U.S. patent application number 12/178280 was filed with the patent office on 2008-12-25 for human coagulation factor vii variants.
This patent application is currently assigned to Novo Nordisk HealthCare A/G. Invention is credited to Ole Hvilsted Olsen, Egon Persson.
Application Number | 20080318276 12/178280 |
Document ID | / |
Family ID | 29554459 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080318276 |
Kind Code |
A1 |
Persson; Egon ; et
al. |
December 25, 2008 |
Human Coagulation Factor VII Variants
Abstract
The invention concerns novel coagulation factor VII variants,
wherein the Phe residue in position 374 of SEQ ID NO 1 has been
replaced by another amino acid residue which can be encoded by
nucleic acid constructs and, optionally, wherein at least one other
amino acid residue in the remaining positions in the protease
domain has been replaced by another amino acid residue which can be
encoded by nucleic acid constructs; with the proviso that the
variant is not FVII(Ala305).
Inventors: |
Persson; Egon; (Malmo,
SE) ; Olsen; Ole Hvilsted; (Bronshoj, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk HealthCare A/G
Zurich
CH
|
Family ID: |
29554459 |
Appl. No.: |
12/178280 |
Filed: |
July 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11111072 |
Apr 21, 2005 |
7416860 |
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12178280 |
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09848107 |
May 3, 2001 |
6905683 |
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11111072 |
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60204712 |
May 16, 2000 |
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60236892 |
Sep 29, 2000 |
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Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/325; 536/23.2; 800/13; 800/295 |
Current CPC
Class: |
C12Y 304/21021 20130101;
C12N 15/8509 20130101; A61P 7/04 20180101; A01K 2267/01 20130101;
C12N 9/6437 20130101 |
Class at
Publication: |
435/69.6 ;
536/23.2; 435/325; 800/13; 435/320.1; 800/295 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 15/57 20060101 C12N015/57; C12N 5/10 20060101
C12N005/10; C12N 15/85 20060101 C12N015/85; A01K 67/027 20060101
A01K067/027; A01H 5/00 20060101 A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2000 |
DK |
PA 2000 00734 |
Sep 13, 2000 |
DK |
PA 2000 01360 |
Claims
1. A nucleic acid construct comprising a nucleotide sequence
encoding a human coagulation Factor VII variant, wherein said
variant comprises a substitution of the Leu in position 305 of SEQ
ID NO 1 with an amino acid residue selected from the group
consisting of Val, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys,
Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln and wherein the ratio
between the activity of the variant and the activity of native
Factor VII polypeptide having a sequence shown in SEQ ID NO 1 is at
least about 1.25 when tested in an in vitro hydrolysis assay.
2. A recombinant vector comprising a nucleic acid construct as
defined in claim 1.
3. A recombinant host cell comprising a nucleic acid construct as
defined in claim 1.
4. A recombinant host cell as defined in claim 3, wherein the cell
is of mammalian origin.
5. A recombinant host cell as defined in claim 4, wherein the cell
is selected from the group consisting of CHO cells and BHK
cells.
6. A transgenic animal comprising the nucleic acid construct
defined in claim 1.
7. A transgenic plant comprising the nucleic acid construct defined
in claim 1.
8. A method for producing a human coagulation Factor VII variant,
which comprises (i) cultivating a cell as defined in claim 3 in an
appropriate growth medium under conditions allowing expression of
the nucleic acid construct and (ii) recovering the resulting
polypeptide from the culture medium.
9. A method for producing a human coagulation Factor VII variant,
which comprises recovering the variant from milk produced by a
transgenic animal as defined in claim 6.
10. A method for producing a human coagulation Factor VII variant,
comprising (i) cultivating a cell of a transgenic plant as defined
in claim 7 under conditions in which the variant is expressed, and
(ii) recovering the variant from the resulting plant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 11/111,072
filed Apr. 21, 2005 which is a continuation of Ser. No. 09/848,107
filed May 3, 2001 and claims the benefit of priority under 35
U.S.C. 119 of Danish application no. PA 2000 00734 filed on May 3,
2000, Danish application no. PA 2000 01360 filed on Sep. 13, 2000,
U.S. provisional application No. 60/204,712 filed on May 16, 2000,
and U.S. provisional application No. 60/236,892 filed on Sep. 29,
2000, the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel human coagulation
Factor VIIa variants having coagulant activity as well as nucleic
acid constructs encoding such variants, vectors and host cells
comprising and expressing the nucleic acid, pharmaceutical
compositions, uses and methods of treatment.
BACKGROUND OF THE INVENTION
[0003] Blood coagulation is a process consisting of a complex
interaction of various blood components (or factors) that
eventually gives raise 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).
[0004] 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
VII 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. In recent years Factor VII and tissue factor have been
found to be the main initiators of blood coagulation.
[0005] 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. Like several other plasma
proteins involved in haemostasis, Factor VII is dependent on
Vitamin K for its activity, which is required for the
gamma-carboxylation of multiple glutamic acid residues that are
clustered close to the amino terminus of the protein. These
gamma-carboxylated glutamic acids are required for the metal
ion-induced interaction of Factor VII with phospholipids. 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. In the presence of tissue factor, phospholipids and
calcium ions, the two-chain Factor VIIa rapidly activates Factor X
or Factor IX by limited proteolysis.
[0006] It is often desirable to stimulate or improve 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.
[0007] 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.
[0008] Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93,
14379-14384) relates to a Factor VII variant wherein Leu305 has
been replaced by Ala (FVII(Ala305)).
[0009] 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.
[0010] There is, however, still a need for variants of Factor VIIa
having coagulant activity, variants with high activity that can be
administered at relatively low doses, and variants which do not
produce the undesirable side effects such as systemic activation of
the coagulation system and bleeding, respectively, associated with
conventional therapies.
SUMMARY OF THE INVENTION
[0011] The invention provides coagulation Factor VIIa variants with
coagulant activity. In a first aspect, the invention provides a
human coagulation Factor VII variant, wherein the Leu residue in
position 305 or the Phe residue in position 374 of SEQ ID NO 1 has
been replaced by another amino acid residue which can be encoded by
nucleic acid constructs and, optionally, wherein at least one other
amino acid residue in the remaining positions in the protease
domain has been replaced by another amino acid residue which can be
encoded by nucleic acid constructs; with the proviso that the
variant is not FVII(Ala305).
[0012] In one embodiment, the Leu residue in position 305 or the
Phe residue in position 374 of SEQ ID NO 1 and at the most 20 amino
acid residues in the remaining positions in the protease domain
(positions 153-406) have been replaced. In one embodiment, at the
most 15 additional amino acid residues are replaced; in another
embodiment, at the most 10 amino acid residues are replaced; in
another embodiment, at the most 5 amino acid residues are
replaced.
[0013] In another embodiment of the invention the Leu residue in
position 305 or the Phe residue in position 374 of SEQ ID NO: 1 and
at least one residue in position 274 and/or 300-304 and/or position
306-312 have been replaced.
[0014] In another embodiment, the Leu residue in position 305 or
the Phe residue in position 374 of SEQ ID NO: 1 and at least the
residue in position 274 have been replaced.
[0015] In another embodiment, the Leu residue in position 305 or
the Phe residue in position 374 of SEQ ID NO: 1 and at least one
residue in position 300-304 have been replaced.
[0016] In another embodiment, the Leu residue in position 305 or
the Phe residue in position 374 of SEQ ID NO: 1 and at least one
residue in position 306-312 have been replaced.
[0017] In another embodiment, the Ala residue in position 274 has
been replaced by Met or Leu or Lys or Arg; and/or the Arg residue
in position 304 has been replaced by Tyr or Phe or Leu or Met;
and/or the Met residue in position 306 has been replaced by Asp or
Asn; and/or the Asp residue in position 309 has been replaced by
Ser or Thr.
[0018] In another embodiment, the Leu residue in position 305 or
the Phe residue in position 374 is the only amino acid residue that
has been replaced.
[0019] In one embodiment, the Leu residue in position 305 has been
replaced. In another embodiment, the Phe residue in position 374
has been replaced.
[0020] In one embodiment, the Phe residue in position 374 is the
only amino acid residue that has been replaced.
[0021] In another embodiment, the Leu residue in position 305 is
the only amino acid residue that has been replaced.
[0022] In a specific embodiment, the Leu residue in position 305
has been replaced by Val.
[0023] In another embodiment, the Leu residue in position 305 has
been replaced by an amino acid residue selected from the group
consisting of Val, Tyr and Ile, or the Phe residue in position 374
has been replaced by Pro.
[0024] In one embodiment of the invention the residues 300-322,
305-322, 300-312, or 305-312 of SEQ ID NO: 1 are replaced by the
corresponding sequences from trypsin (SEQ ID NO: 3, 7, 11, 15,
respectively), thrombin (SEQ ID NO: 4, 8, 12, 16, respectively),
Factor Xa (SEQ ID NO: 5, 9, 13, 17, respectively) or another
constitutively active serine protease. In yet another embodiment,
one or more of residues 313-322 of SEQ ID NO: 1 is/are deleted.
[0025] In one aspect, the amino acid residue at position 305 has
been replaced by an amino acid residue selected from a list of Ala,
Val, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu,
Lys, Arg, His, Asp and Gln, or the amino acid residue at position
374 has been replaced by an amino acid residue selected from a list
of Ala, Val, Leu, Ile, Met, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn,
Glu, Lys, Arg, His, Asp or Gln, with the proviso that the variant
is not FVII(Ala305).
[0026] In another aspect, the amino acid residue at position 305
has been replaced by an amino acid residue selected from a list of
Ala, Val, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn,
Glu, Lys, Arg, His, Asp and Gln, or the amino acid residue at
position 374 has been replaced by an amino acid residue selected
from a list of Ala, Val, Leu, Ile, Met, Trp, Pro, Gly, Ser, Thr,
Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp or Gln.
[0027] In another aspect, the amino acid residue at position 305
has been replaced by an amino acid residue that can be encoded by
nucleic acids, such as Ala, Val, Ile, Met, Phe, Trp, Pro, Gly, Ser,
Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln, and the amino
acid residue at position 374 has been replaced by an amino acid
residue that can be encoded by nucleic acid, such as Ala, Val, Leu,
Ile, Met, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg,
His, Asp or Gln.
[0028] In one embodiment, the amino acid residue at position 305
has been replaced by an amino acid residue selected from a list of
Ala, Val, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn,
Glu, Lys, Arg, His, Asp and Gln, and the amino acid residue at
position 374 has been replaced by an amino acid residue selected
from a list of Ala, Val, Leu, Ile, Met, Trp, Pro, Gly, Ser, Thr,
Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp or Gln.
[0029] The present invention also provides a human coagulation
Factor VII variant, wherein the ratio between the activity of the
variant and the activity of the native Factor VII polypeptide shown
in SEQ ID NO: 1 is at least about 1.25 when tested in the "In Vitro
Hydrolysis Assay" defined herein. In one embodiment, the ratio is
at least about 2.0; in yet another embodiment, at least about
4.0.
[0030] In another aspect, the invention provides human coagulation
Factor VIIa variants that have increased tissue factor-independent
activity compared to native human coagulation Factor VIIa. In
another aspect, the increased activity is not accompanied by
changes in the substrate specificity. In another aspect of the
invention, the binding of the variants to tissue factor should not
be impaired and the variants should have at least the activity of
wild-type Factor VIIa when bound to tissue factor.
[0031] Another aspect of the present invention relates to a nucleic
acid construct, preferably a DNA construct, comprising a nucleotide
sequence encoding a Factor VII variant according to the
invention.
[0032] In another aspect, the invention provides a recombinant
vector comprising the nucleic acid construct.
[0033] Another aspect of the present invention relates to a
recombinant host cell, preferably of mammalian origin, comprising
the nucleic acid construct or the recombinant vector.
[0034] In one embodiment, the recombinant host cells are CHO or BHK
cells.
[0035] Another aspect of the present invention relates to a
transgenic animal or a transgenic plant containing and expressing
the nucleic acid construct.
[0036] Other aspects of the present invention relate to a
pharmaceutical composition comprising a human coagulation Factor
VII variant wherein the Leu residue in position 305 or the Phe
residue in position 374 of SEQ ID NO: 1 has been replaced by
another amino acid residue which can be encoded by nucleic acid
constructs and, optionally, wherein at least one other amino acid
residue in the remaining positions in the protease domain has been
replaced by another amino acid residue which can be encoded by
nucleic acid constructs, optionally in combination with a
pharmaceutically acceptable carrier; to the human coagulation
Factor VII variant for use as a medicament; to the use of the human
coagulation Factor VII variant for the preparation of a composition
for the treatment or prophylaxis of bleeding episodes or for the
enhancement of the normal haemostatic system; to a method for the
treatment or prophylaxis of bleeding episodes in a subject or for
the enhancement of the normal haemostatic system; and to methods
for producing a Factor VII variant according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows the full amino acid sequence of native human
coagulation Factor VII (SEQ ID NO: 1).
[0038] FIG. 2 shows the region 300-322 of human coagulation Factor
VII and the corresponding region of homologous serine
proteases:
TABLE-US-00001 [0039] Region 300-322 of Factor VII (SEQ ID NO: 2)
Corresponding region of trypsin (SEQ ID NO: 3) Corresponding region
of thrombin (SEQ ID NO: 4) Corresponding region of FXa (SEQ ID NO:
5) Region 305-322 of Factor VII (SEQ ID NO: 6) Corresponding region
of trypsin (SEQ ID NO: 7) Corresponding region of thrombin (SEQ ID
NO: 8) Corresponding region of FXa (SEQ ID NO: 9) Region 300-312 of
Factor VII (SEQ ID NO: 10) Corresponding region of trypsin (SEQ ID
NO: 11) Corresponding region of thrombin (SEQ ID NO: 12)
Corresponding region of FXa (SEQ ID NO: 13) Region 305-312 of
Factor VII (SEQ ID NO: 14) Corresponding region of trypsin (SEQ ID
NO: 15) Corresponding region of thrombin (SEQ ID NO: 16)
Corresponding region of FXa (SEQ ID NO: 17)
DETAILED DESCRIPTION OF THE INVENTION
[0040] It has now been found that Factor VIIa variants wherein at
least one of the amino acid residues Leu305 or Phe374 (and
optionally one or more additional residues) is/are replaced by
another amino acid residue have coagulant activity.
[0041] The residues Leu305 and Phe374 are located at each end of an
.alpha.-helix starting at residue 307. This .alpha.-helix is found
in the tissue factor-complexed form of Factor VIIa. In free Factor
VIIa (Factor VIIa not bound to tissue factor) the helix is
distorted and thus possibly unstable. The helix is believed to be
important to the activity. The variants according to the present
invention attain the active conformation, which normally has to be
induced by tissue factor.
[0042] The activity may be due to a stabilisation of the
.alpha.-helix starting at residue 307, a reorientation of the helix
or some other change in conformation. Replacement of one of the
residues Leu305 or Phe374, which are located at each end of the
helix, will induce a reorientation and/or stabilisation of the
helix.
[0043] Due to the higher inherent activity of the described Factor
VIIa variant compared to native Factor VIIa, a lower dose will be
adequate to obtain a functionally adequate concentration at the
site of action and thus it will be possible to administer a lower
dose to the subject having bleeding episodes or needing enhancement
of the normal haemostatic system.
[0044] As discussed briefly above, it has been found by the present
inventors that by replacing either the Leu residue in position 305
or the Phe residue in position 374 with another amino acid, Factor
VIIa will spontaneously attain a more active conformation that
normally has to be induced by tissue factor. Examples of preferred
amino acid residues, which may replace Leu in position 305, include
Val, Tyr and Ile.
[0045] Thus, it is contemplated by the present inventors that such
Factor VIIa variants exhibit an inherent activity which may be
therapeutically useful in situations where the procoagulant
activity is independent of tissue factor (Factor Xa generation on
the platelet surface) such as when high doses of, for example,
NovoSeven.RTM. are administered.
[0046] As said, replacement of other amino acid residues in the
sequence may, in addition to the effect obtained by replacement of
the Leu305 or the Phe374 residue, further facilitate formation of
the active conformation of the molecule. In principle these
remaining positions may be anywhere (except, of course, in position
305 or 374) in the protease domain. It is believed, however, that
the most pronounced effects will be seen when the above-mentioned
mutations are carried out in the vicinity (sequential or
three-dimensional) of residue 305 (or 374).
[0047] It is well established that replacement of a few amino acid
residues in the N-terminal Gla domain (residues 1-37) of Factor VII
can provide the protein with a substantially higher affinity for
membrane phospholipids, such as membrane phospholipids of tissue
factor-bearing cells or of platelets, thereby generating Factor VII
derivatives which have an improved procoagulant effect.
[0048] Thus, the Factor VII variants mentioned above may, in
addition to the already performed amino acid replacement in
positions 305 or 374 and the optional amino acid replacements in
positions 274, 300-304 and 306-310 or elsewhere in the protease
domain, also have some amino acid residues replaced in the
N-terminal Gla domain, thereby obtaining a protein having an
increased activity as well as an increased affinity for membrane
phospholipids compared to native Factor VII.
[0049] Preferably the amino acid residues in positions 10 and 32
(referring to SEQ ID NO: 1) of Factor VII may be replaced with
another amino acid residue that can be encoded by nucleic acid
constructs.
[0050] Examples of preferred amino acid residues to be incorporated
in the above-mentioned positions are:
[0051] The amino acid residue Pro in position 10 is replaced by
Gln, Arg, His, Gln, Asn or Lys; and/or the amino acid residue Lys
in position 32 is replaced by Glu, Gln or Asn.
[0052] Other residues in the Gla domain, based on the different
phospholipid affinities and sequences of the vitamin K-dependent
plasma proteins, may also be considered for substitution.
[0053] In the present context the three-letter or one-letter
indications of the amino acids have been used in their conventional
meaning as indicated in table 1. Unless indicated explicitly, the
amino acids mentioned herein are L-amino acids. 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-00002 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
[0054] The term "N-terminal GLA-domain" means the amino acid
sequence 1-37 of Factor VII.
[0055] The term "protease domain" means the amino acid sequence
153-406 of Factor VII (the heavy-chain of Factor VIIa).
[0056] The three-letter indication "GLA" means 4-carboxyglutamic
acid (.gamma.-carboxyglutamate).
[0057] The indication "FVII(Ala305)" means Factor VII as shown in
SEQ ID NO: 1 wherein the Leu residue in position 305 has been
replaced by Ala.
[0058] The term "Factor VII" or "FVII" as used herein is intended
to comprise the inactive one-chain zymogen Factor VII molecule as
well as the activated two-chain Factor VII molecule, and may, where
appropriate, be used interchangeably with the terms "polypeptide",
"protein", "protease" and "enzyme".
[0059] As used herein the term "nucleic acid construct" is intended
to mean any nucleic acid molecule of cDNA, genomic DNA, synthetic
DNA or RNA origin. The term "construct" is intended to indicate a
nucleic acid segment which may be single- or double-stranded, and
which may be based on a complete or partial naturally occurring
nucleotide sequence encoding the polypeptide of interest. The
construct may optionally contain other nucleic acid segments. In a
similar way, the term "amino acid residue which can be encoded by
nucleic acid constructs" covers amino acid residues which can be
encoded by the nucleic acid constructs defined above, i.e. amino
acids such as Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser,
Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.
[0060] In the present context, the term "treatment" is meant to
include both prevention of an expected bleeding, such as in
surgery, and regulation of an already occurring bleeding, such as
in trauma, with the purpose of inhibiting or minimising the
bleeding. Prophylactic administration of the Factor VIIa variant
according to the invention is thus included in the term
"treatment".
[0061] The term "activity" means the ability to generate thrombin,
the term "inherent activity" also includes the ability to generate
thrombin on the surface of activated platelets in the absence of
tissue factor.
[0062] The term "enhancement of the normal haemostatic system"
means an enhancement of the ability to generate thrombin.
[0063] As used herein the term "bleeding disorder" 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.
[0064] 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 likened 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.
[0065] In one embodiment of the invention, the bleeding is
associated with haemophilia. In another embodiment, the bleeding is
associated with haemophilia with acquired 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.
[0066] 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".
[0067] 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 nucleic acid sequence
encoding the Factor VII variant of the invention.
Preparation of Factor VII Variants
[0068] The Factor VII variants described herein may be produced by
means of recombinant nucleic acid techniques. In general, a cloned
wild-type Factor VII nucleic acid sequence is modified to encode
the desired protein. This modified sequence is then inserted into
an expression vector, which is in turn transformed or transfected
into host cells. Higher eukaryotic cells, in particular cultured
mammalian cells, are preferred as host cells. The complete
nucleotide and amino acid sequences for human Factor VII are known
(see U.S. Pat. No. 4,784,950, 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)).
[0069] The amino acid sequence alterations may be accomplished by a
variety of techniques. Modification of the nucleic acid sequence
may be by site-specific mutagenesis. Techniques for site-specific
mutagenesis are well known in the art and are described in, for
example, Zoller and Smith (DNA 3:479-488, 1984) or "Splicing by
extension overlap", Horton et al., Gene 77, 1989, pp. 61-68. Thus,
using the nucleotide and amino acid sequences of Factor VII, one
may introduce the alteration(s) of choice. Likewise, procedures for
preparing a DNA construct using polymerase chain reaction using
specific primers are well known to persons skilled in the art (cf.
PCR Protocols, 1990, Academic Press, San Diego, Calif., USA).
[0070] The nucleic acid construct encoding the Factor VII variant
of the invention may suitably be of genomic or cDNA origin, for
instance obtained by preparing a genomic or cDNA library and
screening for DNA sequences coding for all or part of the
polypeptide by hybridization using synthetic oligonucleotide probes
in accordance with standard techniques (cf. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989).
[0071] The nucleic acid construct encoding the Factor VII variant
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 synthesised, e.g. in an
automatic DNA synthesiser, purified, annealed, ligated and cloned
in suitable vectors.
[0072] Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed genomic
and cDNA origin prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0073] The nucleic acid construct is preferably a DNA
construct,
[0074] DNA sequences for use in producing Factor VII variants
according to the present invention will typically encode a pre-pro
polypeptide at the amino-terminus of Factor VII to obtain proper
posttranslational processing (e.g. gamma-carboxylation of glutamic
acid residues) and secretion from the host cell. The pre-pro
polypeptide may be that of Factor VII or another vitamin
K-dependent plasma protein, such as Factor IX, Factor X,
prothrombin, protein C or protein S. As will be appreciated by
those skilled in the art, additional modifications can be made in
the amino acid sequence of the Factor VII variants where those
modifications do not significantly impair the ability of the
protein to act as a coagulant. For example, the Factor VII variants
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.
[0075] Expression vectors for use in expressing Factor VIIa
variants will comprise a promoter capable of directing the
transcription of a cloned gene or cDNA. Preferred promoters for use
in cultured mammalian cells include viral promoters and cellular
promoters. Viral promoters include the SV40 promoter (Subramani et
al., Mol. Cell. Biol. 1:854-864, 1981) and the CMV promoter
(Boshart et al., Cell 41:521-530, 1985). A particularly preferred
viral promoter is the major late promoter from adenovirus 2
(Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982). Cellular
promoters include the mouse kappa gene promoter (Bergman et al.,
Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse
V.sub.H promoter (Loh et al., Cell 33:85-93, 1983). A particularly
preferred cellular promoter is the mouse metallothionein-I promoter
(Palmiter et al., Science 222:809-814, 1983). Expression vectors
may also contain a set of RNA splice sites located downstream from
the promoter and upstream from the insertion site for the Factor
VII sequence itself. Preferred RNA splice sites may be obtained
from adenovirus and/or immunoglobulin genes. Also contained in the
expression vectors is a polyadenylation signal located downstream
of the insertion site. Particularly preferred polyadenylation
signals include the early or late polyadenylation signal from SV40
(Kaufman and Sharp, ibid.), the polyadenylation signal from the
adenovirus 5 EIb region, the human growth hormone gene terminator
(DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981) or the
polyadenylation signal from the human Factor VII gene or the bovine
Factor VII gene. The expression vectors may also include a
noncoding viral leader sequence, such as the adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites; and enhancer sequences, such as the SV40 enhancer.
[0076] Cloned DNA sequences are introduced into cultured mammalian
cells by, for example, calcium phosphate-mediated transfection
(Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic
Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology
52d:456-467, 1973) or electroporation (Neumann et al., EMBO J.
1:841-845, 1982). To identify and select cells that express the
exogenous DNA, a gene that confers a selectable phenotype (a
selectable marker) is generally introduced into cells along with
the gene or cDNA of interest. Preferred selectable markers include
genes that confer resistance to drugs such as neomycin, hygromycin,
and methotrexate. The selectable marker may be an amplifiable
selectable marker. A preferred amplifiable selectable marker is a
dihydrofolate reductase (DHFR) sequence. Selectable markers are
reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, Mass., incorporated herein by reference). The
person skilled in the art will easily be able to choose suitable
selectable markers.
[0077] 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.
[0078] After the cells have taken up the DNA, they are grown in an
appropriate growth medium, typically for 1-2 days, to begin
expressing the gene of interest. 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 media are
prepared using procedures known in the art (see, e.g., references
for bacteria and yeast; Bennett, J. W. and LaSure, L., editors,
More Gene Manipulations in Fungi, Academic Press, CA, 1991). Growth
media generally include a carbon source, a nitrogen source,
essential amino acids, essential sugars, vitamins, salts,
phospholipids, proteins and growth factors. For production of
gamma-carboxylated Factor VII variants, the medium will contain
vitamin K, preferably at a concentration of about 0.1 mg/ml to
about 5 mg/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 desired
Factor VII variant.
[0079] Preferred mammalian cell lines include the CHO (ATCC CCL
61), 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), hereinafter referred to as BHK 570 cells. The BHK 570 cell
line is available from the American Type Culture Collection, 12301
Parklawn Dr., Rockville, Md. 20852, under ATCC accession number CRL
10314. A tk.sup.- ts13 BHK cell line is also available from the
ATCC under accession number CRL 1632. In addition, a number of
other cell lines may be used, including Rat Hep I (Rat hepatoma;
ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK
(ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1)
and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA
77:4216-4220, 1980).
[0080] Transgenic animal technology may be employed to produce the
Factor VII variants of the invention. It is preferred to produce
the proteins within the mammary glands of a host female mammal.
Expression in the mammary gland and subsequent secretion of the
protein of interest into the milk overcomes many difficulties
encountered in isolating proteins from other sources. Milk is
readily collected, available in large quantities, and biochemically
well characterized. Furthermore, the major milk proteins are
present in milk at high concentrations (typically from about 1 to
15 g/l).
[0081] From a commercial point of view, it is clearly preferable to
use as the host a species that has a large milk yield. While
smaller animals such as mice and rats can be used (and are
preferred at the proof of principle stage), it is preferred to use
livestock mammals including, but not limited to, pigs, goats, sheep
and cattle. Sheep are particularly preferred due to such factors as
the previous history of transgenesis in this species, milk yield,
cost and the ready availability of equipment for collecting sheep
milk (see, for example, WO 88/00239 for a comparison of factors
influencing the choice of host species). It is generally desirable
to select a breed of host animal that has been bred for dairy use,
such as East Friesland sheep, or to introduce dairy stock by
breeding of the transgenic line at a later date. In any event,
animals of known, good health status should be used.
[0082] To obtain expression in the mammary gland, a transcription
promoter from a milk protein gene is used. Milk protein genes
include those genes encoding caseins (see U.S. Pat. No. 5,304,489),
beta-lactoglobulin, a-lactalbumin, and whey acidic protein. The
beta-lactoglobulin (BLG) promoter is preferred. In the case of the
ovine beta-lactoglobulin gene, a region of at least the proximal
406 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 a .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.
[0083] Other regions of the beta-lactoglobulin gene may also be
incorporated in constructs, as may genomic regions of the gene to
be expressed. It is generally accepted in the art that constructs
lacking introns, for example, express poorly in comparison with
those that contain such DNA sequences (see Brinster et al., Proc.
Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc.
Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al.,
Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318, each
of which is incorporated herein by reference). In this regard, it
is generally preferred, where possible, to use genomic sequences
containing all or some of the native introns of a gene encoding the
protein or polypeptide of interest, thus the further inclusion of
at least some introns from, e.g, the beta-lactoglobulin gene, is
preferred. One such region is a DNA segment that provides for
intron splicing and RNA polyadenylation from the 3' non-coding
region of the ovine beta-lactoglobulin gene. When substituted for
the natural 3' non-coding sequences of a gene, this ovine
beta-lactoglobulin segment can both enhance and stabilize
expression levels of the protein or polypeptide of interest. Within
other embodiments, the region surrounding the initiation ATG of the
variant Factor VII sequence is replaced with corresponding
sequences from a milk specific protein gene. Such replacement
provides a putative tissue-specific initiation environment to
enhance expression. It is convenient to replace the entire variant
Factor VII pre-pro and 5' non-coding sequences with those of, for
example, the BLG gene, although smaller regions may be
replaced.
[0084] For expression of Factor VII variants in transgenic animals,
a DNA segment encoding variant Factor VII is operably linked to
additional DNA segments required for its expression to produce
expression units. Such additional segments include the
above-mentioned promoter, as well as sequences that provide for
termination of transcription and polyadenylation of mRNA. The
expression units will further include a DNA segment encoding a
secretory signal sequence operably linked to the segment encoding
modified Factor VII. The secretory signal sequence may be a native
Factor VII secretory signal sequence or may be that of another
protein, such as a milk protein (see, for example, von Heijne,
Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat.
No. 4,873,316, which are incorporated herein by reference).
[0085] Construction of expression units for use in transgenic
animals is conveniently carried out by inserting a variant Factor
VII sequence into a plasmid or phage vector containing the
additional DNA segments, although the expression unit may be
constructed by essentially any sequence of ligations. It is
particularly convenient to provide a vector containing a DNA
segment encoding a milk protein and to replace the coding sequence
for the milk protein with that of a variant 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 variant Factor VII sequence. Amplification is
conveniently carried out in bacterial (e.g. E. coli) host cells,
thus the vectors will typically include an origin of replication
and a selectable marker functional in bacterial host cells. The
expression unit is then introduced into fertilized eggs (including
early-stage embryos) of the chosen host species. Introduction of
heterologous DNA can be accomplished by one of several routes,
including microinjection (e.g. U.S. Pat. No. 4,873,191), retroviral
infection (Jaenisch, Science 240: 1468-1474 (1988)) or
site-directed integration using embryonic stem (ES) cells (reviewed
by Bradley et al., Bio/Technology 10: 534-539 (1992)). The eggs are
then implanted into the oviducts or uteri of pseudopregnant females
and allowed to develop to term. Offspring carrying the introduced
DNA in their germ line can pass the DNA on to their progeny in the
normal, Mendelian fashion, allowing the development of transgenic
herds. General procedures for producing transgenic animals are
known in the art (see, for example, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory,
1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al.,
Biol. Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8:
140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991);
Krimpenfort et al., Bio/Technology 9: 844-847 (1991); Wall et al.,
J. Cell. Biochem. 49:113-120 (1992); U.S. Pat. No. 4,873,191; U.S.
Pat. No. 4,873,316; WO 88/00239, WO 90/05188, WO 92/11757; and GB
87/00458). Techniques for introducing foreign DNA sequences into
mammals and their germ cells were originally developed in the mouse
(see, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384
(1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter
and Brinster, Cell 41: 343-345 (1985); Brinster et al., Proc. Natl.
Acad. Sci. USA 82: 4438-4442 (1985); and Hogan et al. (ibid.)).
These techniques were subsequently adapted for use with larger
animals, including livestock species (see, e.g., WO 88/00239, WO
90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6:
179-183 (1988)). To summarise, in the most efficient route used to
date in the generation of transgenic mice or livestock, several
hundred linear molecules of the DNA of interest are injected into
one of the pro-nuclei of a fertilized egg according to established
techniques. Injection of DNA into the cytoplasm of a zygote can
also be employed.
[0086] Production in transgenic plants may also be employed.
Expression may be generalised 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 EP 0 255 378).
[0087] The Factor VII variants of the invention are recovered from
cell culture medium or milk. The Factor VII variants of the present
invention may be purified by a variety of procedures known in the
art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing (IEF), differential solubility (e.g., ammonium
sulfate precipitation), or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989). Preferably, they may be purified by affinity
chromatography on an anti-Factor VII antibody column. The use of
calcium-dependent monoclonal antibodies, as described by
Wakabayashi et al., J. Biol. Chem. 261:11097-11108, (1986) and Thim
et al., Biochemistry 27: 7785-7793, (1988), is particularly
preferred. Additional purification may be achieved by conventional
chemical purification means, such as high performance liquid
chromatography. Other methods of purification, including barium
citrate precipitation, are known in the art, and may be applied to
the purification of the novel Factor VII variants described herein
(see, for example, Scopes, R., Protein Purification,
Springer-Verlag, N.Y., 1982).
[0088] For therapeutic purposes it is preferred that the Factor VII
variants of the invention are substantially pure. Thus, in a
preferred embodiment of the invention the Factor VII variants of
the invention is purified to at least about 90 to 95% homogeneity,
preferably to at least about 98% homogeneity. Purity may be
assessed by e.g. gel electrophoresis and amino-terminal amino acid
sequencing.
[0089] The Factor VII variant is cleaved at its activation site in
order to convert it to its two-chain form. Activation may be
carried out according to procedures known in the art, such as those
disclosed by Osterud, et al., Biochemistry 11:2853-2857 (1972);
Thomas, U.S. Pat. No. 4,456,591; Hedner and Kisiel, J. Clin.
Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, Behring Inst.
Mitt. 73:29-42 (1983). Alternatively, as described by Bjoern et al.
(Research Disclosure, 269 September 1986, pp. 564-565), Factor VII
may be activated by passing it through an ion-exchange
chromatography column, such as Mono Q.RTM. (Pharmacia fine
Chemicals) or the like. The resulting activated Factor VII variant
may then be formulated and administered as described below.
Assays
[0090] The invention also provides suitable assays for selecting
preferred Factor VIIa variants according to the invention. These
assays can be performed as a simple preliminary in vitro test.
[0091] Thus, Example 6 herein 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 native Factor VII shown
in FIG. 1 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" defined herein.
[0092] The activity of the variants can also be measured using a
physiological substrate such as factor X (see Example 7), suitably
at a concentration of 100-1000 nM, where the factor Xa generated is
measured after the addition of a suitable chromogenic substrate
(eg. S-2765). In addition, the activity assay may be run at
physiological temperature.
[0093] 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 VII 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).
Administration and Pharmaceutical Compositions
[0094] The Factor VII variants 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.
[0095] 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.
[0096] 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 Factor
VII variants according to the invention may thus also be used in
their activated form to control such excessive bleedings associated
with a normal haemostatic mechanism.
[0097] For treatment in connection with deliberate interventions,
the Factor VII variants 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.
[0098] The dose of the Factor VII variants 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 weight
of the subject and the severity of the condition.
[0099] 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 variant 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 variants 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.
[0100] The concentration of Factor VII variant 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.
[0101] 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 variant. 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).
[0102] The compositions containing the Factor VII variants 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
variant per day for a 70 kg subject, with dosages of from about 1.0
mg to about 200 mg of the Factor VII variant per day being more
commonly used.
[0103] 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 variants 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.
[0104] In prophylactic applications, compositions containing the
Factor VII variant 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.
[0105] 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 variants may be administered by
continuous infusion using e.g. a portable pump system.
[0106] Local delivery of the Factor VII variant 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 variant sufficient to effectively
treat the subject.
The present invention is further illustrated by the following
examples which, however, are not to be construed as limiting the
scope of protection. The features disclosed in the foregoing
description and in the following examples may, both separately and
in any combination thereof, be material for realising the invention
in diverse forms thereof.
EXAMPLES
[0107] The terminology for amino acid substitutions used in the
following examples are 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 [L305V]-FVII, where the leucine
at position 305 of SEQ ID NO: 1 is replaced by a valine. In another
example, [L305V/M306D/D309S]-FVII, the leucine at position 305 of
SEQ ID NO: 1 is replaced by a valine and the methionine at position
306 of SEQ ID NO: 1 is replaced by an aspartic acid and the
aspartic acid at position 309 of SEQ ID NO: 1 is replaced by a
serine, all mutations in the same Factor VII polypeptide.
Example 1
DNA Encoding [L305V/M306D/D309S]-FVII, [L305V]-FVII, [L3051]-FVII,
[L305T]-FVII and [F374P]-FVII
[0108] DNA constructs encoding [L305V/M306D/D309S]-FVII,
[L305V]-FVII, [L3051]-FVII, [L305T]-FVII and [F374P]-FVII were
prepared by site-directed mutagenesis using a supercoiled, double
stranded DNA vector with an insert of interest and two synthetic
primers containing the desired mutation. The following primers were
used:
TABLE-US-00003 For [L305V]-FVII: (SEQ ID NO: 18) 5'-CGT GCC CCG GGT
GAT GAC CCA GGA C-3' (SEQ ID NO: 19) 5'-GTC CTG GGT CAT CAC CCG GGG
CAC G-3' For [M306D/D309S]-FVII: (SEQ ID NO: 20) 5-TCT AGA TAC CCA
GTC TTG CCT GCA GCA GTC ACG GAA-3' (SEQ ID NO: 21) 5'-TTC CGT GAC
TGC TGC AGG CAA GAC TGG GTA TCT AGA-3' For [F374P]-FVII: (SEQ ID
NO: 22) 5'-CCG TGG GCC ACC CTG GGG TGT ACA CC-3' (SEQ ID NO: 23)
5'-GGT GTA CAC CCC AGG GTG GCC CAC GG-3' For [L305I]-FVII: (SEQ ID
NO: 24) 5'-CCT CAA CGT GCC CCG GAT CAT GAC CCA GGA C-3' (SEQ ID NO:
25) 5'-GTC CTG GGT CAT GAT CCG GGG CAC GTT GAG G-3' For
[L305T]-FVII: (SEQ ID NO: 26) 5'-CCT CAA CGT GCC CCG GAC GAT GAC
CCA GGA C-3' (SEQ ID NO: 27) 5'-GTC CTG GGT CAT CGT CCG GGG CAC GTT
GAG G-3'
The oligonucleotide primers, each complementary to opposite strands
of the vector, 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.
[0109] Procedures for preparing a DNA construct using polymerase
chain reaction using specific primers are well known to persons
skilled in the art (cf. PCR Protocols, 1990, Academic Press, San
Diego, Calif., USA).
Example 2
Preparation of [L305V/M306D/D309S]-FVII
[0110] 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 [L305V/M306D/D309S]-FVII. The Factor VII variant was
purified as follows:
[0111] Conditioned medium was loaded onto a 25-ml column of Q
Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM
EDTA, 0.1% Triton X-100 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, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 1 M
NaCl, 5 mM CaCl.sub.2, 0.1% Triton X-100, pH 7.5. The fractions
containing [L305V/M306D/D309S]-FVII were pooled, 10 mM CaCl.sub.2
was added, and applied to a 25-ml column containing monoclonal
antibody F1A2 (Novo Nordisk, Bagsv.ae butted.rd, 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,
100 mM NaCl and 0.02% Triton X-100. 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 [L305V/M306D/D309S]-FVII 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
Preparation of [L305V]-FVII
[0112] 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 [L305V]-FVII. The Factor VII variant was purified as
follows:
[0113] Conditioned medium was loaded onto a 25-ml column of Q
Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM
EDTA, 0.1% Triton X-100 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, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 1 M
NaCl, 5 mM CaCl.sub.2, 0.1% Triton X-100, pH 7.5. The fractions
containing [L305V]-FVII were pooled, 10 mM CaCl.sub.2 was added,
and applied to a 25-ml column containing monoclonal antibody F1A2
(Novo Nordisk, Bagsv.ae butted.rd, 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,
100 mM NaCl and 0.02% Triton X-100. 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 [L305V]-FVII 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 4
Preparation of [F374P]-FVII
[0114] 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 [F374P]-FVII. The Factor VII variant was purified as
follows:
[0115] Conditioned medium was loaded onto a 25-ml column of Q
Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM
EDTA, 0.1% Triton X-100 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, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 1 M
NaCl, 5 mM CaCl.sub.2, 0.1% Triton X-100, pH 7.5. The fractions
containing [F374P]-FVII were pooled, 10 mM CaCl.sub.2 was added,
and applied to a 25-ml column containing monoclonal antibody F1A2
(Novo Nordisk, Bagsv.ae butted.rd, 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,
100 mM NaCl and 0.02% Triton X-100. 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 [F374P]-FVII 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 5
Preparation of [L3051]-FVII and [L305T]-FVII
[0116] BHK cells are 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 [L3051]-FVII or [L305T]-FVII. The Factor VII variant is
purified as follows:
[0117] Conditioned medium is loaded onto a 25-ml column of Q
Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM
EDTA, 0.1% Triton X-100 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 is accomplished by a gradient from 10 mM
Tris, 50 mM NaCl, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 1 M
NaCl, 5 mM CaCl.sub.2, 0.1% Triton X-100, pH 7.5. The fractions
containing [L3051]-FVII or [L305T]-FVII are pooled, 10 mM
CaCl.sub.2 is added, and applied to a 25-ml column containing
monoclonal antibody F1A2 (Novo Nordisk, Bagsv.ae butted.rd,
Denmark) coupled to CNBr-activated Sepharose 4B (Pharmacia
Biotech). The column is equilibrated with 50 mM Hepes, pH 7.5,
containing 10 mM CaCl.sub.2, 100 mM NaCl and 0.02% Triton X-100.
After washing with equilibration buffer and equilibration buffer
containing 2 M NaCl, bound material is eluted with equilibration
buffer containing 10 mM EDTA instead of CaCl.sub.2. Before use or
storage, excess CaCl.sub.2 over EDTA is added or [L3051]-FVII or
[L305T]-FVII are transferred to a Ca.sup.2+-containing buffer. The
yield of each step is followed by factor VII ELISA measurements and
the purified protein is analysed by SDS-PAGE.
Example 6
In Vitro Hydrolysis Assay
[0118] Native (wild-type) Factor Vila 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 Factor VIIa
wild-type).
Example 7
In Vitro Proteolysis Assay
[0119] Native (wild-type) Factor Vila 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 8
Relative Activities of FVIIa Variants Measured in the Assays
Described in Examples 6 and 7
TABLE-US-00004 [0120] Variant Ratio in example 6 Ratio in example 7
L305V/M306D/D309S-FVIIa 3.0 .+-. 0.1 6.3 .+-. 0.9 L305V-FVIIa 3.2
.+-. 0.2 3.3 .+-. 0.2 F374P-FVIIa 1.4 <1 wt-FVIIa 1.0 1.0
Sequence CWU 1
1
271406PRTHumanMISC_FEATURE(1)..(406)Xaa=gamma carboxyglutamic acid
1Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa1 5
10 15Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe
Lys20 25 30Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp
Gly Asp35 40 45Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly Gly Ser Cys
Lys Asp Gln50 55 60Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pro Ala Phe
Glu Gly Arg Asn65 70 75 80Cys Glu Thr His Lys Asp Asp Gln Leu Ile
Cys Val Asn Glu Asn Gly85 90 95Gly Cys Glu Gln Tyr Cys Ser Asp His
Thr Gly Thr Lys Arg Ser Cys100 105 110Arg Cys His Glu Gly Tyr Ser
Leu Leu Ala Asp Gly Val Ser Cys Thr115 120 125Pro Thr Val Glu Tyr
Pro Cys Gly Lys Ile Pro Ile Leu Glu Lys Arg130 135 140Asn Ala Ser
Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val Cys Pro145 150 155
160Lys Gly Glu Cys Pro Trp Gln Val Leu Leu Leu Val Asn Gly Ala
Gln165 170 175Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile Trp Val Val
Ser Ala Ala180 185 190His Cys Phe Asp Lys Ile Lys Asn Trp Arg Asn
Leu Ile Ala Val Leu195 200 205Gly Glu His Asp Leu Ser Glu His Asp
Gly Asp Glu Gln Ser Arg Arg210 215 220Val Ala Gln Val Ile Ile Pro
Ser Thr Tyr Val Pro Gly Thr Thr Asn225 230 235 240His Asp Ile Ala
Leu Leu Arg Leu His Gln Pro Val Val Leu Thr Asp245 250 255His Val
Val Pro Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Arg Thr260 265
270Leu Ala Phe Val Arg Phe Ser Leu Val Ser Gly Trp Gly Gln Leu
Leu275 280 285Asp Arg Gly Ala Thr Ala Leu Glu Leu Met Val Leu Asn
Val Pro Arg290 295 300Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg
Lys Val Gly Asp Ser305 310 315 320Pro Asn Ile Thr Glu Tyr Met Phe
Cys Ala Gly Tyr Ser Asp Gly Ser325 330 335Lys Asp Ser Cys Lys Gly
Asp Ser Gly Gly Pro His Ala Thr His Tyr340 345 350Arg Gly Thr Trp
Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gly Cys355 360 365Ala Thr
Val Gly His Phe Gly Val Tyr Thr Arg Val Ser Gln Tyr Ile370 375
380Glu Trp Leu Gln Lys Leu Met Arg Ser Glu Pro Arg Pro Gly Val
Leu385 390 395 400Leu Arg Ala Pro Phe Pro405223PRTHuman 2Leu Asn
Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg1 5 10 15Lys
Val Gly Asp Ser Pro Asn20318PRTHuman 3Leu Lys Ala Pro Ile Leu Asp
Asn Ser Ser Cys Lys Ser Ala Tyr Pro1 5 10 15Gly Gln418PRTHuman 4Val
Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg1 5 10
15Ile Arg518PRTHuman 5Leu Glu Val Pro Tyr Val Asp Arg Asn Ser Cys
Lys Leu Ser Ser Ser1 5 10 15Phe Ile618PRTHuman 6Leu Met Thr Gln Asp
Cys Leu Gln Gln Ser Arg Lys Val Gly Asp Ser1 5 10 15Pro
Asn713PRTHuman 7Leu Asp Asn Ser Ser Cys Lys Ser Ala Tyr Pro Gly
Gln1 5 10813PRTHuman 8Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg
Ile Arg1 5 10913PRTHuman 9Val Asp Arg Asn Ser Cys Lys Leu Ser Ser
Ser Phe Ile1 5 101013PRTHuman 10Leu Asn Val Pro Arg Leu Met Thr Gln
Asp Cys Leu Gln1 5 101113PRTHuman 11Leu Lys Ala Pro Ile Leu Asp Asn
Ser Ser Cys Lys Ser1 5 101213PRTHuman 12Val Asn Leu Pro Ile Val Glu
Arg Pro Val Cys Lys Asp1 5 101313PRTHuman 13Leu Glu Val Pro Tyr Val
Asp Arg Asn Ser Cys Lys Leu1 5 10148PRTHuman 14Leu Met Thr Gln Asp
Cys Leu Gln1 5158PRTHuman 15Leu Asp Asn Ser Ser Cys Lys Ser1
5168PRTHuman 16Val Glu Arg Pro Val Cys Lys Asp1 5178PRTHuman 17Val
Asp Arg Asn Ser Cys Lys Leu1 51825DNAArtificialSynthetic
18cgtgccccgg gtgatgaccc aggac 251925DNAArtificial SequenceSynthetic
19gtcctgggtc atcacccggg gcacg 252036DNAArtificial SequenceSynthetic
20tctagatacc cagtcttgcc tgcagcagtc acggaa 362136DNAArtificial
SequenceSynthetic 21ttccgtgact gctgcaggca agactgggta tctaga
362226DNAArtificialSynthetic 22ccgtgggcca ccctggggtg tacacc
262326DNAArtificial SequenceSynthetic 23ggtgtacacc ccagggtggc
ccacgg 262431DNAArtificial SequenceSynthetic 24cctcaacgtg
ccccggatca tgacccagga c 312531DNAArtificial SequenceSynthetic
25gtcctgggtc atgatccggg gcacgttgag g 312631DNAArtificial
SequenceSynthetic 26cctcaacgtg ccccggacga tgacccagga c
312731DNAArtificial SequenceSynthetic 27gtcctgggtc atcgtccggg
gcacgttgag g 31
* * * * *