000 nM, where the factor Xa generated is measured after the
addition of a suitable chromogenic substrate (e.g. S-2765). In
addition, the activity assay may be run at physiological
temperature.
[0195] The ability of the Factor VIIa variants to generate thrombin
can also be measured in an assay comprising all relevant
coagulation factors and inhibitors at physiological concentrations
(minus factor VIII when mimicking hemophilia A conditions) 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).
[0196] The term "identity" as known in the art, refers to a
relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
between polypeptides, as the case may be, as determined by the
number of matches between strings of two or more nucleotide
residues or two or more amino acid residues. "Identity" measures
the percent of identical matches between the smaller of two or more
sequences with gap alignments (if any) addressed by a particular
mathematical model or computer program (i.e., "algorithms").
[0197] The term "similarity" is a related concept, but in contrast
to "identity", refers to a sequence relationship that includes both
identical matches and conservative substitution matches. If two
polypeptide sequences have, for example, 10/20 identical amino
acids, and the remainder are all non-conservative substitutions,
then the percent identity and similarity would both be 50%. If, in
the same example, there are 5 more positions where there are
conservative substitutions, then the percent identity remains 50%,
but the percent similarity would be 75% 15/20). Therefore, in cases
where there are conservative substitutions, the degree of
similarity between two polypeptides will be higher than the percent
identity between those two polypeptides.
[0198] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates or other
materials (i.e., contaminants) with which it is naturally
associated, (2) is not covalently linked to all or a portion of a
polypeptide to which the "isolated polypeptide" is linked in
nature, (3) is operably linked covalently to a polypeptide to which
it is not covalently linked in nature, or (4) does not occur in
nature. Preferably, the isolated polypeptide is substantially free
from any other contaminating polypeptides or other contaminants
that are found in its natural environment which would interfere
with its therapeutic, diagnostic, prophylactic or research use.
[0199] Conservative modifications to the amino acid sequence of SEQ
ID NO:1 (and the corresponding modifications to the encoding
nucleotides) outside the positions for substitutions claimed will
produce FVII polypeptides having functional and chemical
characteristics similar to those of naturally occurring FVII
polypeptide. In contrast, substantial modifications in the
functional and/or chemical characteristics of FVII polypeptides may
be accomplished by selecting substitutions in the amino acid
sequence of SEQ ID NO:1 that differ significantly in their effect
on maintaining (a) the structure of the molecular backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain.
[0200] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
non-native residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with almandine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,
Adv. Biophys. 35:1-24, which discuss alanine scanning
mutagenesis).
[0201] Identity and similarity of related polypeptides can be
readily calculated by known methods. Such methods include, but are
not limited to, those described in Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math., 48:1073 (1988).
[0202] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410
(1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well known Smith Waterman algorithm
may also be used to determine identity.
[0203] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least 50 contiguous amino acids of
the target polypeptide.
[0204] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
1/10 times the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix (see Dayhoff et al., Atlas
of Protein Sequence and Structure, vol. 5, supp. 3 (1978) for the
PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci
USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is
also used by the algorithm.
[0205] Preferred parameters for a polypeptide sequence comparison
include the following:
[0206] Algorithm: Needleman et al., J. Mol. Biol, 48:443-453
(1970); Comparison matrix: BLOSUM 62 from Henikoff et al., Proc.
Natl. Acad. Sci. USA, 89:10915-10919 (1992); Gap Penalty: 12, Gap
Length Penalty: 4, Threshold of Similarity: 0.
[0207] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0208] Preferred parameters for nucleic acid molecule sequence
comparisons include the following: Algorithm: Needleman et al., J.
Mol Biol., 48:443-453 (1970); Comparison matrix: matches=+10,
mismatch=0, Gap Penalty: 50, Gap Length Penalty: 3.
[0209] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0210] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, thresholds of similarity,
etc. may be used, including those set forth in the Program Manual,
Wisconsin Package, Version 9, September, 1997. The particular
choices to be made will be apparent to those of skill in the art
and will depend on the specific comparison to be made, such as DNA
to DNA, protein to protein, protein to DNA; and additionally,
whether the comparison is between given pairs of sequences (in
which case GAP or BestFit are generally preferred) or between one
sequence and a large database of sequences (in which case FASTA or
BLASTA are preferred).
[0211] The term "Tissue Factor binding affinity", as used herein,
means the strength of the binding of a FVII polypeptide to human
Tissue Factor. The affinity of a FVII polypeptide is measured by
the dissociation constant K.sub.d, defined as
[FVII].times.[TF]/[FVII-TF] where [FVII-TF] is the molar
concentration of the FVII/TF complex, [FVII] is the molar
concentration of the unbound FVII polypeptide and [TF] is the molar
concentration of the unbound human Tissue Factor. The affinity
constant K.sub.a is defined by 1/K.sub.d.
[0212] The phrase "substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa", as
used herein, means an activity more than 50% of the activity of
recombinant wild type human Factor VIIa. In one embodiment the
activity is more than 60% of the activity of recombinant wild type
human Factor VIIa. In one embodiment the activity is more than 70%
of the activity of recombinant wild type human Factor VIIa. In one
embodiment the activity is more than 80% of the activity of
recombinant wild type human Factor VIIa. In another embodiment the
activity is more than 90% of the activity of recombinant wild type
human Factor VIIa. In a further embodiment the activity is more
than 100% of the activity of recombinant wild type human Factor
VIIa. In a further embodiment the activity is more than 120% of the
activity of recombinant wild type human Factor VIIa. In a further
embodiment the activity is more than 200% of the activity of
recombinant wild type human Factor VIIa. In a further embodiment
the activity is more than 400% of the activity of recombinant wild
type human Factor VIIa.
[0213] The term "activity" as used herein means the ability of a
Factor VII polypeptide to convert its substrate Factor X to the
active Factor Xa. The activity of a Factor VI I polypeptide may be
measured with the "In Vitro Proteolysis Assay".
[0214] The phrase "a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa", as used herein, means an
binding affinity lower than that of recombinant wild type human
Factor VIIa as measured in a TF binding affinity assay, such as the
Biosensor assay as described in Example 5. In one embodiment the
Tissue Factor binding affinity is less than 90% of the Tissue
Factor binding affinity of recombinant wild type human Factor VIIa.
In one embodiment the Tissue Factor binding affinity is less than
80% of the Tissue Factor binding affinity of recombinant wild type
human Factor VIIa. In one embodiment the Tissue Factor binding
affinity is less than 70% of the Tissue Factor binding affinity of
recombinant wild type human Factor VIIa. In one embodiment the
Tissue Factor binding affinity is less than 60% of the Tissue
Factor binding affinity of recombinant wild type human Factor VIIa.
In one embodiment the Tissue Factor binding affinity is less than
50% of the Tissue Factor binding affinity of recombinant wild type
human Factor VIIa. In one embodiment the Tissue Factor binding
affinity is less than 40% of the Tissue Factor binding affinity of
recombinant wild type human Factor VIIa. In one embodiment the
Tissue Factor binding affinity is less than 30% of the Tissue
Factor binding affinity of recombinant wild type human Factor VIIa.
In one embodiment the Tissue Factor binding affinity is less than
20% of the Tissue Factor binding affinity of recombinant wild type
human Factor VIIa. In one embodiment the Tissue Factor binding
affinity is less than 10% of the Tissue Factor binding affinity of
recombinant wild type human Factor VIIa. In one embodiment the
Tissue Factor binding affinity is less than 5% of the Tissue Factor
binding affinity of recombinant wild type human Factor VIIa. In one
embodiment the Tissue Factor binding affinity is less than 1% of
the Tissue Factor binding affinity of recombinant wild type human
Factor VIIa. In one embodiment the Tissue Factor binding affinity
is less than 0.1% of the Tissue Factor binding affinity of
recombinant wild type human Factor VIIa. In one embodiment the
Tissue Factor binding affinity is less than 0.01% of the Tissue
Factor binding affinity of recombinant wild type human Factor VIIa.
In one embodiment the Tissue Factor binding affinity is less than
0.001% of the Tissue Factor binding affinity of recombinant wild
type human Factor VIIa.
[0215] The term "polyethylene glycol" or "PEG" means a polyethylene
glycol compound or a derivative thereof, with or without coupling
agents, coupling or activating moieties (e.g., with thiol,
triflate, tresylate, azirdine, oxirane, or preferably with a
maleimide moiety). Compounds such as maleimido monomethoxy PEG are
exemplary of activated PEG compounds of the invention.
[0216] In one embodiment of the invention, the Factor VII
polypeptides are Factor VII polypeptides, wherein one or more amino
acid corresponding to amino acids at positions selected from K18,
R36, S43, K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85,
Q88, N93, F275, R277, M306, T307, Q308, D309, or R379 of SEQ ID NO:
1 have been substituted with a different amino acid, which
different amino acid decreases the Tissue Factor binding
affinity.
[0217] The term "a different amino acid" as used herein means one
or more amino acids that are different from that amino acid
naturally present at that position. This includes but is not
limited to amino acids that can be encoded by a polynucleotide.
Preferably the different amino acid is in natural L-form and can be
encoded by a polynucleotide. A specific example being L-Glutamic
acid (L-Glu).
[0218] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position K18 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position K18 of SEQ ID NO: 1 is
selected from the list consisting of E, D, A, and F.
[0219] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position R36 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position R36 of SEQ ID NO: 1 is
selected from the list consisting of E, D, A, and F.
[0220] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position S43 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position S43 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, F, and N. In
one embodiment this different amino acid is selected from the list
consisting of E, D, K, R, A, and F. In one embodiment this
different amino acid is an N.
[0221] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position K62 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position K62 of SEQ ID NO: 1 is
selected from the list consisting of E, D, A, and F. In one
embodiment this different amino acid is selected from the list
consisting of D, A, and F.
[0222] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position Q64 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position Q64 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0223] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position L65 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position L65 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0224] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position 169 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position 169 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F. In one
embodiment this different amino acid is selected from the list
consisting of E, D, K, R, and F.
[0225] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position F71 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position F71 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, and A. In one
embodiment this different amino acid is selected from the list
consisting of K, R, and A.
[0226] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position L73 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position L73 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0227] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position P74 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position P74 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0228] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position E77 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position E77 of SEQ ID NO: 1 is
selected from the list consisting of K, R, and A.
[0229] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position G78 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position G78 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0230] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position R79 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position R79 of SEQ ID NO: 1 is
selected from the list consisting of E, D, and A.
[0231] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position K85 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position K85 of SEQ ID NO: 1 is
selected from the list consisting of E, D, and A.
[0232] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position Q88 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position Q88 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0233] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position N93 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position N93 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F.
[0234] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position F275 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position F275 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, and A.
[0235] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position R277 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position R277 of SEQ ID NO: 1 is
selected from the list consisting of E, D, A, and F.
[0236] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position T307 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position T307 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, F, and N.
[0237] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position Q308 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position Q308 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, F, and N.
[0238] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position R379 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position R379 of SEQ ID NO: 1 is
selected from the list consisting of E, D, A, and F.
[0239] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position M306 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position M306 of SEQ ID NO: 1 is
selected from the list consisting of E, D, K, R, A, and F. In one
embodiment this different amino acid is selected from the list
consisting of E, K, R, A, and F.
[0240] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the amino acid
corresponding to the amino acid at a position D309 of SEQ ID NO: 1
has been substituted with a different amino acid. In one embodiment
this different amino acid at a position D309 of SEQ ID NO: 1 is
selected from the list consisting of K, R, and A.
[0241] In a further embodiment, the Factor VII polypeptide of the
invention is a Factor VII polypeptide selected from the list
consisting of S43N-FVII, K62E-FVII, Q64E-FVII, I69F-FVII,
K62E/I69A-FVII, Q64E/I69A-FVII, K62E/Q64E/I69A-FVII,
K62E/I69F-FVII, Q64E/I69F-FVII, and K62E/Q64E/I69F-FVII.
[0242] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides further comprising amino acid
substitutions selected from the list consisting of L305V,
L305V/M306D/D309S, L3051, L305T, F374P, V158T/M298Q,
V158D/E296V/M298Q, K337A, M298Q, V158D/M298Q, L305V/K337A,
V158D/E296V/M298Q/L305V, V158D/E296V/M298Q/K337A,
V158D/E296V/M298Q/L305V/K337A, K157A, E296V, E296V/M298Q,
V158D/E296V, V158D/M298K, and S336G, L305V/K337A, L305V/V158D,
L305V/E296V, L305V/M298Q, L305V/V158T, L305V/K337A/V158T,
L305V/K337A/M298Q, L305V/K337A/E296V, L305V/K337A/V158D,
L305V/V158D/M298Q, L305V/V158D/E296V, L305V/V158T/M298Q,
L305V/V158T/E296V, L305V/E296V/M298Q, L305V/V158D/E296V/M298Q,
L305V/V158T/E296V/M298Q, L305V/V158T/K337A/M298Q,
L305V/V158T/E296V/K337A, L305V/V158D/K337A/M298Q,
L305V/V158D/E296V/K337A, L305V/V158D/E296V/M298Q/K337A,
L305V/V158T/E296V/M298Q/K337A, S314E/K316H, S314E/K316Q,
S314E/L305V, S314E/K337A, S314E/V158D, S314E/E296V, S314E/M298Q,
S314E/V158T, K316H/L305V, K316H/K337A, K316H/V158D, K316H/E296V,
K316H/M298Q, K316H/V158T, K316Q/L305V, K316Q/K337A, K316Q/V158D,
K316Q/E296V, K316Q/M298Q, K316Q/V158T, S314E/L305V/K337A,
S314E/L305V/V158D, S314E/L305V/E296V, S314E/L305V/M298Q,
S314E/L305V/V158T, S314E/L305V/K337A/V158T,
S314E/L305V/K337A/M298Q, S314E/L305V/K337A/E296V,
S314E/L305V/K337A/V158D, S314E/L305V/V158D/M298Q,
S314E/L305V/V158D/E296V, S314E/L305V/V158T/M298Q,
S314E/L305V/V158T/E296V, S314E/L305V/E296V/M298Q,
S314E/L305V/V158D/E296V/M298Q, S314E/L305V/V158T/E296V/M298Q,
S314E/L305V/V158T/K337A/M298Q, S314E/L305V/V158T/E296V/K337A,
S314E/L305V/V158D/K337A/M298Q, S314E/L305V/V158D/E296V/K337A,
S314E/L305V/V158D/E296V/M298Q/K337A,
S314E/L305V/V158T/E296V/M298Q/K337A, K316H/L305V/K337A,
K316H/L305V/V158D, K316H/L305V/E296V, K316H/L305V/M298Q,
K316H/L305V/V158T, K316H/L305V/K337A/V158T,
K316H/L305V/K337A/M298Q, K316H/L305V/K337A/E296V,
K316H/L305V/K337A/V158D, K316H/L305V/V158D/M298Q,
K316H/L305V/V158D/E296V, K316H/L305V/V158T/M298Q,
K316H/L305V/V158T/E296V, K316H/L305V/E296V/M298Q,
K316H/L305V/V158D/E296V/M298Q, K316H/L305V/V158T/E296V/M298Q,
K316H/L305V/V158T/K337A/M298Q, K316H/L305V/V158T/E296V/K337A,
K316H/L305V/V158D/K337A/M298Q, K316H/L305V/V158D/E296V/K337A,
K316H/L305V/V158D/E296V/M298Q/K337A,
K316H/L305V/V158T/E296V/M298Q/K337A, K316Q/L305V/K337A,
K316Q/L305V/V158D, K316Q/L305V/E296V, K316Q/L305V/M298Q,
K316Q/L305V/V158T, K316Q/L305V/K337A/V158T,
K316Q/L305V/K337A/M298Q, K316Q/L305V/K337A/E296V,
K316Q/L305V/K337A/V158D, K316Q/L305V/V158D/M298Q,
K316Q/L305V/V158D/E296V, K316Q/L305V/V158T/M298Q,
K316Q/L305V/V158T/E296V, K316Q/L305V/E296V/M298Q,
K316Q/L305V/V158D/E296V/M298Q, K316Q/L305V/V158T/E296V/M298Q,
K316Q/L305V/V158T/K337A/M298Q, K316Q/L305V/V158T/E296V/K337A,
K316Q/L305V/V158D/K337A/M298Q, K316Q/L305V/V158D/E296V/K337A,
K316Q/L305V/V158D/E296V/M298Q/K337A,
K316Q/L305V/V158T/E296V/M298Q/K337A, F374Y/K337A, F374Y/V158D,
F374Y/E296V, F374Y/M298Q, F374Y/V158T, F374Y/S314E, F374Y/L305V,
F374Y/L305V/K337A, F374Y/L305V/V158D, F374Y/L305V/E296V,
F374Y/L305V/M298Q, F374Y/L305V/V158T, F374Y/L305V/S314E,
F374Y/K337A/S314E, F374Y/K337A/V158T, F374Y/K337A/M298Q,
F374Y/K337A/E296V, F374Y/K337A/V158D, F374Y/V158D/S314E,
F374Y/V158D/M298Q, F374Y/V158D/E296V, F374Y/V158T/S314E,
F374Y/V158T/M298Q, F374Y/V158T/E296V, F374Y/E296V/S314E,
F374Y/S314E/M298Q, F374Y/E296V/M298Q, F374Y/L305V/K337A/V158D,
F374Y/L305V/K337A/E296V, F374Y/L305V/K337A/M298Q,
F374Y/L305V/K337A/V158T, F374Y/L305V/K337A/S314E,
F374Y/L305V/V158D/E296V, F374Y/L305V/V158D/M298Q,
F374Y/L305V/V158D/S314E, F374Y/L305V/E296V/M298Q,
F374Y/L305V/E296V/V158T, F374Y/L305V/E296V/S314E,
F374Y/L305V/M298Q/V158T, F374Y/L305V/M298Q/S314E,
F374Y/L305V/V158T/S314E, F374Y/K337A/S314E/V158T,
F374Y/K337A/S314E/M298Q, F374Y/K337A/S314E/E296V,
F374Y/K337A/S314E/V158D, F374Y/K337A/V158T/M298Q,
F374Y/K337A/V158T/E296V, F374Y/K337A/M298Q/E296V,
F374Y/K337A/M298Q/V158D, F374Y/K337A/E296V/V158D,
F374Y/V158D/S314E/M298Q, F374Y/V158D/S314E/E296V,
F374Y/V158D/M298Q/E296V, F374Y/V158T/S314E/E296V,
F374Y/V158T/S314E/M298Q, F374Y/V158T/M298Q/E296V,
F374Y/E296V/S314E/M298Q, F374Y/L305V/M298Q/K337A/S314E,
F374Y/L305V/E296V/K337A/S314E, F374Y/E296V/M298Q/K337A/S314E,
F374Y/L305V/E296V/M298Q/K337A, F374Y/L305V/E296V/M298Q/S314E,
F374Y/V158D/E296V/M298Q/K337A, F374Y/V158D/E296V/M298Q/S314E,
F374Y/L305V/V158D/K337A/S314E, F374Y/V158D/M298Q/K337A/S314E,
F374Y/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q,
F374Y/L305V/V158D/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A,
F374Y/L305V/V158D/M298Q/S314E, F374Y/L305V/V158D/E296V/S314E,
F374Y/V158T/E296V/M298Q/K337A, F374Y/V158T/E296V/M298Q/S314E,
F374Y/L305V/V158T/K337A/S314E, F374Y/V158T/M298Q/K337A/S314E,
F374Y/V158T/E296V/K337A/S314E, F374Y/L305V/V158T/E296V/M298Q,
F374Y/L305V/V158T/M298Q/K337A, F374Y/L305V/V158T/E296V/K337A,
F374Y/L305V/V158T/M298Q/S314E, F374Y/L305V/V158T/E296V/S314E,
F374Y/E296V/M298Q/K337A/V158T/S314E,
F374Y/V158D/E296V/M298Q/K337A/S314E,
F374Y/L305V/V158D/E296V/M298Q/S314E,
F374Y/L305V/E296V/M298Q/V158T/S314E,
F374Y/L305V/E296V/M298Q/K337A/V158T,
F374Y/L305V/E296V/K337A/V158T/S314E,
F374Y/L305V/M298Q/K337A/V158T/S314E,
F374Y/L305V/V158D/E296V/M298Q/K337A,
F374Y/L305V/V158D/E296V/K337A/S314E,
F374Y/L305V/V158D/M298Q/K337A/S314E,
F374Y/L305V/E296V/M298Q/K337A/V158T/S314E,
F374Y/L305V/V158D/E296V/M298Q/K337A/S314E, S52A, S60A; R152E,
S344A, P11Q/K33E, T106N, K143N/N145T, V253N, R290N/A292T, G291N,
R315N/V317T, and K143N/N145T/R315N/V317T; or having substitutions,
additions or deletions in the amino acid sequence from 233Thr to
240Asn, or having substitutions, additions or deletions in the
amino acid sequence from 304Arg to 329Cys.
[0243] In a further embodiment, the Factor VII polypeptides of the
invention are Factor VII polypeptides, wherein the dissociation
constant K.sub.d of said Factor VII polypeptide is higher than 5
nM, such as higher than 7 nM, such as higher than 10 nM, such as
higher than 20 nM, such as higher than 30 nM, such as higher than
50 nM, such as higher than 100 nM, such as higher than 200 nM, such
as higher than 300 nM, such as higher than 400 nM, such as higher
than 500 nM, such as higher than 1 .mu.M.
[0244] In a further embodiment, the Factor VII polypeptides of the
invention comprises one or more amino acid substitution selected
from K18X.sub.1, R36X.sub.2, S43X.sub.3, K62X.sub.4, Q64X.sub.5,
L65X.sub.6, I69X.sub.7, F71X.sub.8, L73X.sub.9, P74X.sub.10,
E77X.sub.11, G78X.sub.12, R79X.sub.13, K85X.sub.14, Q88X.sub.15,
N93X.sub.16, F275X.sub.17, R277X.sub.18, M306X.sub.19,
T307X.sub.20, Q308X.sub.21, D309X.sub.22, or R379X.sub.23
corresponding to amino acids at positions of SEQ ID NO: 1, which
different amino acid decreases the Tissue Factor binding affinity,
wherein
[0245] X.sub.1 is E, D, A, or F;
[0246] X.sub.2 is E, D, K, A, F, or N;
[0247] X.sub.3 is E, D, K, R, A, or F;
[0248] X.sub.4 is D, A, or F;
[0249] X.sub.5 is E, D, K, R, A, F, or N;
[0250] X.sub.6 is E, D, K, R, A, or F;
[0251] X.sub.7 is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,
W, or Y;
[0252] X.sub.8 is A, G, H, I, K, L, M, P, R, S, T, V, or W;
[0253] X.sub.9 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,
W, or Y;
[0254] X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V,
W, or Y;
[0255] X.sub.11 is A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y;
[0256] X.sub.12 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,
W, or Y;
[0257] X.sub.13 is A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V,
W, or Y;
[0258] X.sub.14 is A, D, E, F, G, I, L, M, P, S, T, V, W, or Y;
[0259] X.sub.15 is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V,
W, or Y;
[0260] X.sub.16 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,
W, or Y;
[0261] X.sub.17 is A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W,
or Y;
[0262] X.sub.18 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y;
[0263] X.sub.19 is A, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, or
Y;
[0264] X.sub.20 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V,
W, or Y;
[0265] X.sub.21 is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V,
W, or Y;
[0266] X.sub.22 is A, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y;
and
[0267] X.sub.23 is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W,
or Y.
[0268] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein one or more amino
acid corresponding to amino acids at positions selected from K18,
R36, K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85, Q88,
N93, F275, R277, M306, Q308, D309, or R379 of SEQ ID NO: 1 have
been substituted with a cysteine amino acid residue, which cysteine
amino acid residue decreases the Tissue Factor binding affinity,
and which cysteine amino acid residue is optionally conjugated with
a chemical group, which chemical group increases the molecular
weight of said Factor VII polypeptide. In one embodiment K18 of SEQ
ID NO: 1 has been substituted with a cysteine amino acid residue.
In one embodiment R36 of SEQ ID NO: 1 has been substituted with a
cysteine amino acid residue. In one embodiment K62 of SEQ ID NO: 1
has been substituted with a cysteine amino acid residue. In one
embodiment Q64 of SEQ ID NO: 1 has been substituted with a cysteine
amino acid residue. In one embodiment L65 of SEQ ID NO: 1 has been
substituted with a cysteine amino acid residue. In one embodiment
I69 of SEQ ID NO: 1 has been substituted with a cysteine amino acid
residue. In one embodiment F71 of SEQ ID NO: 1 has been substituted
with a cysteine amino acid residue. In one embodiment L73 of SEQ ID
NO: 1 has been substituted with a cysteine amino acid residue. In
one embodiment P74 of SEQ ID NO: 1 has been substituted with a
cysteine amino acid residue. In one embodiment E77 of SEQ ID NO: 1
has been substituted with a cysteine amino acid residue. In one
embodiment G78 of SEQ ID NO: 1 has been substituted with a cysteine
amino acid residue. In one embodiment R79 of SEQ ID NO: 1 has been
substituted with a cysteine amino acid residue. In one embodiment
K85 of SEQ ID NO: 1 has been substituted with a cysteine amino acid
residue. In one embodiment Q88 of SEQ ID NO: 1 has been substituted
with a cysteine amino acid residue. In one embodiment N93 of SEQ ID
NO: 1 has been substituted with a cysteine amino acid residue. In
one embodiment F275 of SEQ ID NO: 1 has been substituted with a
cysteine amino acid residue. In one embodiment R277 of SEQ ID NO: 1
has been substituted with a cysteine amino acid residue. In one
embodiment M306 of SEQ ID NO: 1 has been substituted with a
cysteine amino acid residue. In one embodiment Q308 of SEQ ID NO: 1
has been substituted with a cysteine amino acid residue. In one
embodiment D309 of SEQ ID NO: 1 has been substituted with a
cysteine amino acid residue. In one embodiment R379 of SEQ ID NO: 1
has been substituted with a cysteine amino acid residue.
[0269] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide comprising one or more
N-glycosylation site N-Xaa-S/T introduced by amino acid
substitutions corresponding to amino acids starting at positions
selected from R36, Q64, I69, F71, P74, E77, G78, Q88, N93, F275,
M306, T307, or D309 of SEQ ID NO: 1, wherein Xaa is any amino acid
except P, which introduced N-glycosylation site decreases the
Tissue Factor binding affinity. In one embodiment an
N-glycosylation site N-Xaa-S/T is introduced at a position starting
at R36 of SEQ ID NO: 1. In one embodiment an N-glycosylation site
N-Xaa-S/T is introduced at a position starting at R36 of SEQ ID NO:
1. In one embodiment an N-glycosylation site N-Xaa-S/T is
introduced at a position starting at Q64 of SEQ ID NO: 1.
[0270] In one embodiment an N-glycosylation site N-Xaa-S/T is
introduced at a position starting at 169 of SEQ ID NO: 1. In one
embodiment an N-glycosylation site N-Xaa-S/T is introduced at a
position starting at F71 of SEQ ID NO: 1. In one embodiment an
N-glycosylation site N-Xaa-S/T is introduced at a position starting
at P74 of SEQ ID NO: 1. In one embodiment an N-glycosylation site
N-Xaa-S/T is introduced at a position starting at E77 of SEQ ID NO:
1.
[0271] In one embodiment an N-glycosylation site N-Xaa-S/T is
introduced at a position starting at G78 of SEQ ID NO: 1.In one
embodiment an N-glycosylation site N-Xaa-S/T is introduced at a
position starting at Q88 of SEQ ID NO: 1.In one embodiment an
N-glycosylation site N-Xaa-S/T is introduced at a position starting
at N93 of SEQ ID NO: 1.In one embodiment an N-glycosylation site
N-Xaa-S/T is introduced at a position starting at F275 of SEQ ID
NO: 1.
[0272] In one embodiment an N-glycosylation site N-Xaa-S/T is
introduced at a position starting at M306 of SEQ ID NO: 1.In one
embodiment an N-glycosylation site N-Xaa-S/T is introduced at a
position starting at T307 of SEQ ID NO: 1. In one embodiment an
N-glycosylation site N-Xaa-S/T is introduced at a position starting
at D309 of SEQ ID NO: 1.
[0273] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position K18 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of E, D, A, and F.
[0274] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position R36 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of E, D, K, A, F, and N.
[0275] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position S43 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of E, D, K, R, A, and F.
[0276] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position K62 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of D, A, and F.
[0277] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position Q64 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of E, D, K, R, A, F, and N.
[0278] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position L65 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of E, D, K, R, A, and F.
[0279] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position 169 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W,
and Y.
[0280] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position F71 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, G, H, I, K, L, M, P, R, S, T, V, and W.
[0281] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position L73 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W,
and Y.
[0282] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position P74 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,
and Y.
[0283] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position E77 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y.
[0284] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position G78 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W,
and Y.
[0285] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position R79 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W,
and Y.
[0286] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position K85 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, I, L, M, P, S, T, V, W, and Y.
[0287] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position Q88 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W,
and Y.
[0288] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position N93 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,
and Y.
[0289] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position F275 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, and
Y.
[0290] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position R277 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, and
Y.
[0291] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position M306 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, and
Y.
[0292] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position T307 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W,
and Y.
[0293] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position Q308 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W,
and Y.
[0294] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position D309 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, F, G, H, I, K, L, M, N, P, Q, R, V, W, and Y.
[0295] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, wherein the amino acid
corresponding to the amino acid at a position R379 of SEQ ID NO: 1
has been substituted with an amino acid selected from the list
consisting of A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, or
Y.
[0296] In a further embodiment, the Factor VII polypeptides of the
invention is a Factor VII polypeptide, which is selected from the
list consisting of FVII-(S43N), FVII-(K62E), FVII-(Q64E),
FVII-(I69F), FVII-(K62E/I69A), FVII-(Q64E/I69A),
FVII-(Q62E/Q64E/I69A), FVII-(K62E/I69F), FVII-(Q64E/I69F),
FVII-(K62E/Q64E/I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C),
FVII-(M306C), FVII-(R277C), FVII-(I69N/F71T), FVII-(F71N/L73T),
FVII-(R277N), and FVII-(D309N/L311T).
[0297] In one embodiment the polynucleotide construct of the
invention is a vector.
[0298] The term "a polynucleotide" denotes a single- or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. Polynucleotides include RNA
and DNA, and may be isolated from natural sources, synthesized in
vitro, or prepared from a combination of natural and synthetic
molecules. The length of a polynucleotide molecule is given herein
in terms of nucleotides (abbreviated "nt") or base pairs
(abbreviated "bp"). The term "nucleotides" is used for both single-
and double-stranded molecules where the context permits. When the
term is applied to double-stranded molecules it is used to denote
overall length and will be understood to be equivalent to the term
"base pairs". It will be recognized by those skilled in the art
that the two strands of a double-stranded polynucleotide may differ
slightly in length and that the ends thereof may be staggered as a
result of enzymatic cleavage; thus all nucleotides within a
double-stranded polynucleotide molecule may not be paired. Such
unpaired ends will in general not exceed 20 nt in length.
[0299] The term "vector", as used herein, means any nucleic acid
entity capable of the amplification in a host cell. Thus, the
vector may be an autonomously replicating vector, i.e. a vector,
which exists as an extrachromosomal entity, the replication of
which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a
host cell, is integrated into the host cell genome and replicated
together with the chromosome(s) into which it has been integrated.
The choice of vector will often depend on the host cell into which
it is to be introduced. Vectors include, but are not limited to
plasmid vectors, phage vectors, viruses or cosmid vectors. Vectors
usually contain a replication origin and at least one selectable
gene, i.e., a gene which encodes a product which is readily
detectable or the presence of which is essential for cell
growth.
[0300] In one embodiment, the eukaryotic host cell of the invention
is of mammalian origin.
[0301] In a further embodiment, the eukaryotic host cell of the
invention is selected from the group consisting of CHO cells, BHK
cells or HEK cells.
[0302] The term "a eukaryotic host cell", as used herein, represent
any cell, including hybrid cells, in which heterologous DNA can be
expressed. Typical host cells includes, but are not limited to
insect cells, yeast cells, mammalian cells, including human cells,
such as BHK, CHO, HEK, and COS cells. In practicing the present
invention, the host cells being cultivated are preferably mammalian
cells, more preferably an established mammalian cell line,
including, without limitation, CHO (e.g., ATCC CCL 61), COS-1
(e.g., ATCC CRL 1650), baby hamster kidney (BHK) and HEK293 (e.g.,
ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell
lines.
[0303] 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.
[0304] Other suitable cell lines include, without limitation, 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 (Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA 77:4216-4220, 1980). Also useful are 3T3 cells,
Namalwa cells, myelomas and fusions of myelomas with other cells.
In one embodiment the eucaryotic host cell is of mammalian origin.
In a further embodiment the eucaryotic host cell is selected from
the group consisting of CHO cells, BHK cells or HEK cells.
[0305] The term "treatment", as used herein, means the
administration of an effective amount of a therapeutically active
compound of the invention with the purpose of preventing any
symptoms or disease state to develop or with the purpose of curing
or easing such symptoms or disease states already developed. The
term "treatment" is thus meant to include prophylactic
treatment.
[0306] The term "enhancement of the normal haemostatic system"
means an enhancement of the ability to generate thrombin.
[0307] As used herein the term "bleeding disorders" reflects any
defect, congenital, acquired or induced, of cellular or molecular
origin that is manifested in bleedings. Examples are clotting
factor deficiencies (e.g. haemophilia A and B or deficiency of
coagulation Factors XI or VII), clotting factor inhibitors,
defective platelet function, thrombocytopenia or von Willebrand's
disease.
[0308] The term "bleeding episodes" is meant to include
uncontrolled and excessive bleeding which is a major problem both
in connection with surgery and other forms of tissue damage.
Uncontrolled and excessive bleeding may occur in subjects having a
normal coagulation system and subjects having coagulation or
bleeding disorders. Clotting factor deficiencies (haemophilia A and
B, deficiency of coagulation factors XI or VII) or clotting factor
inhibitors may be the cause of bleeding disorders. Excessive
bleedings also occur in subjects with a normally functioning blood
clotting cascade (no clotting factor deficiencies or -inhibitors
against any of the coagulation factors) and may be caused by a
defective platelet function, thrombocytopenia or von Willebrand's
disease. In such cases, the bleedings may be similar to those
bleedings caused by haemophilia because the haemostatic system, as
in haemophilia, lacks or has abnormal essential clotting
"compounds" (such as platelets or von Willebrand factor protein)
that causes major bleedings. In subjects who experience extensive
tissue damage in association with surgery or vast trauma, the
normal haemostatic mechanism may be overwhelmed by the demand of
immediate haemostasis and they may develop bleeding in spite of a
normal haemostatic mechanism. Achieving satisfactory haemostasis
also is a problem when bleedings occur in organs such as the brain,
inner ear region and eyes with limited possibility for surgical
haemostasis. The same problem may arise in the process of taking
biopsies from various organs (liver, lung, tumour tissue,
gastrointestinal tract) as well as in laparoscopic surgery. Common
for all these situations is the difficulty to provide haemostasis
by surgical techniques (sutures, clips, etc.) which also is the
case when bleeding is diffuse (haemorrhagic gastritis and profuse
uterine bleeding). Acute and profuse bleedings may also occur in
subjects on anticoagulant therapy in whom a defective haemostasis
has been induced by the therapy given. Such subjects may need
surgical interventions in case the anticoagulant effect has to be
counteracted rapidly. Radical retropubic prostatectomy is a
commonly performed procedure for subjects with localized prostate
cancer. The operation is frequently complicated by significant and
sometimes massive blood loss. The considerable blood loss during
prostatectomy is mainly related to the complicated anatomical
situation, with various densely vascularized sites that are not
easily accessible for surgical haemostasis, and which may result in
diffuse bleeding from a large area. Another situation that may
cause problems in the case of unsatisfactory haemostasis is when
subjects with a normal haemostatic mechanism are given
anticoagulant therapy to prevent thromboembolic disease. Such
therapy may include heparin, other forms of proteoglycans, warfarin
or other forms of vitamin K-antagonists as well as aspirin and
other platelet aggregation inhibitors.
[0309] In one embodiment of the invention, the bleeding is
associated with haemophilia A or B. In another embodiment, the
bleeding is associated with haemophilia with aquired inhibitors. In
another embodiment, the bleeding is associated with
thrombocytopenia. In another embodiment, the bleeding is associated
with von Willebrand's disease. In another embodiment, the bleeding
is associated with severe tissue damage. In another embodiment, the
bleeding is associated with severe trauma. In another embodiment,
the bleeding is associated with surgery. In another embodiment, the
bleeding is associated with laparoscopic surgery. In another
embodiment, the bleeding is associated with haemorrhagic gastritis.
In another embodiment, the bleeding is profuse uterine bleeding. In
another embodiment, the bleeding is occurring in organs with a
limited possibility for mechanical haemostasis. In another
embodiment, the bleeding is occurring in the brain, inner ear
region or eyes. In another embodiment, the bleeding is associated
with the process of taking biopsies. In another embodiment, the
bleeding is associated with anticoagulant therapy.
[0310] The term "subject" as used herein is intended to mean any
animal, in particular mammals, such as humans, and may, where
appropriate, be used interchangeably with the term "patient".
[0311] The terminology for amino acid substitutions used herein is
as follows. The first letter represent the amino acid naturally
present at a position of SEQ ID NO: 1. The following number
represent the position in SEQ ID NO: 1. The second letter represent
the different amino acid substituting for the natural amino acid.
An example is S43N, where a serine at position 43 of SEQ ID NO: 1
is replaced by an Asparagine. In another example, K62E/I69A, the
lysine in position 62 of SEQ ID NO: 1 is replaced by a glutamic
acid and the isoleucine in position 69 of SEQ ID NO: 1 is replaced
by an alanine in the same Factor VII polypeptide. I69A-FVII thus
means human wild type FVII, wherein a isoleucine in position 69 of
SEQ ID NO: 1 has been replaced by an alanine.
[0312] In one aspect the present invention relates to a composition
comprising wild type human FVIIa and a Factor VII polypeptide with
a Tissue Factor binding affinity lower than recombinant wild type
human Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa.
[0313] In a further aspect the present invention relates to a
method for the treatment of bleeding episodes in a subject or for
the enhancement of the normal haemostatic system, the method
comprising administering to a subject in need thereof a
therapeutically or prophylactically effective amount of:
[0314] a) composition comprising wild type human FVIIa and a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa; or
[0315] b) a first composition comprising wild type human FVIIa and
a second composition comprising a Factor VII polypeptide with a
Tissue Factor binding affinity lower than recombinant wild type
human Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa.
[0316] In a further aspect the present invention relates to a
process for preparing a composition comprising wild type human
FVIIa and a Factor VII polypeptide with a Tissue Factor binding
affinity lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa, wherein the process
comprises the step of: mixing wild type human FVIIa with a Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa in an aqueous medium.
[0317] In a further aspect the present invention relates to a use
of a composition comprising wild type human FVIIa and a Factor VII
polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa, for the preparation of a medicament for the
treatment of bleeding episodes in a subject or for the enhancement
of the normal haemostatic system.
[0318] In a further aspect the present invention relates to the use
of a Factor VII polypeptide with a Tissue Factor binding affinity
lower than recombinant wild type human Factor VIIa and
substantially the same activity or increased activity compared to
recombinant wild type human Factor VIIa; in the therapeutic
treatment of bleedings, where the exposure of TF is high, such as
indications where there is a risk of plaque rupture, plaque
disruption after percutaneous transluminal coronary angioplasty
(PTCA) procedures, sepsis, or other bleeding complications, where
inflammatory conditions where increased quantities of tissue factor
is seen. In one embodiment the bleedings, where the exposure of TF
is high is a coagulation disorder, such as disseminated
intravascular coagulation (DIC).
[0319] The present invention also encompasses methods and
compositions that provide combination therapies in which the Factor
VII polypeptide with a Tissue Factor binding affinity lower than
recombinant wild type human Factor VIIa and substantially the same
activity or increased activity compared to recombinant wild type
human Factor VIIa is administered with another procoagulant
compound, antifibrinolytic compound or regulator of anticoagulant
compounds. Suitable compounds include, without limitation, FVIII
(such as Refacto.RTM. (Genetics Institute), Kogenate FS.RTM.
(Bayer), Monoclate-P.RTM. (Aventis Behring)), Factor XIII (see,
e.g., WO 01/85198); inhibitors of tissue factor pathway inhibitor
(TFPI inhibitors) (see, e.g., WO 01/85199); Factor IX (see, e.g.,
WO 02/062376); thrombin activatable fibrinolysis inhibitor (TAFI)
(see, e.g., PCT/DK02/00734; PAI-1 (see, e.g., PCT/DK02/00735;
Factor V (see, e.g., PCT/DK02/00736); protein C inhibitors (see,
e.g., PCT/DK02/00737); thrombomodulin (see, e.g., PCT/DK02/00738);
protein S inhibitors (see, e.g., PCT/DK02/00739); tissue
plasminogen activator inhibitors (see, e.g., PCT/DK02/00740);
alpha2-antiplasmin (see, e.g., PCT/DK02/00741); aprotinin (see,
e.g., PCT/DK02/00742); tranexamic acid (see, e.g., PCT/DK02/00751);
epsilon-aminocaproic acid (see, e.g., PCT/DK02/00752); prothrombin,
thrombin, Factor VII, Factor X, Factor XI and fibrinogen,
artificial oxygen carriers (such as POLYHEME.RTM., Northfield lab),
colloids (such as Hextend, BioTime, Inc), Desmopressin (such as
DDAVP.RTM. Tablets (desmopressin acetate) Adventis
Pharmaceuticals), activated prothrombin complex concentrates (i.e.,
Bebulin, Proplex-T, Profiline, Autoplex, FEIBA), Clopidogrel,
Ticlopidine, Glycoprotein IIB/IIIA antagonists (Abciximab), LMWH,
Warfarin. Streptokinase, Tissue plasminogen activator (tPA)/tPA
mutants.
[0320] In one embodiment the Factor VII polypeptide with a Tissue
Factor binding affinity lower than recombinant wild type human
Factor VIIa and substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa is as
disclosed in any of international patent applications WO 99/20767,
WO 00/66753, WO 01/58935, WO 03/93465 and WO 04/029091, each of
which is hereby incorporated by reference in its entirety.
[0321] In the present specification, amino acids are represented
using abbreviations, as indicated in table 1, approved by IUPAC-IUB
Commission on Biochemical Nomenclature (CBN). Amino acid and the
like having isomers represented by name or the following
abbreviations are in natural L-form unless otherwise indicated.
Further, the left and right ends of an amino acid sequence of a
peptide are, respectively, the N- and C-termini unless otherwise
specified. TABLE-US-00001 TABLE 1 Abbreviations for amino acids:
Amino acid Tree-letter code One-letter code Glycine Gly G Proline
Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I
Methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y
Tryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R
Glutamine Gln Q Asparagine Asn N Glutamic Acid Glu E Aspartic Acid
Asp D Serine Ser S Threonine Thr T
[0322] The invention also relates to a method of preparing human
Factor VII polypeptides as mentioned above. The human Factor VII
polypeptides are preferably produced by recombinant DNA techniques.
To this end, DNA sequences encoding human Factor VII may be
isolated by preparing a genomic or cDNA library and screening for
DNA sequences coding for all or part of the protein by
hybridization using synthetic oligonucleotide probes in accordance
with standard techniques (cf. Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989). For the present purpose, the DNA sequence
encoding the protein is preferably of human origin, i.e. derived
from a human genomic DNA or cDNA library.
[0323] The DNA sequences encoding the human Factor VII polypeptides
may also be prepared synthetically by established standard methods,
e.g. the phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22 (1981), 1859-1869, or the method described
by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the
phosphoamidite method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated and cloned
in suitable vectors.
[0324] The DNA sequences may also be prepared by polymerase chain
reaction using specific primers, for instance as described in U.S.
Pat. No. 4,683,202, Saiki et al., Science 239 (1988), 487-491, or
Sambrook et al., supra.
[0325] The DNA sequences encoding the human Factor VII polypeptides
are usually inserted into a recombinant vector which may be any
vector, which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e. a vector, which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0326] The vector is preferably an expression vector in which the
DNA sequence encoding the human Factor VII polypeptides is operably
linked to additional segments required for transcription of the
DNA. In general, the expression vector is derived from plasmid or
viral DNA, or may contain elements of both. The term, "operably
linked" indicates that the segments are arranged so that they
function in concert for their intended purposes, e.g. transcription
initiates in a promoter and proceeds through the DNA sequence
coding for the polypeptide.
[0327] The promoter may be any DNA sequence, which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
[0328] Examples of suitable promoters for directing the
transcription of the DNA encoding the human Factor VII polypeptide
in mammalian cells are the SV40 promoter (Subramani et al., Mol.
Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene)
promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV
promoter (Boshart et al., Cell 41:521-530, 1985) or the adenovirus
2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol,
2:1304-1319, 1982).
[0329] An example of a suitable promoter for use in insect cells is
the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al.,
FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al.,
J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica
polyhedrosis virus basic protein promoter (EP 397 485), the
baculovirus immediate early gene 1 promoter (U.S. Pat. No.
5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K
delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.
5,162,222).
[0330] Examples of suitable promoters for use in yeast host cells
include promoters from yeast glycolytic genes (Hitzeman et al., J.
Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol.
Appl Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young
et al., in Genetic Engineering of Microorganisms for Chemicals
(Hollaender et al, eds.), Plenum Press, New York, 1982), or the
TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature
304 (1983), 652-654) promoters.
[0331] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter (McKnight et al.,
The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of
other useful promoters are those derived from the gene encoding A.
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.
niger neutral .alpha.-amylase, A. niger acid stable
.alpha.-amylase, A. niger or A. awamori glucoamylase (gluA),
Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae
triose phosphate isomerase or A. nidulans acetamidase. Preferred
are the TAKA-amylase and gluA promoters. Suitable promoters are
mentioned in, e.g. EP 238 023 and EP 383 779.
[0332] The DNA sequences encoding the human Factor VII polypeptides
may also, if necessary, be operably connected to a suitable
terminator, such as the human growth hormone terminator (Palmiter
et al., Science 222, 1983, pp. 809-814) or the TPI1 (Alber and
Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3
(McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) terminators.
The vector may also contain a set of RNA splice sites located
downstream from the promoter and upstream from the insertion site
for the Factor VII sequence itself. Preferred RNA splice sites may
be obtained from adenovirus and/or immunoglobulin genes. Also
contained in the expression vectors is a polyadenylation signal
located downstream of the insertion site. Particularly preferred
polyadenylation signals include the early or late polyadenylation
signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation
signal from the adenovirus 5 E1b region, the human growth hormone
gene terminator (DeNoto et al. Nuc. Acids Res. 9:3719-3730, 1981)
or the polyadenylation signal from the human Factor VII gene or the
bovine Factor VII gene. The expression vectors may also include a
noncoding viral leader sequence, such as the adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites; and enhancer sequences, such as the SV40 enhancer.
[0333] The recombinant vector may further comprise a DNA sequence
enabling the vector to replicate in the host cell in question. An
example of such a sequence (when the host cell is a mammalian cell)
is the SV40 origin of replication.
[0334] When the host cell is a yeast cell, suitable sequences
enabling the vector to replicate are the yeast plasmid 2.mu.
replication genes REP 1-3 and origin of replication.
[0335] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
such as the gene coding for dihydrofolate reductase (DHFR) or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 1985, pp. 125-130), or one which confers resistance to a
drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol,
neomycin, hygromycin or methotrexate. For filamentous fungi,
selectable markers include amdS, pyrG, argB, niaD or sC.
[0336] To direct the human Factor VII polypeptides of the present
invention into the secretory pathway of the host cells, a secretory
signal sequence (also known as a leader sequence, prepro sequence
or pre sequence) may be provided in the recombinant vector. The
secretory signal sequence is joined to the DNA sequences encoding
the human Factor VII polypeptides in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA
sequence encoding the peptide. The secretory signal sequence may be
that, normally associated with the protein or may be from a gene
encoding another secreted protein.
[0337] For secretion from yeast cells, the secretory signal
sequence may encode any signal peptide, which ensures efficient
direction of the expressed human Factor VII polypeptides into the
secretory pathway of the cell. The signal peptide may be naturally
occurring signal peptide, or a functional part thereof, or it may
be a synthetic peptide. Suitable signal peptides have been found to
be the .alpha.-factor signal peptide (cf. U.S. Pat. No. 4,870,008),
the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et
al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase
signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp.
887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the
yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani
et al., Yeast 6, 1990, pp. 127-137).
[0338] For efficient secretion in yeast, a sequence encoding a
leader peptide may also be inserted downstream of the signal
sequence and upstream of the DNA sequence encoding the human Factor
VII polypeptides. The function of the leader peptide is to allow
the expressed peptide to be directed from the endoplasmic reticulum
to the Golgi apparatus and further to a secretory vesicle for
secretion into the culture medium (i.e. exportation of the human
Factor VII polypeptides across the cell wall or at least through
the cellular membrane into the periplasmic space of the yeast
cell). The leader peptide may be the yeast alpha-factor leader (the
use of which is described in e.g. U.S. Pat. No. 4,546,082, U.S.
Pat. No. 4,870,008, EP 16 201, EP 123 294, EP 123 544 and EP 163
529). Alternatively, the leader peptide may be a synthetic leader
peptide, which is to say a leader peptide not found in nature.
Synthetic leader peptides may, for instance, be constructed as
described in WO 89/02463 or WO 92/11378.
[0339] For use in filamentous fungi, the signal peptide may
conveniently be derived from a gene encoding an Aspergillus sp.
amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase
or protease or a Humicola lanuginosa lipase. The signal peptide is
preferably derived from a gene encoding A. oryzae TAKA amylase, A.
niger neutral .alpha.-amylase, A. niger acid-stable amylase, or A.
niger glucoamylase. Suitable signal peptides are disclosed in, e.g.
EP 238 023 and EP 215 594.
[0340] For use in insect cells, the signal peptide may conveniently
be derived from an insect gene (cf. WO 90/05783), such as the
lepidopteran Manduca sexta adipokinetic hormone precursor signal
peptide (cf. U.S. Pat. No. 5,023,328).
[0341] The procedures used to ligate the DNA sequences coding for
the human Factor VII polypeptides, the promoter and optionally the
terminator and/or secretory signal sequence, respectively, and to
insert them into suitable vectors containing the information
necessary for replication, are well known to persons skilled in the
art (cf., for instance, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
[0342] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in e.g. Kaufman and
Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.
Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978),
725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603,
Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al.,
EMBO J. 1 (1982), 841-845.
[0343] Selectable markers may be introduced into the cell on a
separate plasmid at the same time as the gene of interest, or they
may be introduced on the same plasmid. If on the same plasmid, the
selectable marker and the gene of interest may be under the control
of different promoters or the same promoter, the latter arrangement
producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Pat. No.
4,713,339). It may also be advantageous to add additional DNA,
known as "carrier DNA," to the mixture that is introduced into the
cells.
[0344] After the cells have taken up the DNA, they are grown in an
appropriate growth medium, typically 1-2 days, to begin expressing
the gene of interest. As used herein the term "appropriate growth
medium" means a medium containing nutrients and other components
required for the growth of cells and the expression of the human
Factor VII polypeptides of interest. Media generally include a
carbon source, a nitrogen source, essential amino acids, essential
sugars, vitamins, salts, phospholipids, protein and growth factors.
For production of gamma-carboxylated proteins, the medium will
contain vitamin K, preferably at a concentration of about 0.1
.mu.g/ml to about 5 .mu.g/ml. Drug selection is then applied to
select for the growth of cells that are expressing the selectable
marker in a stable fashion. For cells that have been transfected
with an amplifiable selectable marker the drug concentration may be
increased to select for an increased copy number of the cloned
sequences, thereby increasing expression levels. Clones of stably
transfected cells are then screened for expression of the human
Factor VII polypeptide of interest.
[0345] The host cell into which the DNA sequences encoding the
human Factor VII polypeptides is introduced may be any cell, which
is capable of producing the posttranslational modified human Factor
VII polypeptides and includes yeast, fungi and higher eucaryotic
cells.
[0346] Examples of mammalian cell lines for use in the present
invention are the COS-1 (ATCC CRL 1650), baby hamster kidney (BHK)
and 293 (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, incorporated herein by reference), hereinafter
referred to as BHK 570 cells. The BHK 570 cell line has been
deposited with 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 within the present invention, including Rat Hep I
(Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL
1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469
(ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
[0347] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous polypeptides there from are described, e.g.
in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos.
4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are
hereby incorporated by reference. Transformed cells are selected by
a phenotype determined by a selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient, e.g. leucine. A preferred vector for use in yeast is the
POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences
encoding the human Factor VII polypeptides may be preceded by a
signal sequence and optionally a leader sequence, e.g. as described
above. Further examples of suitable yeast cells are strains of
Kluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, or
Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol.
132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).
[0348] Examples of other fungal cells are cells of filamentous
fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or
Trichoderma spp., in particular strains of A. oryzae, A. nidulans
or A. niger. The use of Aspergillus spp. for the expression of
proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438
The transformation of F. oxysporum may, for instance, be carried
out as described by Malardier et al., 1989, Gene 78: 147-156. The
transformation of Trichoderma spp. may be performed for instance as
described in EP 244 234.
[0349] When a filamentous fungus is used as the host cell, it may
be transformed with the DNA construct of the invention,
conveniently by integrating the DNA construct in the host
chromosome to obtain a recombinant host cell. This integration is
generally considered to be an advantage as the DNA sequence is more
likely to be stably maintained in the cell. Integration of the DNA
constructs into the host chromosome may be performed according to
conventional methods, e.g. by homologous or heterologous
recombination.
[0350] Transformation of insect cells and production of
heterologous polypeptides therein may be performed as described in
U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos.
5,155,037; 5,162,222; EP 397,485) all of which are incorporated
herein by reference. The insect cell line used as the host may
suitably be a Lepidoptera cell line, such as Spodoptera frugiperda
cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214).
Culture conditions may suitably be as described in, for instance,
WO 89/01029 or WO 89/01028, or any of the aforementioned
references.
[0351] The transformed or transfected host cell described above is
then cultured in a suitable nutrient medium under conditions
permitting expression of the human Factor VII polypeptide after
which all or part of the resulting peptide may be recovered from
the culture. The medium used to culture the cells may be any
conventional medium suitable for growing the host cells, such as
minimal or complex media containing appropriate supplements.
Suitable media are available from commercial suppliers or may be
prepared according to published recipes (e.g. in catalogues of the
American Type Culture Collection). The human Factor VII polypeptide
produced by the cells may then be recovered from the culture medium
by conventional procedures including separating the host cells from
the medium by centrifugation or filtration, precipitating the
proteinaqueous components of the supernatant or filtrate by means
of a salt, e.g. ammonium sulphate, purification by a variety of
chromatographic procedures, e.g. ion exchange chromatography,
gelfiltration chromatography, affinity chromatography, or the like,
dependent on the type of polypeptide in question.
[0352] For the preparation of recombinant human Factor VII
polypeptides, a cloned wild-type Factor VII DNA sequence is used.
This sequence may be modified to encode a desired Factor VII
variant. The complete nucleotide and amino acid sequences for human
Factor VII are known. See U.S. Pat. No. 4,784,950, which is
incorporated herein by reference, where the cloning and expression
of recombinant human Factor VII is described. The bovine Factor VII
sequence is described in Takeya et al., J. Biol. Chem,
263:14868-14872 (1988), which is incorporated by reference
herein.
[0353] The amino acid sequence alterations may be accomplished by a
variety of techniques. Modification of the DNA sequence may be by
site-specific mutagenesis. Techniques for site-specific mutagenesis
are well known in the art and are described by, for example, Zoller
and Smith (DNA 3:479-488, 1984). Thus, using the nucleotide and
amino acid sequences of Factor VII, one may introduce the
alterations of choice.
[0354] DNA sequences for use within the present invention will
typically encode a pre-pro peptide at the amino-terminus of the
Factor VII protein to obtain proper post-translational processing
(e.g. gamma-carboxylation of glutamic acid residues) and secretion
from the host cell. The pre-pro peptide may be that of Factor VII
or another vitamin K-dependent plasma protein, such as factor IX,
factor X, prothrombin, protein C or protein S. As will be
appreciated by those skilled in the art, additional modifications
can be made in the amino acid sequence of Factor VII where those
modifications do not significantly impair the ability of the
protein to act as a coagulation factor. For example, Factor VII in
the catalytic triad can also be modified in the activation cleavage
site to inhibit the conversion of zymogen Factor VII into its
activated two-chain form, as generally described in U.S. Pat. No.
5,288,629, incorporated herein by reference.
[0355] Within the present invention, transgenic animal technology
may be employed to produce the human Factor VII polypeptide. It is
preferred to produce the proteins within the mammary glands of a
host female mammal. Expression in the mammary gland and subsequent
secretion of the protein of interest into the milk overcomes many
difficulties encountered in isolating proteins from other sources.
Milk is readily collected, available in large quantities, and well
characterized biochemically. Furthermore, the major milk proteins
are present in milk at high concentrations (typically from about 1
to 15 g/l). From a commercial point of view, it is clearly
preferable to use as the host a species that has a large milk
yield. While smaller animals such as mice and rats can be used (and
are preferred at the proof of principle stage), within the present
invention it is preferred to use livestock mammals including, but
not limited to, pigs, goats, sheep and cattle. Sheep are
particularly preferred due to such factors as the previous history
of transgenesis in this species, milk yield, cost and the ready
availability of equipment for collecting sheep milk. See WIPO
Publication WO 88/00239 for a comparison of factors influencing the
choice of host species. It is generally desirable to select a breed
of host animal that has been bred for dairy use, such as East
Friesland sheep, or to introduce dairy stock by breeding of the
transgenic line at a later date. In any event, animals of known,
good health status should be used.
[0356] To obtain expression in the mammary gland, a transcription
promoter from a milk protein gene is used. Milk protein genes
include those genes encoding caseins (see U.S. Pat. No. 5,304,489,
incorporated herein by reference), beta-lactoglobulin,
alpha-lactalbumin, and whey acidic protein. The beta-lactoglobulin
(BLG) promoter is preferred. In the case of the ovine
beta-lactoglobulin gene, a region of at least the proximal 406 bp
of 5' flanking sequence of the gene will generally be used,
although larger portions of the 5' flanking sequence, up to about 5
kbp, are preferred, such as about 4.25 kbp DNA segment encompassing
the 5' flanking promoter and non-coding portion of the
beta-lactoglobulin gene. See Whitelaw et al., Biochem J. 286: 31-39
(1992). Similar fragments of promoter DNA from other species are
also suitable.
[0357] Other regions of the beta-lactoglobulin gene may also be
incorporated in constructs, as may genomic regions of the gene to
be expressed. It is generally accepted in the art that constructs
lacking introns, for example, express poorly in comparison with
those that contain such DNA sequences (see Brinster et al., Proc.
Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc.
Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al.,
Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318, each
of which is incorporated herein by reference). In this regard, it
is generally preferred, where possible, to use genomic sequences
containing all or some of the native introns of a gene encoding the
protein or polypeptide of interest, thus the further inclusion of
at least some introns from, e.g, the beta-lactoglobulin gene, is
preferred. One such region is a DNA segment which provides for
intron splicing and RNA polyadenylation from the 3' non-coding
region of the ovine beta-lactoglobulin gene. When substituted for
the natural 3' non-coding sequences of a gene, this ovine
beta-lactoglobulin segment can both enhance and stabilize
expression levels of the protein or polypeptide of interest. Within
other embodiments, the region surrounding the initiation ATG of the
sequence encoding the human Factor VII polypeptide is replaced with
corresponding sequences from a milk specific protein gene. Such
replacement provides a putative tissue-specific initiation
environment to enhance expression. It is convenient to replace the
entire pre-pro sequence of the human Factor VII polypeptide and 5'
non-coding sequences with those of, for example, the BLG gene,
although smaller regions may be replaced.
[0358] For expression of a human Factor VII polypeptide in
transgenic animals, a DNA segment encoding the human Factor VII
polypeptide is operably linked to additional DNA segments required
for its expression to produce expression units. Such additional
segments include the above-mentioned promoter, as well as sequences
which provide for termination of transcription and polyadenylation
of mRNA. The expression units will further include a DNA segment
encoding a secretory signal sequence operably linked to the segment
encoding the human Factor VII polypeptide. The secretory signal
sequence may be a native secretory signal sequence of the human
Factor VII polypeptide or may be that of another protein, such as a
milk protein. See, for example, von Heinje, Nuc. Acids Res. 14:
4683-4690 (1986); and Meade et al., U.S. Pat. No. 4,873,316, which
are incorporated herein by reference.
[0359] Construction of expression units for use in transgenic
animals is conveniently carried out by inserting a sequence
encoding the human Factor VII polypeptide into a plasmid or phage
vector containing the additional DNA segments, although the
expression unit may be constructed by essentially any sequence of
ligations. It is particularly convenient to provide a vector
containing a DNA segment encoding a milk protein and to replace the
coding sequence for the milk protein with that of the human Factor
VII polypeptide, thereby creating a gene fusion that includes the
expression control sequences of the milk protein gene. In any
event, cloning of the expression units in plasmids or other vectors
facilitates the amplification of the human Factor VII polypeptide.
Amplification is conveniently carried out in bacterial (e.g. E.
coli) host cells, thus the vectors will typically include an origin
of replication and a selectable marker functional in bacterial host
cells.
[0360] The expression unit is then introduced into fertilized eggs
(including early-stage embryos) of the chosen host species.
Introduction of heterologous DNA can be accomplished by one of
several routes, including microinjection (e.g. U.S. Pat. No.
4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474
(1988)) or site-directed integration using embryonic stem (ES)
cells (reviewed by Bradley et al., Bio/Technology 10: 534-539
(1992)). The eggs are then implanted into the oviducts or uteri of
pseudopregnant females and allowed to develop. Offspring carrying
the introduced DNA in their germ line can pass the DNA on to their
progeny in the normal, Mendelian fashion, allowing the development
of transgenic herds.
[0361] General procedures for producing transgenic animals are
known in the art. See, for example, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory,
1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al.,
Biol. Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8:
140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991);
Krimpenfort et al., Bio/Technology 9: 844-847 (1991); Wall et al.,
J. Cell. Biochem. 49: 113-120 (1992); U.S. Pat. Nos. 4,873,191 and
4,873,316; WIPO publications WO 88/00239, WO 90/05188, WO 92/11757;
and GB 87/00458, which are incorporated herein by reference.
Techniques for introducing foreign DNA sequences into mammals and
their germ cells were originally developed in the mouse. See, e.g.,
Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980);
Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and
Brinster, Cell 41: 343-345 (1985); and Brinster et al., Proc. Natl.
Acad. Sci. USA 82: 4438-4442 (1985). These techniques were
subsequently adapted for use with larger animals, including
livestock species (see e.g., WIPO publications WO 88/00239, WO
90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6:
179-183 (1988). To summarize, in the most efficient route used to
date in the generation of transgenic mice or livestock, several
hundred linear molecules of the DNA of interest are injected into
one of the pro-nuclei of a fertilized egg according to established
techniques. Injection of DNA into the cytoplasm of a zygote can
also be employed. Production in transgenic plants may also be
employed. Expression may be generalized or directed to a particular
organ, such as a tuber. See, Hiatt, Nature 344:469-479 (1990);
Edelbaum et al., J. Interferon Res. 12:449-453 (1992); Sijmons et
al., Bio/Technology 8:217-221 (1990); and European Patent Office
Publication EP 255,378.
[0362] Factor VII produced according to the present invention may
be purified by affinity chromatography on an anti-Factor VII
antibody column. It is preferred that the immunoadsorption column
comprise a high-specificity monoclonal antibody. The use of
calcium-dependent monoclonal antibodies, as described by
Wakabayashi et al., J. Biol. Chem, 261:11097-11108, (1986) and Thim
et al., Biochem. 27: 7785-7793, (1988), incorporated by reference
herein, is particularly preferred. Additional purification may be
achieved by conventional chemical purification means, such as high
performance liquid chromatography. Other methods of purification,
including barium citrate precipitation, are known in the art, and
may be applied to the purification of the Factor VII described
herein (see, generally, Scopes, R., Protein Purification,
Springer-Verlag, N.Y., 1982). Substantially pure Factor VII of at
least about 90 to 95% homogeneity is preferred, and 98 to 99% or
more homogeneity most preferred, for pharmaceutical uses. Once
purified, partially or to homogeneity as desired, the Factor VII
may then be used therapeutically.
[0363] Conversion of single-chain Factor VII to active two-chain
Factor VIIa may be achieved using factor XIIa as described by
Hedner and Kisiel (1983, J. Clin. Invest. 71: 1836-1841), or with
other proteases having trypsin-like specificity (Kisiel and
Fujikawa, Behring Inst. Mitt. 73: 29-42, 1983). Alternatively
Factor VII may be autoactivated by passing it through an
ion-exchange chromatography column, such as mono Q.RTM.. (Pharmacia
Fire Chemicals) or the like (Bjoern et al., 1986, Research
Disclosures 269:564-565). The Factor VII molecules of the present
invention and pharmaceutical compositions thereof are particularly
useful for administration to humans to treat a variety of
conditions involving intravascular coagulation.
[0364] The invention also provides suitable assays for selecting
preferred Factor VIIa polypeptides and Factor VIIa derivatives
according to the invention. These assays can be performed as a
simple preliminary in vitro test.
[0365] Thus, Example 3 herein discloses a simple test (entitled "In
Vitro Hydrolysis Assay") for the activity of Factor VIIa
polypeptides of the invention. Based thereon, Factor VIIa
polypeptides which are of particular interest are such polypeptides
where the ratio between the activity of the variant and the
activity of native human Factor VII shown in FIG. 1 is about 1.0 or
higher, when tested in the "In Vitro Hydrolysis Assay" defined
herein.
[0366] The activity of the polypeptides can also be measured using
a physiological substrate such as factor X ("In Vitro Proteolysis
Assay") (see Example 4), suitably at a concentration of 100-1000
nM, where the factor Xa generated is measured after the addition of
a suitable chromogenic substrate (e.g. S-2765). In addition, the
activity assay may be run at physiological temperature.
[0367] The ability of the procoagulant Factor VIIa polypeptides to
generate thrombin can also be measured in an assay comprising all
relevant coagulation factors and inhibitors at physiological
concentrations (minus factor VIII when mimicking haemophilia A
conditions) 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).
[0368] The procoagulant Factor VII polypeptides according to the
present invention may be used to control bleeding disorders which
have several causes such as clotting factor deficiencies (e.g.
haemophilia A and B or deficiency of coagulation factors XI or VII)
or clotting factor inhibitors, or they may be used to control
excessive bleeding occurring in subjects with a normally
functioning blood clotting cascade (no clotting factor deficiencies
or inhibitors against any of the coagulation factors). The
bleedings may be caused by a defective platelet function,
thrombocytopenia or von Willebrand's disease. They may also be seen
in subjects in whom an increased fibrinolytic activity has been
induced by various stimuli.
[0369] In subjects who experience extensive tissue damage in
association with surgery or vast trauma, the haemostatic mechanism
may be overwhelmed by the demand of immediate haemostasis and they
may develop bleedings in spite of a normal haemostatic mechanism.
Achieving satisfactory haemostasis is also a problem when bleedings
occur in organs such as the brain, inner ear region and eyes and
may also be a problem in cases of diffuse bleedings (haemorrhagic
gastritis and profuse uterine bleeding) when it is difficult to
identify the source. The same problem may arise in the process of
taking biopsies from various organs (liver, lung, tumour tissue,
gastrointestinal tract) as well as in laparoscopic surgery. These
situations share the difficulty of providing haemostasis by
surgical techniques (sutures, clips, etc.). Acute and profuse
bleedings may also occur in subjects on anticoagulant therapy in
whom a defective haemostasis has been induced by the therapy given.
Such subjects may need surgical interventions in case the
anticoagulant effect has to be counteracted rapidly. Another
situation that may cause problems in the case of unsatisfactory
haemostasis is when subjects with a normal haemostatic mechanism
are given anticoagulant therapy to prevent thromboembolic disease.
Such therapy may include heparin, other forms of proteoglycans,
warfarin or other forms of vitamin K-antagonists as well as aspirin
and other platelet aggregation inhibitors.
[0370] A systemic activation of the coagulation cascade may lead to
disseminated intravascular coagulation (DIC). However, such
complications have not been seen in subjects treated with high
doses of recombinant Factor VIIa because of a localised haemostatic
process of the kind induced by the complex formation between Factor
VIIa and TF exposed at the site of vessel wall injury. The
procoagulant Factor VII polypeptides according to the invention may
thus also be used in their activated form to control such excessive
bleedings associated with a normal haemostatic mechanism.
[0371] For treatment in connection with deliberate interventions,
the procoagulant Factor VII polypeptides of the invention will
typically be administered within about 24 hours prior to performing
the intervention, and for as much as 7 days or more thereafter.
Administration as a coagulant can be by a variety of routes as
described herein.
[0372] The dose of the Factor VII polypeptides ranges from about
0.05 mg to 500 mg/day, preferably from about 1 mg to 200 mg/day,
and more preferably from about 10 mg to about 175 mg/day for a 70
kg subject as loading and maintenance doses, depending on the
severity of the condition.
[0373] The pharmaceutical compositions 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, or it may be administered by continuous or
pulsatile infusion. The compositions for parenteral administration
comprise the Factor VII polypeptide of the invention in combination
with, preferably dissolved in, a pharmaceutically acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, such as water, buffered water, 0.4% saline,
0.3% glycine and the like. The Factor VII polypeptides 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. No. 4,837,028, U.S.
Pat. No. 4,501,728, and U.S. Pat. No. 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. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, etc.
[0374] The concentration of Factor VII polypeptide 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.
[0375] 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 Factor VII polypeptide. 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).
[0376] The compositions containing the Factor VII polypeptides 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". As will be understood by the person skilled in
the art amounts effective for this 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 Factor VII
polypeptide per day for a 70 kg subject, with dosages of from about
1.0 mg to about 200 mg of the Factor VII polypeptide per day being
more commonly used.
[0377] It must be kept in mind that the materials of the present
invention may generally be employed in serious disease or injury
states, that is, life threatening or potentially life threatening
situations. In such cases, in view of the minimisation of
extraneous substances and general lack of immunogenicity of human
Factor VII polypeptides in humans, it is possible and may be felt
desirable by the treating physician to administer a substantial
excess of these variant Factor VII compositions.
[0378] In prophylactic applications, compositions containing the
Factor VII polypeptide of the invention are administered to a
subject susceptible to or otherwise at risk of a disease state or
injury to enhance the subject's own coagulative capability. Such an
amount is defined to be a "prophylactically effective dose." In
prophylactic applications, the precise amounts once again depend on
the subject's state of health and weight, but the dose generally
ranges from about 0.05 mg to about 500 mg per day for a 70-kilogram
subject, more commonly from about 1.0 mg to about 200 mg per day
for a 70-kilogram subject.
[0379] Single or multiple administrations of the compositions can
be carried out with dose levels and patterns being selected by the
treating physician. For ambulatory subjects requiring daily
maintenance levels, the Factor VII polypeptides may be administered
by continuous infusion using e.g. a portable pump system.
[0380] Local delivery of the Factor VII polypeptide of the present
invention, such as, for example, topical application may be carried
out, for example, by means of a spray, perfusion, double balloon
catheters, stent, incorporated into vascular grafts or stents,
hydrogels used to coat balloon catheters, or other well established
methods. In any event, the pharmaceutical compositions should
provide a quantity of Factor VII polypeptide sufficient to
effectively treat the subject.
[0381] Inactivated Factor VII polypeptides of the present invention
are able to bind to cell-surface tissue factor. For example,
DEGR-Factor VIIa binds cell-surface tissue factor with an
equivalent or higher affinity than wild-type Factor VIIa.
DEGR-Factor VIIa, however, has no enzymatic activity, yet it binds
to tissue factor and acts as a competitive antagonist for wild-type
Factor VIIa, thereby inhibiting the subsequent steps in the
extrinsic pathway of coagulation leading to the generation of
thrombin.
[0382] Inactivated Factor VII polypeptides are particularly useful
for administration to humans to treat a variety of conditions
involving intravascular coagulation. For example, although deep
vein thrombosis and pulmonary embolism can be treated with
conventional anticoagulants, the inactivated Factor VII
polypeptides described herein may be used to prevent the occurrence
of thromboembolic complications in identified high risk patients,
such as those undergoing surgery or those with congestive heart
failure. In addition, inactivated Factor VII polypeptides may act
as an antagonist for tissue factor-mediated induction of
coagulation, thus blocking the production of thrombin and the
subsequent deposition of fibrin. As such, inactivated Factor VII
polypeptides may be useful for inhibiting tissue factor activity
resulting in, for example, the inhibition of blood coagulation,
thrombosis or platelet deposition.
[0383] The inactivated Factor VII polypeptides may be particularly
useful in the treatment of intimal hyperplasia, restenosis due to
acute vascular injury, deep venous thrombosis, arterial thrombosis,
post surgical thrombosis, coronary artery bypass graft (CABG),
percutaneous transdermal coronary angioplastry (PTCA), stroke,
cancer, tumour metastasis, angiogenesis, ischemia/reperfusion,
rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis
following angioplastry, acute and chronic indications such as
inflammation, septic chock, septicemia, hypotension, adult
respiratory distress syndrome (ARDS), disseminated intravascular
coagulopathy (DIC), pulmonary embolism, platelet deposition,
myocardial infarction, or the prophylactic treatment of mammals
with atherosclerotic vessels at risk for thrombosis. Acute vascular
injuries are those which occur rapidly (i.e. over days to months),
in contrast to chronic vascular injuries (e.g. atherosclerosis)
which develop over a lifetime. Acute vascular injuries often result
from surgical procedures such as vascular reconstruction, wherein
the techniques of angioplasty, endarterectomy, atherectomy,
vascular graft emplacement or the like are employed. Hyperplasia
may also occur as a delayed response in response to, e.g., graft
emplacement or organ transplantation. Since inactivated Factor VII
polypeptides is more selective than heparin, generally binding only
tissue factor which has been exposed at sites of injury, and
because inactivated Factor VII polypeptides does not destroy other
coagulation proteins, it will be more effective and less likely to
cause bleeding complications than heparin when used
prophylactically for the prevention of deep vein thrombosis.
[0384] Inactivated Factor VII polypeptides which maintain tissue
factor binding inhibit platelet accumulation at the site of
vascular injury by blocking the production of thrombin and the
subsequent deposition of fibrin.
[0385] Due to the ability of DEGR-Factor VII to block thrombin
generation and limit platelet deposition at sites of acute vascular
injury, inactivated Factor VII polypeptides which maintain tissue
factor binding activity but lack Factor VIIa enzymatic activity can
be used to inhibit vascular restenosis.
[0386] Compositions comprising inactivated Factor VII polypeptides
are particularly useful in methods for treating patients when
formulated into pharmaceutical compositions, where they may be
given to individuals suffering from a variety of disease states to
treat coagulation-related conditions. Such inactivated Factor VII
polypeptides, capable of binding tissue factor but having a
substantially reduced ability to catalyze activation of other
factors in the clotting cascade, may possess a longer plasma
half-life and thus a correspondingly longer period of
anticoagulative activity when compared to other anticoagulants.
Among the medical indications for the subject compositions are
those commonly treated with anticoagulants, such as, for example,
deep vein thrombosis, pulmonary embolism, stroke, disseminated
intravascular coagulation (DIC), fibrin deposition in lungs and
kidneys associated with gram-negative endotoxemia, and myocardial
infarction. The compositions can be used to inhibit vascular
restenosis as occurs following mechanical vascular injury, such as
injury caused by balloon angioplasty, endarterectomy, reductive
atherectomy, stent placement, laser therapy or rotablation, or as
occurs secondary to vascular grafts, stents, bypass grafts or organ
transplants. The compositions can thus be used to inhibit platelet
deposition and associated disorders. Thus, a method of inhibiting
coagulation, vascular restenosis or platelet deposition, for
example, comprises administering to a patient a composition
comprising inactivated Factor VII polypeptides, such as that having
at least one amino acid substitution in a catalytic triad of
Ser344, Asp242 and His193, in an amount sufficient to effectively
inhibit coagulation, vascular restenosis or platelet deposition.
The methods also find use in the treatment of acute closure of a
coronary artery in an individual (e.g. acute myocardial
infarction), which comprises administering the inactivated Factor
VII polypeptides, which includes DEGR-Factor VII and FFR-Factor
VII, in conjunction with tissue plasminogen activator or
streptokinase, and can accelerate tPA induced thrombolysis. The
inactivated Factor VII polypeptides is given prior to, in
conjunction with, or shortly following administration of a
thrombolytic agent, such as tissue plasminogen activator.
[0387] Compositions of inactivated Factor VII polypeptides will
also have substantial utility in the prevention of cardiogenic
emboli and in the treatment of thrombotic strokes. Because of its
low potential for causing bleeding complications and its
selectivity, Factor VII polypeptides can be given to stroke victims
and may prevent the extension of the occluding arterial thrombus.
The amount of Factor VII polypeptides administered will vary with
each patient depending on the nature and severity of the stroke,
but doses will generally be in the range of those suggested
below.
[0388] Inactivated Factor VII polypeptides and compositions thereof
can also be used to inhibit deleterious events associated with
ischemic reperfusion. Severe ischemia to a tissue, organ or limb
may be due to a decrease in blood flow and may be associated with
trauma, surgical manipulation, or lowered blood pressure. One of
the complications associated with severe ischemia is the
up-regulation of tissue factor in the arterial system. This
increased expression of tissue factor is believed to stimulate a
procoagulant response, primarily in the capillary bed. Following
reperfusion to the ischemic tissue, thrombi can be generated which
may be either occlusive or non-occlusive. The generation of thrombi
in the arterial bed, and the deposition of platelets along the
thrombus, lead to the secondary generation of ischemia to the
tissue. The generation of the thrombi and the presence of platelets
can then cause the generation and release of multiple bioactive
factors, including those generated from the coagulation pathway,
such as thrombin and Factor X, as well as factors released from
activated platelets. In turn, these factors may induce the
generation of additional factors by the underlying endothelial and
smooth muscle cells, or by adjacent mononuclear cells, such as
TNF-alpha and IL-1. These factors, in turn, can then activate the
endothelial cells leading to the up-regulation of various adhesion
molecules associated with monocyte and neutrophil binding. The
binding and transmigration of monocytes and neutrophils, the
release of bioactive compounds by these cells, including the
generation of free-oxygen radicals, can exacerbate the level of
endothelial cell activation and damage. Ultimately, if the cascade
of events goes unchecked, this can lead to systemic complications
and the potential to stimulate multiple organ failure. By blocking
tissue factor according to the present invention by administering a
specific inhibitor for tissue factor/Factor VII binding (e.g.,
FFR-FVIIa), and thereby blocking the initiation of the extrinsic
pathway of coagulation, the initiation of the cascade of events may
be prevented, thereby eliminating, or minimizing the deleterious
events associated with ischemia/reperfusion.
[0389] The dose of inactivated Factor VII polypeptides for
prevention of deep vein thrombosis is in the range of about 50
.mu.g to 500 mg/day, more typically 1 mg to 200 mg/day, and more
preferably 10 to about 175 mg/day for a 70 kg patient, and
administration should begin at least about 6 hours prior to surgery
and continue at least until the patient becomes ambulatory. The
dose of inactivated Factor VII polypeptides in the treatment for
restenosis will vary with each patient but will generally be in the
range of those suggested above.
[0390] Compositions comprising inactivated Factor VII polypeptides
will typically be administered within about 24 hours prior to
performing an intervention, and for as much as 7 days or more
thereafter. Administration can be by a variety of routes as further
described herein. The compositions comprising inactivated Factor
VII polypeptides can also be administered systemically or locally
for the placement of vascular grafts (e.g., by coating synthetic or
modified natural arterial vascular grafts), at sites of
anastomosis, surgical endarterectomy (typically carotid artery
endarterectomy), bypass grafts, and the like.
[0391] In the treatment of established deep vein thrombosis and/or
pulmonary embolism, the dose of Factor VII polypeptides ranges from
about 50 .mu.g to 500 mg/day, more typically 1 mg to 200 mg/day,
and more preferably 10 mg to about 175 mg/day for a 70 kg patient
as loading and maintenance doses, depending on the weight of the
patient and the severity of the condition. Because of the lower
likelihood of bleeding complications from infusions of inactivated
Factor VII polypeptides, inactivated Factor VII polypeptides can
replace or lower the dose of heparin during or after surgery in
conjunction with thrombectomies or embolectomies.
[0392] In cases of acute bacteremia, endotoxemia or DIC, the
patient is given a loading dose of a Factor VII polypeptide of at
least about 50 .mu.g to 500 mg/day, more typically 1 mg to 200
mg/day, and more preferably 10 mg to about 175 mg/day for a 70 kg
patient, with maintenance doses thereafter in the range of 50 .mu.g
to 500 mg/day, typically 1 mg to 200 mg/day for a 70 kg
patient.
[0393] Preferably, the Factor VII polypeptide has a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unconjugated Factor VII from which it was derived. Preferably, the
half-life of the Factor VII polypeptide is enhanced by at least
1.5-fold to 2-fold, more preferably by about 2-fold to 3-fold, even
more preferably by about 5-fold to 10-fold, optimally about
100-fold, usually about 6-fold relative to the half-life of the
unmodified parent Factor VII.
[0394] General methods of attaching polyethylene glycol to proteins
are disclosed within U.S. Pat. No. 4,179,337 issued Dec. 18, 1979
(incorporated herein by reference to disclose methods of attaching
polyethylene glycol to proteins). Further, other methods of
attaching polyethylene glycol are disclosed within U.S. Pat. No.
5,122,614 issued Jun. 16, 1992, also incorporated herein by
reference to disclose methods of attaching polyethylene glycol to
proteins. Maleimido-PEG is perhaps the most useful reagent for
cysteine-PEGylation, but other chemistries are available for
specific cysteine modification.
[0395] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realizing the invention in diverse forms thereof.
EXAMPLES
Example 1
[0396] Construction of DNA Encoding FVII-(S43N), FVII-(K62E),
FVII-(Q64E), FVII-(I69F), FVII-(K62E/I69A), FVII-(Q64E/I69A),
FVII-(Q62E/Q64E/I69A), FVII-(K62E/I69F), FVII-(Q64E/I69F),
FVII-(K62E/Q64E/I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C),
FVII-(M306C), FVII-(R277C), FVII-(I69N/F71T), FVII-(F71N/L73T),
FVII-(R277N), and FVII-(D309N/L311T):
[0397] DNA constructs encoding FVII-(S43N), FVII-(K62E),
FVII-(Q64E), FVII-(I69F), FVII-(K62E/I69A), FVII-(Q64E/I69A),
FVII-(Q62E/Q64E/I69A), FVII-(K62E/I69F), FVII-(Q64E/I69F),
FVII-(K62E/Q64E/I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C),
FVII-(M306C), FVII-(R277C), FVII-(I69N/F71T), FVII-(F71N/L73T),
FVII-(R277N), and FVII-(D309N/L311T) were prepared by site-directed
mutagenesis using a supercoiled, double stranded DNA vector with
insert of human FVII and two synthetic primers containing the
desired mutation. The following primers were used: TABLE-US-00002
For FVII-(S43N): 5'-GGACGAAGCTGTTCTGGATTAACTACAGTGATGGGGACCAG-3'
(SEQ ID NO:2) 5'-CTGGTCCCCATCACTGTAGTTAATCCAGAACAGCTTCGTCC-3' (SEQ
ID NO:3) For FVII-(K62E): 5'-GGGGGCTCCTGCGAGGACCAGCTCCAG-3' (SEQ ID
NO:4) 5'-CTGGAGCTGGTCCTCGCAGGAGCCCCC-3' (SEQ ID NO:5) For
FVII-(Q64E): 5'-GGGCTCCTGCAAGGACGAGCTCCAGTCCTATATCTGC-3' (SEQ ID
NO:6) 5'-GCAGATATAGGACTGGAGCTCGTCCTTGCAGGAGCCC-3' (SEQ ID NO:7) For
FVII-(I69F): 5'-CCAGCTCCAGTCCTATTTCTGCTTCTGCCTCCC-3' (SEQ ID NO:8)
5'-GGGAGGCAGAAGCAGAAATAGGACTGGAGCTGG-3' (SEQ ID NO:9) For
FVII-(K62E/I69A):
5'-GGGGGCTCCTGCGAGGACCAGCTCCAGTCCTATGCCTGCTTCTGCCTC-3' (SEQ ID
NO:10) 5'-GAGGCAGAAGCAGGCATAGGACTGGAGCTGGTCCTCGCAGGAGCCCCC-3' (SEQ
ID NO:11) For FVII-(Q64E/I69A):
5'-GGGCTCCTGCAAGGACGAGCTCCAGTCCTATGCCTGCTTCTGCCTCC-3' (SEQ ID
NO:12) 5'-GGAGGCAGAAGCAGGCATAGGACTGGAGCTCGTCCTTGCAGGAGCCC-3' (SEQ
ID NO:13) For FVII-(K62E/Q64E/I69A):
5'-GGGGGCTCCTGCGAGGACGAGCTCCAGTCCTATGCCTGCTTCTGCCTCC-3' (SEQ ID
NO:14) 5'-GGAGGCAGAAGCAGGCATAGGACTGGAGCTCGTCCTCGCAGGAGCCCCC-3' (SEQ
ID NO:15) For FVII-(S43C):
5'-GGACGAAGCTGTTCTGGATTTGCTACAGTGATGGGGAC-3' (SEQ ID NO:16)
5'-GTCCCCATCACTGTAGCAAATCCAGAACAGCTTCGTCC-3' (SEQ ID NO:17) For
FVII-(I69C) 5'-CCAGCTCCAGTCCTATTGCTGCTTCTGCCTCCCTG-3' (SEQ ID
NO:18) 5'-CAGGGAGGCAGAAGCAGCAATAGGACTGGAGCTGG-3' (SEQ ID NO:19) For
FVII-(Q64C) 5'-GGGCTCCTGCAAGGACTGCCTCCAGTCCTATATCTG-3' (SEQ ID
NO:20) 5'-CAGATATAGGACTGGAGGCAGTCCTTGCAGGAGCCC-3' (SEQ ID NO:21)
For FVII-(R277C) 5'-GGACGCTGGCCTTCGTGTGCTTCTCATTGGTCAGCG-3' (SEQ ID
NO:22) 5'-CGCTGACCAATGAGAAGCACACGAAGGCCAGCGTCC-3' (SEQ ID NO:23)
For FVII-(M306C) 5'-CAACGTGCCCCGGCTGTGCACCCAGGACTGCCTGC-3' (SEQ ID
NO:24) 5'-GCAGGCAGTCCTGGGTGCACAGCCGGGGCACGTTG-3' (SEQ ID NO:25)
FVII-(I69N/F71T): 5'-CCAGCTCCAGTCCTATAACTGCACCTGCCTCCCTGCCTTCG-3'
(SEQ ID NO:26) 5'-CGAAGGCAGGGAGGCAGGTGCAGTTATAGGACTGGAGCTGG-3' (SEQ
ID NO:27) FVII-(F71N/L73T):
5'-CCAGTCCTATATCTGCAACTGCACCCCTGCCTTCGAGGGCCG-3' (SEQ ID NO:28)
5'-CGGCCCTCGAAGGCAGGGGTGCAGTTGCAGATATAGGACTGG-3' (SEQ ID NO:29)
FVII-(R277N) 5'-GGACGCTGGCCTTCGTGAACTTCTCATTGGTCAGCGG-3' (SEQ ID
NO:30) 5'-CCGCTGACCAATGAGAAGTTCACGAAGGCCAGCGTCC-3' (SEQ ID NO:31)
FVII-(D309N/L311T) 5'-CGGCTGATGACCCAGAACTGCACCCAGCAGTCACGGAAGG-3'
(SEQ ID NO:32) 5'-CCTTCCGTGACTGCTGGGTGCAGTTCTGGGTCATCAGCCG-3' (SEQ
ID NO:33)
[0398] The oligonucleotide primers, each complementary to opposite
strands of the vector insert, were extended during temperature
cycling by means of Pfu DNA polymerase. On incorporation of the
primers, a mutated plasmid containing staggered nicks was
generated. Following temperature cycling, the product was treated
with DpnI which is specific for methylated and hemimethylated DNA
to digest the parental DNA template and to select for
mutation-containing synthesized DNA.
[0399] Procedures for preparing a DNA construct using polymerase
chain reaction using specific primers are well known to persons
skilled in the art (cf. PCR Protocols, 1990, Academic ress, San
Diego, Calif., USA).
Example 2
[0400] Preparation of FVII-(S43N), FVII-(K62E), FVII-(Q64E),
FVII-(I69F), FVII-(K62E/I69A), FVII-(Q64E/I69A),
FVII-(Q62E/Q64E/I69A), FVII-(K62E/I69F), FVII-(Q64E/I69F),
FVII-(K62E/Q64E/I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C),
FVII-(M306C), FVII-(R277C), FVII-(I69N/F71T), FVII-(F71N/L73T),
FVII-(R277N), and FVII-(D309N/L311T).
[0401] BHK cells were transfected essentially as previously
described (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson
and Nielsen (1996) FEBS Lett. 385, 241-243) to obtain expression of
the variant FVII polypeptide. The Factor VII polypeptide was
purified as follows:
[0402] Conditioned medium was loaded onto a 50-ml column of Q
Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM
EDTA, and 10 mM Tris, adjustment of pH to 8.0 and adjustment of the
conductivity to 10-11 mS/cm by adding water. Elution of the protein
was accomplished by a gradient from 10 mM Tris, 50 mM NaCl, pH 8.0
to 10 mM Tris, 50 mM NaCl, 25 mM CaCl.sub.2, pH 8.0. The fractions
containing the variant FVII polypeptide were pooled, and applied to
a 25-ml column containing the monoclonal antibody F1A2 (Novo
Nordisk, Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B
(Pharmacia Biotech). The column was equilibrated with 50 mM Hepes,
pH 7.5, containing 10 mM CaCl.sub.2, and 100 mM NaCl. After washing
with equilibration buffer and equilibration buffer containing 2 M
NaCl, bound material was eluted with equilibration buffer
containing 10 mM EDTA instead of CaCl.sub.2. Before use or storage,
excess CaCl.sub.2 over EDTA was added or the variant FVII
polypeptide was transferred to a Ca.sup.2+-containing buffer. The
yield of each step was followed by factor VII ELISA measurements
and the purified protein was analysed by SDS-PAGE.
Example 3
[0403] In Vitro Hydrolysis Assay
[0404] 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). 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 nm Factor VIIa wild-type).
Example 4
[0405] In Vitro Proteolysis Assay
[0406] 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=(A.sub.405 nm Factor VIIa variant)/(A.sub.405 nm Factor VIIa
wild-type).
Example 5
[0407] TF Binding Affinity Assay--Biosensor Assay:
[0408] FVII polypeptides are tested on the Biacore instrument by
passing a standard solution of the FVII polypeptide over a chip
with immobilized TF. This is followed by different concentrations
of sTF in 10 mM hepes pH 7.4 containing 150 mM NaCl, 10 mM
CaCl.sub.2 and 0.0003% polysorbate 20. Kd's are calculated from the
sensorgrams using the integrated Biacore evaluation software.
Sequence CWU 1
1
1 1 406 PRT Homo sapiens MISC_FEATURE (1)..(406) Xaa=
4-carboxyglutamic acid (gamma-carboxyglutamate) 1 Ala Asn Ala Phe
Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys
Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Lys 20 25 30
Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp Gly Asp 35
40 45 Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly Gly Ser Cys Lys Asp
Gln 50 55 60 Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pro Ala Phe Glu
Gly Arg Asn 65 70 75 80 Cys Glu Thr His Lys Asp Asp Gln Leu Ile Cys
Val Asn Glu Asn Gly 85 90 95 Gly Cys Glu Gln Tyr Cys Ser Asp His
Thr Gly Thr Lys Arg Ser Cys 100 105 110 Arg Cys His Glu Gly Tyr Ser
Leu Leu Ala Asp Gly Val Ser Cys Thr 115 120 125 Pro Thr Val Glu Tyr
Pro Cys Gly Lys Ile Pro Ile Leu Glu Lys Arg 130 135 140 Asn Ala Ser
Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val Cys Pro 145 150 155 160
Lys Gly Glu Cys Pro Trp Gln Val Leu Leu Leu Val Asn Gly Ala Gln 165
170 175 Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile Trp Val Val Ser Ala
Ala 180 185 190 His Cys Phe Asp Lys Ile Lys Asn Trp Arg Asn Leu Ile
Ala Val Leu 195 200 205 Gly Glu His Asp Leu Ser Glu His Asp Gly Asp
Glu Gln Ser Arg Arg 210 215 220 Val Ala Gln Val Ile Ile Pro Ser Thr
Tyr Val Pro Gly Thr Thr Asn 225 230 235 240 His Asp Ile Ala Leu Leu
Arg Leu His Gln Pro Val Val Leu Thr Asp 245 250 255 His Val Val Pro
Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Arg Thr 260 265 270 Leu Ala
Phe Val Arg Phe Ser Leu Val Ser Gly Trp Gly Gln Leu Leu 275 280 285
Asp Arg Gly Ala Thr Ala Leu Glu Leu Met Val Leu Asn Val Pro Arg 290
295 300 Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg Lys Val Gly Asp
Ser 305 310 315 320 Pro Asn Ile Thr Glu Tyr Met Phe Cys Ala Gly Tyr
Ser Asp Gly Ser 325 330 335 Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly
Pro His Ala Thr His Tyr 340 345 350 Arg Gly Thr Trp Tyr Leu Thr Gly
Ile Val Ser Trp Gly Gln Gly Cys 355 360 365 Ala Thr Val Gly His Phe
Gly Val Tyr Thr Arg Val Ser Gln Tyr Ile 370 375 380 Glu Trp Leu Gln
Lys Leu Met Arg Ser Glu Pro Arg Pro Gly Val Leu 385 390 395 400 Leu
Arg Ala Pro Phe Pro 405
* * * * *