U.S. patent application number 15/837471 was filed with the patent office on 2018-04-05 for covalent complex of von willebrand factor and factor viii, compositions, and uses relating thereto.
The applicant listed for this patent is CSL LIMITED. Invention is credited to Hubert Metzner, Stefan Schulte, Thomas Weimer.
Application Number | 20180092966 15/837471 |
Document ID | / |
Family ID | 48143163 |
Filed Date | 2018-04-05 |
United States Patent
Application |
20180092966 |
Kind Code |
A1 |
Metzner; Hubert ; et
al. |
April 5, 2018 |
COVALENT COMPLEX OF VON WILLEBRAND FACTOR AND FACTOR VIII,
COMPOSITIONS, AND USES RELATING THERETO
Abstract
The present invention relates to a covalent complex of von
Willebrand Factor (VWF) and Factor VIII, wherein the complex is
modified such that it has an extended half-life in vivo. The
invention further relates to a method of producing the complex, as
well as the therapeutic or prophylactic use of the complex for
treating or preventing bleeding events.
Inventors: |
Metzner; Hubert; (Marburg,
DE) ; Schulte; Stefan; (Marburg, DE) ; Weimer;
Thomas; (Gladenbach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSL LIMITED |
Parkville |
|
AU |
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|
Family ID: |
48143163 |
Appl. No.: |
15/837471 |
Filed: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14785776 |
Oct 20, 2015 |
9878017 |
|
|
PCT/EP2014/058093 |
Apr 22, 2014 |
|
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15837471 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/37 20130101;
C07K 2319/31 20130101; C07K 14/755 20130101; C07K 2319/50 20130101;
A61K 47/60 20170801; A61P 7/04 20180101; A61K 47/55 20170801; A61K
47/62 20170801; A61K 47/65 20170801; A61K 38/36 20130101; A61K
47/64 20170801 |
International
Class: |
A61K 38/37 20060101
A61K038/37; C07K 14/755 20060101 C07K014/755; A61K 38/36 20060101
A61K038/36 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2013 |
EP |
13164728.1 |
Claims
1. A complex comprising a von Willebrand factor (VWF) covalently
linked to a Factor VIII (FVIII), wherein the complex comprises a
half-life extending moiety, and wherein the FVIII is modified by
substitution of a naturally occurring amino acid with a cysteine
residue or insertion of a cysteine residue which forms a disulphide
bridge with a cysteine residue in VWF.
2. The complex of claim 1, wherein the covalent link is not
provided by the half-life extending moiety.
3.-4. (canceled)
5. The complex of claim 1, wherein the naturally occurring amino
acid in FVIII that is substituted is selected from an amino acid in
the FVIII a3 domain.
6. The complex of claim 1, wherein the naturally occurring amino
acid in FVIII that is substituted is located within amino acids
1653 to 1660 or within amino acids 1667 to 1674 or within amino
acids 1675 to 1688 of the FVIII a3 domain, or the inserted cysteine
is introduced into the sequence of amino acids 1653 to 1660 or
amino acids 1667 to 1674 or amino acids 1675 to 1688 of the FVIII
a3 domain.
7. The complex of claim 1, wherein the naturally occurring amino
acid that is substituted in FVIII is in the C-terminal domain.
8. The complex of claim 1, wherein the VWF is modified by
substitution of a naturally occurring amino acid with a cysteine
residue or insertion of a cysteine residue which forms the
disulphide bridge with the substituted cysteine residue in the
FVIII or the cysteine residue inserted into the FVIII.
9. The complex of claim 8, wherein the naturally occurring amino
acid in VWF is an amino acid in the D' or D3 domain, or wherein the
inserted cysteine residue in the VWF is in the D' or D3 domain.
10. The complex of claim 8, wherein the inserted cysteine residue
in the VWF is introduced in the TIL' domain, the E' domain, the D3
domain, the C8-3 domain, the TIL-3 domain or the E-3 domain of VWF,
or the naturally occurring amino acid in the VWF is in the TIL'
domain, the E' domain, the D3 domain, the C8-3 domain, the TIL-3
domain or the E-3 domain of VWF.
11. The complex of claim 1, wherein the VWF comprises a FVIII
binding domain.
12. The complex of claim 1, wherein the FVIII is modified to
comprise one or more domains of VWF.
13. The complex of claim 12, wherein the FVIII is modified to
comprise the C-terminal domain CK of VWF.
14. The complex of claim 13, wherein the FVIII is modified to
further comprise any one or more of VWF domains C1 to C6, or
variants thereof.
15. The complex of claim 12, wherein the FVIII comprises amino
acids 2724 to 2812 of SEQ ID NO: 2 or a variant thereof, provided
that cysteine residue 2773 (or equivalent thereof) is preserved in
the variant.
16. The complex of claim 13, wherein the C-terminal VWF domain is
attached to FVIII by a cleavable linker.
17. The complex of claim 16, wherein the cleavable linker comprises
a thrombin cleavage site of FVIII.
18. The complex of claim 16, wherein the linker sequence comprises
additional amino acids providing a peptide of sufficient length to
permit an intramolecular interaction of FVIII and VWF via the a3
and D'D3 domains, respectively.
19. The complex of claim 12, wherein the FVIII is modified at its
C-terminus or at its N-terminus, or within the B-domain of FVIII,
or partially or completely replacing the B-domain of FVIII.
20. The complex of claim 12, wherein the FVIII is modified to
comprise the D'D3 domain of VWF or a fragment or a variant
thereof.
21. The complex of claim 20, wherein the FVIII is modified by
replacing its B domain partially or completely by the VWF D'D3
domain or a fragment or a variant thereof.
22. The complex of claim 21, wherein the FVIII comprises within its
B domain or instead of its B domain or instead of part of its B
domain amino acids 764 to 1241 of SEQ ID NO: 2.
23. The complex of claim 19, wherein the FVIII is expressed as a
two-chain molecule with the VWF D'D3 domain representing the
N-terminus of the FVIII light chain.
24. The complex of claim 19, wherein an additional linker sequence
is introduced between the D'D3 region of VWF and the FVIII light
chain domains.
25. The complex of claim 1, wherein the FVIII is a genetically
engineered FVIII.
26. The complex of claim 25, wherein the engineered FVIII has a
full or partial B-domain deletion, or is a mutated FVIII comprising
one or more amino acid substitutions, insertions, deletions or
combinations thereof, or is a fusion polypeptide with a half-life
extending moiety.
27. The complex of claim 1, wherein the VWF is a half-life extended
form of VWF.
28. The complex of claim 27, wherein the half-life extended form of
VWF is a genetically engineered fusion protein of VWF with a
half-life extending moiety.
29. The complex of claim 27, wherein the half-life extending moiety
is selected from albumin or a variant or fragment thereof, an
immunoglobulin constant region or a portion or variant thereof, an
Fc fragment or variant thereof, a solvated random chain with large
hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein
or a variant thereof, Vitamin D binding protein or a variant
thereof, transferrin or a variant thereof, a carboxyl-terminal
peptide (CTP) of human chorionic gonadotropin-.beta. subunit, and a
polypeptide or lipid capable of binding under physiological
conditions to albumin or an immunoglobulin constant region.
30. The complex of claim 1, wherein the plasma half-life of VWF is
extended by one or more chemical modifications selected from
polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl
starch (HESylation), polysialic acids, heparosan polymers,
elastin-like polypeptides, and hyaluronic acid.
31. The complex of claim 1, wherein the VWF is expressed as a
monomer or a dimer.
32. The complex of claim 1, wherein the VWF forms a multimer.
33. The complex of claim 1, wherein the covalent link is obtained
by one or more chemically synthesized cross-linkers.
34. The complex of claim 1, wherein the FVIII and VWF are connected
by more than one covalent disulfide bond or peptide or
proteinaceous linker.
35. A method of producing the complex of claim 1, comprising
co-expressing the FVIII and VWF in a eukaryotic cell line.
36. A method for treatment of hemophilia A or von Willebrand
disease, comprising administering to a subject in need thereof an
effective amount of the complex of claim 1.
37. (canceled)
38. A pharmaceutical composition comprising the complex of claim
1.
39. The complex of claim 1, wherein the FVIII is modified by
substitution of a naturally occurring amino acid with a cysteine
residue.
40. The complex of claim 1, wherein the FVIII is modified by
insertion of a cysteine residue.
41. A method for reducing or preventing bleeding events in a
subject suffering from hemophilia A or von Willebrand disease,
comprising administering to the subject an effective amount of the
complex of claim 1.
42. The method of claim 41, wherein the VWF is modified by
substitution of a naturally occurring amino acid with a cysteine
residue or insertion of a cysteine residue which forms the
disulphide bridge with the substituted cysteine residue in the
FVIII or the cysteine residue inserted into the FVIII.
43. The method of claim 41, wherein the VWF comprises a FVIII
binding domain.
44. The method of claim 41, wherein the FVIII is modified to
comprise one or more domains of VWF.
45. The method of claim 41, wherein the FVIII has a full or partial
B-domain deletion, or is a mutated FVIII comprising one or more
amino acid substitutions, insertions, deletions or combinations
thereof, or is a fusion polypeptide with a half-life extending
moiety.
46. The complex of claim 45, wherein the half-life extending moiety
is selected from albumin or a variant or fragment thereof, an
immunoglobulin constant region or a portion or variant thereof, an
Fc fragment or variant thereof, a solvated random chain with large
hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein
or a variant thereof, Vitamin D binding protein or a variant
thereof, transferrin or a variant thereof, a carboxyl-terminal
peptide (CTP) of human chorionic gonadotropin-.beta. subunit, and a
polypeptide or lipid capable of binding under physiological
conditions to albumin or an immunoglobulin constant region.
47. The method of claim 41, wherein the VWF is fused to a half-life
extending moiety.
48. The method of claim 47, wherein the half-life extending moiety
is selected from albumin or a variant or fragment thereof, an
immunoglobulin constant region or a portion or variant thereof, an
Fc fragment or variant thereof, a solvated random chain with large
hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein
or a variant thereof, Vitamin D binding protein or a variant
thereof, transferrin or a variant thereof, a carboxyl-terminal
peptide (CTP) of human chorionic gonadotropin-.beta. subunit, and a
polypeptide or lipid capable of binding under physiological
conditions to albumin or an immunoglobulin constant region.
49. The method of claim 41, wherein the VWF is expressed as a
monomer, a dimer, or a multimer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/785,776, filed Oct. 20, 2015, which is the
United States national stage entry under 35 U.S.C. .sctn. 371 of
International Application No. PCT/EP2014/058093, filed on Apr. 22,
2014 and published as WO 2014/173873 A1, which claims priority to
European Patent Application No. 13164728.1, filed on Apr. 22, 2013.
The contents of these applications are each incorporated herein by
reference in their entirety.
[0002] The present invention relates to a covalent complex of von
Willebrand Factor or variants thereof (VWF) and Factor VIII or
variants thereof (Factor VIII), wherein the complex is modified
such that it has an extended half-life in vivo. The invention
further relates to a method of producing the complex, as well as
the therapeutic or prophylactic use of the complex for treating or
preventing bleeding events.
[0003] There are various bleeding disorders caused by deficiencies
of blood coagulation factors. The most common disorders are
hemophilia A and B, resulting from deficiencies of blood
coagulation factor VIII and IX, respectively. Another known
bleeding disorder is von Willebrand disease.
[0004] In plasma factor VIII (FVIII) exists mostly as a noncovalent
complex with von Willebrand Factor. Mature FVIII, a polypeptide of
up to 2332 amino acids after pro-peptide cleavage, is composed of
several domains as depicted in FIGS. 1A-1C. FVIII's function in
coagulation is to accelerate factor IXa-dependent conversion of
factor X to Xa. Due to the complex formation of FVIII and von
Willebrand Factor it was assumed for a long time that FVIII and von
Willebrand Factor functions are two functions of the same molecule.
Only in the seventies it became clear that FVIII and von Willebrand
Factor are separate molecules that form a complex under physiologic
conditions. In the eighties, a dissociation constant of FVIII and
von Willebrand Factor of about 0.2 nmol/L was determined (Leyte et
al., Biochem J 1989, 257: 679-683) and the DNA sequence of both
molecules was determined.
[0005] Classic hemophilia or hemophilia A is an inherited bleeding
disorder. It results from a chromosome X-linked deficiency of blood
coagulation FVIII, and affects almost exclusively males with an
incidence of between one and two individuals per 10,000. The
X-chromosome defect is transmitted by female carriers who are not
themselves hemophiliacs. The clinical manifestation of hemophilia A
is an increased bleeding tendency. Prior to the introduction of
treatment with FVIII concentrates, the mean life span for a person
with severe hemophilia was less than 20 years. The use of
concentrates of FVIII from plasma has considerably improved the
situation for the hemophilia A patients, increasing the mean life
span extensively, giving most of them the possibility to live a
more or less normal life. However, there have been certain problems
with the plasma derived concentrates and their use, the most
serious of which have been the transmission of viruses such as
viruses causing hepatitis B, non-A non-B hepatitis and HIV.
However, different virus inactivation methods and new highly
purified FVIII concentrates have recently been developed which
established a very high safety standard for plasma-derived
FVIII.
[0006] In severe hemophilia A patients undergoing prophylactic
treatment FVIII has to be administered intravenously (i.v.) about 3
times per week due to the short plasma half-life of FVIII of about
12 hours. Each i.v. administration is cumbersome, associated with
pain, and entails the risk of an infection, especially as this is
mostly done at home by the patients themselves or by the parents of
children being diagnosed with hemophilia A.
[0007] It would thus be highly desirable to create a FVIII with
increased functional half-life allowing the manufacturing of
pharmaceutical compositions containing FVIII, which have to be
administered less frequently.
[0008] Several attempts have been made to prolong the functional
half-life of FVIII either by reducing its interaction with cellular
receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching
polymers to FVIII (WO 94/15625, WO 97/11957 and U.S. Pat. No.
4,970,300), by encapsulation of FVIII (WO 99/55306), by the
introduction of novel metal binding sites (WO 97/03193), by
covalently attaching the A2 domain to the A3 domain either by
peptidic (WO 97/40145 and WO 03/087355) or disulfide linkage (WO
02/103024A2) or by introducing mutations that prevent thrombin
cleavage between the A1 and A2 domains and therefore keep the A1
domain covalently attached to the A2 domain after thrombin
activation (WO2006/108590).
[0009] Another approach to enhance the functional half-life of
FVIII or von Willebrand Factor is by PEGylation of FVIII (WO
2007/126808, WO 2006/053299, WO 2004/075923) or by PEGylation of
von Willebrand Factor (WO 2006/071801), with the idea that
pegylated von Willebrand Factor, by having an increased half-life,
would indirectly also enhance the half-life of FVIII present in
plasma. In addition fusion proteins of FVIII with half-life
enhancing polypeptides like albumin or the constant region Fc of
immunoglobulins have been described (WO 2004/101740, WO2008/077616
and WO 2009/156137).
[0010] Von Willebrand Factor, which is missing, functionally defect
or only available in reduced quantity in different forms of von
Willebrand disease (VWD), is a multimeric adhesive glycoprotein
present in the plasma of mammals, which has multiple physiological
functions. During primary hemostasis von Willebrand Factor acts as
a mediator between specific receptors on the platelet surface and
components of the extracellular matrix such as collagen. Moreover,
von Willebrand Factor serves as a carrier and stabilizing protein
for procoagulant FVIII. Von Willebrand Factor is synthesized in
endothelial cells and megakaryocytes as a 2813 amino acid precursor
molecule. The amino acid sequence and the cDNA sequence of
wild-type VWF are disclosed in Collins et al. 1987, Proc Natl.
Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-von
Willebrand Factor, consists of a 22-residue signal peptide, a
741-residue pro-peptide and the 2050-residue polypeptide found in
mature plasma von Willebrand Factor (Fischer et al., FEBS Lett.
351: 345-348, 1994), see also FIG. 2A for pro-von Willebrand Factor
and FIG. 2B for mature von Willebrand Factor monomer unit. After
cleavage of the signal peptide in the endoplasmatic reticulum a
C-terminal disulfide bridge is formed between two monomers of von
Willebrand Factor. During further transport through the secretory
pathway 12 N-linked and 10 O-linked carbohydrate side chains are
added. More importantly, von Willebrand Factor dimers are
multimerized via N-terminal disulfide bridges and the propeptide of
741 amino acids length is cleaved off by the enzyme PACE/furin in
the late Golgi apparatus. The propeptide as well as the
high-molecular-weight multimers of von Willebrand Factor (VWF-HMWM)
are stored in the Weibel-Pallade bodies of endothelial cells or in
the .alpha.-Granules of platelets.
[0011] Once secreted into plasma the protease ADAMTS13 cleaves
ultra-large von Willebrand Factor multimers within the A2 domain of
von Willebrand Factor. Plasma von Willebrand Factor consists of a
whole range of multimers ranging from single dimers of approx. 500
kDa to multimers consisting of up to or even more than 20 dimers of
a molecular weight of over 10,000 kDa. The VWF-HMWM have the
strongest hemostatic activity, which can be measured by a
ristocetin cofactor activity assay (VWF:RCo). The higher the ratio
of VWF:RCo/von Willebrand Factor antigen, the higher the relative
amount of high molecular weight multimers.
[0012] Defects in von Willebrand Factor are the cause of von
Willebrand disease (VWD), which is characterized by a more or less
pronounced bleeding phenotype. VWD type 3 is the most severe form
in which von Willebrand Factor is essentially completely missing,
VWD type 1 relates to a reduced level of von Willebrand Factor and
its phenotype can be very mild. VWD type 2 relates to qualitative
defects of von Willebrand Factor and can be as severe as VWD type
3. VWD type 2 has many sub-forms, some of them being associated
with the loss or the decrease of high molecular weight multimers.
VWD type 2A is characterized by a loss of both intermediate and
large multimers, and is therefore characterised by qualitatively
defective VWF with a decreased ability to bind platelet
glycoprotein 1 receptor. VWD type 2B is characterized by a loss of
highest-molecular-weight multimers. The ability of the
qualitatively defective VWF to bind to glycoprotein 1 receptor on
the platelet membrane is abnormally enhanced, leading to its
spontaneous binding to platelets and subsequent clearance of the
bound platelets and of the large von Willebrand Factor multimers.
VWD type 2M is also a qualitative defect in von Willebrand Factor
characterized by its decreased ability to bind to glycoprotein 1
receptor on the platelet membrane, but a normal multimer
distribution, as are von Willebrand Factor antigen levels. VWD type
2N (Normandy) is a qualitative defect in von Willebrand Factor,
where there is a deficiency of von Willebrand Factor binding to
coagulation factor FVIII. Although the quantity of von Willebrand
Factor and von Willebrand Factor multimers is normal, patients show
a decreased level in FVIII, leading to a similar phenotype as
haemophilia A.
[0013] VWD is the most frequent inherited bleeding disorder in
humans and can be--depending on the type of VWD--treated by therapy
with 1-Desamino-8-D-Arginin-Vasopressin (DDAVP) to release von
Willebrand Factor from intra-cellular storage pools or by
replacement therapy with concentrates containing von Willebrand
Factor of plasmatic or recombinant origin. Von Willebrand Factor
can be prepared from human plasma as for example described in EP
0503991. EP 0784632 describes a method for isolating recombinant
von Willebrand Factor.
[0014] In plasma FVIII binds with high affinity to von Willebrand
Factor, which protects it from premature catabolism and thus plays,
in addition to its role in primary hemostasis, a crucial role in
the regulation of plasma levels of FVIII. As a consequence von
Willebrand Factor is also a central factor in the control of
secondary hemostasis. The half-life of non-activated FVIII bound to
von Willebrand Factor in plasma is about 12 hours. In VWD type 3,
where no or almost no von Willebrand Factor is present, the
half-life of FVIII is only about 2 hours, leading to symptoms of
mild to moderate hemophilia A in such patients due to decreased
concentrations of FVIII.
[0015] The stabilizing effect of von Willebrand Factor on FVIII has
also been used to aid recombinant expression of FVIII in CHO cells
(Kaufman et al. 1989, Mol Cell Biol). Other recent attempts to use
von Willebrand Factor for stabilizing FVIII have been disclosed in
several recent patent applications (WO2011060242, WO2013083858,
WO2013106787, WO2014011819).
[0016] There is still a need for further, better approaches to
increase the half-life of FVIII. It has been found by the inventors
of this application that a covalent attachment of a FVIII molecule
to a half-life extended von Willebrand Factor molecule will provide
a half-life extension to the FVIII moiety such that its half-life
will be similar to that of the unfused half-life extended von
Willebrand Factor molecule. With this method an about 3-fold
half-life extension was seen in a rat PK model over free FVIII. The
present invention provides a covalent complex of von Willebrand
Factor or variants thereof (VWF) and Factor VIII, in particular
using methods to increase the half-life of the VWF-component in the
complex, which allows the provision of stable complexes having a
prolonged half-life which are advantageous in therapy and
prophylaxis of bleeding disorders.
SUMMARY OF THE INVENTION
[0017] In a first aspect the present invention relates to a
covalent complex comprising von Willebrand factor or variants
thereof (VWF) and Factor VIII (FVIII) or variants thereof (Factor
VIII), wherein the complex is modified such that it has an extended
half-life in vivo. Preferably it is modified to comprise a
half-life extending moiety. The VWF and the Factor VIII form a
covalent complex; attached to any part of this complex, preferably
to the VWF moiety, is a half-life extending moiety. Preferably, the
VWF and the Factor VIII are linked by a direct covalent bond, e.g.
via a disulphide bridge of a cysteine that is part of the VWF with
a cysteine that is part of the Factor VIII, or by fusing VWF with
Factor VIII, optionally via a peptide linker, with the proviso that
the covalent complex is not an Fc fusion protein, where one of the
Fc chains is fused to VWF and the other Fc chain is fused to FVIII
or variants thereof. Preferably, the covalent link is not provided
by the half-life extending moiety.
[0018] In a first embodiment, Factor VIII is modified so that it
forms a disulphide bridge with VWF (with the proviso that the
disulphide bridge is not between the two chains of an Fc molecule
that are fused to VWF and Factor VIII respectively). Preferably,
Factor VIII is modified by substitution of a naturally occurring
amino acid with a cysteine residue or by insertion of a cysteine
residue that forms a disulphide bridge with a cysteine residue in
VWF. Preferably, the naturally occurring amino acid that is
substituted in Factor VIII is selected from an amino acid in the a3
domain, or a cysteine residue is inserted into the a3 domain
(residues 1649 to 1689 of SEQ ID No. 6). More preferably, the
naturally occurring amino acid is an acidic residue, preferably a
conserved acidic residue, or a residue involved in a hemophilic
phenotype, or a Tyr residue which may be sulphated in the FVIII a3
domain. More preferably, the naturally occurring amino acid that is
substituted in Factor VIII is located within amino acids 1653 to
1660 or within amino acids 1667 to 1674 or within amino acids 1675
to 1688 of the Factor VIII a3 domain or a cysteine is introduced
into the sequence of amino acids 1653 to 1660 or amino acids 1667
to 1674 or amino acids 1675 to 1688 of the Factor VIII a3 domain.
Even more preferably, the naturally occurring amino acid in the
Factor VIII a3 domain that is substituted with cysteine is selected
from T1653, L1655, D1658, E1660, S1669, V1670, N1672, K1673, K1674,
E1675, D1676 and/or N1685,in SEQ ID NO: 6 or equivalent position in
a genetically engineered form of Factor VIII. Most preferably, the
naturally occurring amino acid in the a3 domain that is substituted
with cysteine is selected from T1654, Q1656, F1677, D1678, I1679,
Y1680, D1681, E1682, D1683, E1684, Q1686, S1687 and/or P1688 in SEQ
ID NO: 6 or equivalent position in a genetically engineered form of
Factor VIII.
[0019] In another embodiment, a cysteine residue is inserted in the
C-terminal domain, or the naturally occurring amino acid that is
substituted with cysteine is in the C-terminal domain of Factor
VIII, preferably the residue is selected from I2098, S2119, N2129,
R2150, P2153, W2229, Q2246 in SEQ ID NO: 6 or equivalent position
in an engineered form of Factor VIII.
[0020] In a further, preferred embodiment of the first aspect of
the invention, VWF is also modified by substitution of a naturally
occurring amino acid with a cysteine residue or the insertion of a
cysteine residue which forms a disulphide bridge with a cysteine
residue introduced into Factor VIII. Preferably, a cysteine residue
is inserted into the D' or D3 domain (see FIGS. 2A and 2B), or the
naturally occurring amino acid in VWF that is substituted with a
cysteine residue is a residue in the D' or D3 domain or a basic or
a highly conserved residue in the D' or D3 domain or a residue
involved in type N-VWD or an amino acid exposed on the surface of
the VWF molecule. In a preferred embodiment of the invention a
cysteine residue is inserted into the TIL' domain, the E' domain,
the VWD3 domain, the C8-3 domain, the TIL-3 domain or the E-3
domain or the naturally occurring amino acid in VWF that is
substituted with a cysteine residue is a residue in the TIL'
domain, the E' domain, the VWD3 domain, the C8-3 domain, the TIL-3
domain or the E-3 domain (all as defined by Zhou et al (2012) Blood
120 (2), 449-458). For example, the naturally occurring amino acid
in VWF is selected from K773, G785, E787, A/T789, K790, T791, Q793,
N794, M800, R820, R826, F830, H831, K834, E835, P838, K843, R852,
R854, K855, W856, H861, H874, K882, L884, R906, K912, H916, K920,
K923, R924, K940, R945, K948, H952, R960, K968, R976, H977, K985,
K991, K1026, R1035, K1036, K1052, Q1053, K1073 or H1074.
Preferably, the naturally occurring amino acid in VWF is selected
from Y795, R816, H817, P828, D853, D879, K922, D951, E1078, E1161,
and/or R1204 in SEQ ID NO: 2 or equivalent position in an
engineered form of VWF. More preferably, the naturally occurring
amino acid in VWF is selected from R768, R782, H817, D853, E933,
L984, E1015, D1076, E1078, P1079, K1116 and/or N1134 in SEQ ID NO:
2 or equivalent position, e.g. in an engineered form of VWF.
[0021] More preferably, one or more of the following combinations
of substitutions of naturally occurring amino acid residues in VWF
and FVIII are introduced:
A/T789C:D1658C, M800C:D1658C, P828C:D1658C, F830C:D1658C,
P838C:D1658C, D853C:D1658C, R924C:D1658C, E1078C:D1658C,
F830C:D1663C, P838C:D1663C, D853C:D1663C, E1078C:D1663C,
E1078C:Y1664C, P838C:D1665C, R816C:D1666C, F830C:D1666C,
E835C:D1666C, T791C:E1671C, F830C:E1671C, E835C:E1671C,
D879C:E1671C, A/T789C:E1675C, T791C:E1675C, N794C:E1675C,
P828C:E1675C, F830C:E1675C, E835C:E1675C, P838C:E1675C,
D879CE1675C, R924C:E1675C, E1078C:E1675C, A/T789C:D1676C,
T791C:D1676C, N794C:D1676C, F830C:D1676C, E835C:D1676C,
A/T789C:D1678C, F830C:D1678C, E835C:D1678C, A/T789C:I1679C,
M800C:I1679C, F830C:I1679C, E835C:I1679C, R854C:I1679C,
D879C:I1679C, A/T789C:Y1680C, T791C:Y1680C, Y795C:Y1680C,
M800C:Y1680C, R816C:Y1680C, F830C:Y1680C, E835C:Y1680C,
R854C:Y1680C, D879C:Y1680C, A/T789C:E1682C, Y795C:E1682C,
R816C:E1682C, P828C:E1682C, E835C:E1682C, P838C:E1682C,
R854C:E1682C, D879C:E1682C, Q1053C:E1682C.
[0022] Even more preferably, one or more of the following
combinations of substitutions of naturally occurring amino acid
residues in VWF and FVIII are introduced:
F1677C:R768C, I1679C:R768C, Y1680C:R768C, N1685C:R768C,
T1654C:R782C, E1675C:R782C, N1685C:R782C, Q1686C:Y795C,
S1687C:Y795C, P1688C:Y795C, P1688C:Y795C, E1675C:H816C,
D1676C:R816C, Y1680C:R816C, E1682C:R816C, P1688C:R816C,
Y1680C:H817C, N1685C:H817C, Q1686C:H817C, S1687C:H817C,
I1679C:P828C, Y1680C:D853C, N1685C:D853C, T1654C:D879C,
P1688C:E933C, P1688:T951C, T1653C:L984C, T1654C:L984C,
L1655C:L984C, S1669C:L984C, K1673C:L984C, D1683C:L984C,
T1653C:E1015C, L1655C:E1015C, S1669C:E1015C, V1670C:E1015C,
N1672C:E1015C, K1673C:E1015C, D1678C:E1015C, I1679C:E1015C,
E1684C:E1015C, S1687C:E1015C, F1677C:V1027C, I1679C:V1027C,
P1688C:V1027C, S1657C:D1076C, K1673C:D1076C, D1676C:D1076C,
F1677C:D1076C, I1679C:D1076C, E1682C:D1076C, D1683C:D1076C,
Q1686C:D1076C, D1676C:E1078C, I1679C:E1078C, Y1680C:E1078C,
T1653C:P1079C, L1655C:P1079C, S1657C:P1079C, D1658C:P1079C,
E1682C:P1079C, V1670C:K1116C, K1673C:K1116C, D1676C:K1116C,
D1678C:K1116C, D1681C:K1116C, Q1686C:K1116C, P1688C:K1116C,
T1653C:N1134C, L1655C:N1134C, E1660C:N1134C, D1678C:N1134C,
D1683C:N1134C, E1684C:N1134C, Q1686C:N1134C, T1653C:E1161C,
L1655C:E1161C, K1674C:E1161C, D1676C:E1161C, E1684C:E1161C,
S1687C:E1161C, P1688C:R1204C.
[0023] Most preferably, one or more of the following combinations
of substitutions of naturally occurring amino acid residues in
FVIII and VWF are introduced:
T1654:P1079, T1654:N1134, Q1656:D1076, F1677:K1116, D1678:R782,
1679:K1116, Y1680:H817, Y1680:D853, Y1680:E1078, D1681:R768,
E1682:R768, D1683:R768, E1684:R768, Q1686:R768, Q1686:E1015,
51687:R768, 51687:N1134, P1688:R768, P1688:H817, P1688:E933,
P1688:L984, P1688:E1015, P1688:D1076 and P1688:N1134.
[0024] Preferably, the naturally occurring amino acids of the
combination of one or more inserted cysteine residues in VWF and
Factor VIII are selected by a relative ratio higher than 0.5 of
covalently bound Factor VIII to VWF as experimentally assessed
shown in example 6 and activity of Factor VIII as experimentally
assessed shown in example 9. Most preferably, the naturally
occurring amino acids of the combination of one or more inserted
cysteine residues in VWF and Factor VIII are selected by a said
ratio of higher than 1.0.
[0025] Preferably, the Factor VIII in the complex of the invention
is a genetically engineered Factor VIII. The engineered Factor VIII
may have a partial or complete B-domain deletion, it may be a
mutated Factor VIII comprising one or more amino acid
substitutions, insertions, deletions or combinations thereof, or it
may be a fusion polypeptide with a half-life extending moiety or a
chemically modified Factor VIII e.g. modified by attachment of a
half-life extending moiety such as polyethylene glycol
(PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation),
polysialic acids, elastin-like polypeptides, heparosan polymers or
hyaluronic acid.
[0026] In a preferred embodiment, the VWF in the complex of the
invention is a half-life extended form of VWF, preferably it is a
genetically engineered form of VWF. More preferably, the
genetically engineered VWF is a fusion protein of VWF with a
half-life extending moiety. Preferably, the half-life extending
moiety is a half-life extending polypeptide (HLEP), more preferably
HLEP is selected from albumin or fragments thereof, immunoglobulin
constant region and portions thereof, e.g. the Fc fragment,
solvated random chains with large hydrodynamic volume (e.g. XTEN
(Schellenberger et al. 2009), homo-amino acid repeats (HAP) or
proline-alanine-serine repeats (PAS)), afamin, alpha-fetoprotein,
Vitamin D binding protein, transferrin or variants thereof,
carboxyl-terminal peptide (CTP) of human chorionic
gonadotropin-.beta. subunit, polypeptides or lipids capable of
binding under physiological conditions to albumin or immunoglobulin
constant regions. In another preferred embodiment, the VWF of the
complex is expressed as a dimer. In a further preferred embodiment,
the VWF of the complex forms multimers.
[0027] In another embodiment of the invention, the half-life of the
complex of the invention is extended by chemical modification, e.g.
attachment of a half-life extending moiety such as polyethylene
glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch
(HESylation), polysialic acids, elastin-like polypeptides,
heparosan polymers or hyaluronic acid.
[0028] A second embodiment of the invention is a covalent complex
comprising VWF and Factor VIII, wherein the complex is modified
such that it has an extended half-life in vivo, and wherein Factor
VIII is modified to comprise one or more VWF domains. Preferably,
the extended half-life of the complex is obtained by using a
half-life extended form of VWF in the complex.
[0029] Preferably, the Factor VIII is fused with one or more of the
C-terminal domains of VWF (see FIGS. 4A-4J), preferably the one or
more C-terminal domains of VWF are fused to the C-terminus of
Factor VIII. Such C-terminal domains of VWF comprise the C-terminal
cystine knot (CK) domain of VWF and may additionally comprise,
besides C or CK domains, one or more additional domains of VWF,
e.g. A or D domains. More preferably, the FVIII comprises,
preferably at its C-terminus, residues 2723-2813, 2724-2813,
2722-2813, 2578-2813, 2580-2813, 2497-2813, 2429-2813, 2400-2813,
2334-2813, 2255-2813, 1873-2813, 1683-2813, 1277-2813, 1264-2813 or
764-2813 of SEQ ID NO: 2 or variants thereof, provided that
cysteine residue 2773 (or equivalent thereof) is preserved.
[0030] Preferably, the C-terminal CK domain of VWF, optionally
comprising further VWF domains as disclosed above, is attached to
FVIII by a cleavable linker. More preferably, the cleavable linker
comprises a cleavage site cleavable by proteases related to blood
coagulation, even more preferably, the cleavable linker comprises a
thrombin cleavage site, preferably one of the thrombin cleavage
sites of FVIII. Preferably, the linker sequence also comprises
additional amino acid residues, preferably the additional amino
acid residues are inserted between the C-terminal domain(s) of VWF
and the cleavable part of the linker. Preferably, the additional
amino acid residues provide a peptide of sufficient length to
permit the interaction of FVIII and VWF via the a3 region of FVIII
and the D'D3 regions of VWF, respectively. The additional amino
acid residues may be more than 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 120 or 150 amino acids. Preferably the additional amino acid
residues form a flexible, "non-structural" peptide, and more
preferably comprise or even consist of glycine-serine repeats,
proline-alanine-serine repeats, homo-amino acid repeats, or
sequences of the FVIII B-domain.
[0031] In another embodiment, the Factor VIII is N-terminally fused
with one or more of the C-terminal domains of VWF. Such C-terminal
domains may be derived from the C-terminal cystine knot (CK) domain
of VWF and may additionally comprise one or more further domains of
VWF. More preferably, the Factor VIII comprises, preferably at its
N-terminus, residues 2723-2813, 2724-2813, 2722-2813, 2580-2813,
2578-2813, 2497-2813, 2429-2813, 2400-2813, 2334-2813, 2255-2813,
1873-2813, 1683-2813, 1277-2813, 1264-2813 or 764-2813 of SEQ ID
NO: 2 or variants thereof, provided that cysteine residue 2773 (or
equivalent thereof) is preserved. For these embodiments, the
expression product would comprise, from N- to C-terminus, a signal
peptide, the CK domain of VWF, optionally with additional domains
of VWF, preferably a cleavable (optionally flexible) linker, and
Factor VIII.
[0032] Another embodiment of the invention is a covalent complex
comprising VWF and Factor VIII, wherein the VWF is a half-life
extended form of VWF, and wherein Factor VIII is modified to
comprise the D'D3 or D1D2D'D3 region of VWF, or fragments thereof
which maintain at least 10% of the FVIII binding activity of
wild-type von Willebrand Factor. Preferably, the Factor VIII is so
modified that its partial or complete B-domain is replaced by the
VWF D'D3 region or fragments thereof (see FIGS. 5A-5E). More
preferably, the Factor VIII comprises, preferably instead of its
(or part of its) B domain, residues 764 to 1241, 764 to 1242, 764
to 1247, 764 to 1270 or any sequence between 764 and 1241 to 1270,
respectively, of SEQ ID NO: 2 or a variant or a fragment
thereof.
[0033] In a preferred embodiment, the D'D3 domain of VWF is
attached to Factor VIII such that a two-chain molecule is generated
upon secretion of the molecule into the cell culture medium and
that the D'D3 domain is located at the N-terminus of the Factor
VIII light chain. This can be achieved by introducing a cleavable
linker comprising, for example, a cleavage site for PACE/furin
between the Factor VIII a2 domain or the remainders of the B domain
and the VWF D'D3 domain (FIGS. 5D and 5E). Preferably, the linker
comprises additional residues between the D'D3 domain of VWF and
the Factor VIII a3 domain, the additional residues comprising a
peptide of sufficient length to permit the intramolecular
interaction of Factor VIII and VWF via the a3 and D'D3 domains,
respectively (FIG. 5E). Preferably, the additional residues
comprise less than 200 amino acids, more preferably less than 100
amino acids, even more preferably less than 90, 80, 70, 60, 50
amino acids, less than 40 amino acids, less than 30 amino acids,
less than 20 amino acids, most preferably less than 10 amino acids.
Preferably the additional residues comprise a flexible,
"non-structural" peptide, more preferably they comprise or even
consist of glycine-serine repeats, proline-alanine-serine repeats
or homo-amino acid repeats. The protein to be expressed to obtain
the mature form as described may be constructed to include
additional sequences, e.g. a signal sequence at the N-terminus.
[0034] Alternatively, the N-terminus of Factor VIII is connected to
the C-terminus of the VWF D'D3 domains or fragments thereof, which
is preferably N-terminally extended by further domains of VWF (e.g.
the D1 and D2 domains); this will aid in the expression and
intracellular formation of covalent bonds with half-life extended
VWF. More preferably, the Factor VIII comprises N-terminally
residues 1 to 1241 or residues 764 to 1241 (after cleavage of the
propeptide) of SEQ ID NO: 2 or a variant or a fragment thereof.
[0035] Preferably, the D'D3 or D1D2D'D3 domains of VWF,
respectively, are attached to the N-terminus of Factor VIII by a
cleavable linker. More preferably, the cleavable linker comprises a
cleavage site cleavable by a protease related to blood coagulation,
even more preferably, the cleavable linker comprises a thrombin
cleavage site, preferably one of the thrombin cleavage sites of
FVIII. Preferably, the linker comprises additional residues between
the D'D3 or D1D2D'D3 domains of VWF and the Factor VIII molecule,
the additional residues comprising a peptide of sufficient length
to permit the interaction of Factor VIII and VWF via the a3 and
D'D3 regions, respectively (FIG. 5E). Preferably, more than 20, 30,
50, 100, or 150 additional amino acids are added. Preferably the
additional amino acids comprise a flexible, non-structural peptide,
more preferably they comprise or even consist of glycine-serine
repeats, proline-alanine-serine repeats, homo-amino acid repeats,
or sequences of the FVIII B-domain.
[0036] Furthermore, the D'D3 or D1D2D'D3 domains of VWF fused to
the N-terminus of FVIII via a linker as described above may be
extended between D3 and the described linker by additional VWF
domains derived from VWF should further VWF-related functionalities
be incorporated into the construct.
[0037] A second aspect of the invention is a method of producing
the covalent complex of Factor VIII and VWF described above,
comprising co-expressing the Factor VIII and VWF molecules in a
eukaryotic cell line. Preferably, the eukaryotic cell line is
modified to express PACE/furin to ensure efficient processing.
Alternatively, the proteins (Factor VIII and VWF) may be produced
separately and then combined in vitro, e.g. in a moderately
oxidizing environment to enable disulphide bridge formation, but
leaving intact the functionalities of Factor VIII and VWF.
[0038] In another embodiment of this aspect of the invention the
(modified) Factor VIII and the (modified) VWF are covalently
connected by chemical crosslinking (see FIG. 7).
[0039] A third aspect of the invention is a covalent complex as
described above for use in medicine, preferably for use in the
treatment or prophylaxis of a bleeding disorder. Preferably, the
bleeding disorder is hemophilia A or VWD.
[0040] A fourth aspect of the invention is a pharmaceutical
composition comprising the covalent complex described above.
[0041] A further aspect of the invention is a method of treating or
preventing a bleeding disorder by administering an effective amount
of a complex described above to a subject in need thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0042] As mentioned above, it would be highly beneficial to have a
FVIII with a long half-life for the chronic treatment of patients
with haemophilia, in particular with haemophilia A. The inventors
have now surprisingly found that a covalent complex of von
Willebrand factor (VWF) and FVIII can be produced, which provides a
longer half-life for FVIII. In particular when the complex is
modified to extend its half-life in vivo, e.g. when a half-life
extended form of VWF or of FVIII is used, the half-life of FVIII is
significantly enhanced, making this an attractive approach for an
improved prophylaxis and treatment of patients with bleeding
disorders such as haemophilia A. Preferably, in vivo recovery may
also be increased by this approach.
[0043] Therefore, in a first aspect the present invention relates
to a covalent complex comprising von Willebrand factor or variants
thereof (VWF) and Factor VIII or variants thereof (Factor VIII),
wherein the complex is modified such that it has an extended
half-life in vivo. For example, VWF may be a half-life-extended
form of VWF; alternatively (or additionally) Factor VIII may be a
half-life extended form of FVIII, or a half-life-extending moiety
may be attached to the covalent complex via a linker. Preferably,
the VWF in the complex comprises a half-life extending moiety.
Preferably, the covalent complex is not a heterodimeric Fc fusion
with one Fc monomer linked to VWF and the other Fc monomer linked
to Factor VIII. More preferably, the covalent link is not provided
by the half-life extending moiety.
[0044] The term "von Willebrand Factor" or "VWF", as used herein,
refers to any polypeptide having a biological activity of wild type
VWF, including variants such as VWF with one or more amino acid
substitutions, insertions, minor or major deletions (e.g. deletions
of one or more domains), or fusion proteins thereof with another
peptide or protein moiety, e.g. a half-life increasing polypeptide,
or non-protein moiety, as long as at least a partial activity of
von Willebrand Factor is retained. VWF activity may be collagen
binding activity, and/or platelet binding activity, and/or FVIII
binding activity. FVIII binding activity would be determined for
the VWF without FVIII covalently bound via the binding sites on
VWF. Assays to measure VWF activity are well established, for
example collagen binding assays, Ristocetin cofactor activity
assays, or FVIII binding assays. The biological activity is
retained in the sense of the invention if the VWF with deletions
and/or other modifications retains at least 10%, preferably 15%,
20%, 25%, or 30%, more preferably at least 40% or 50%, even more
preferably at least 60%, 70% or 75% of any activity measured for
the wild-type VWF. The term "Factor VIII binding domain" refers to
a fragment or portion of VWF that retains at least 10%, preferably
15%, 20%, 25%, or 30%, more preferably at least 40% or 50%, even
more preferably at least 60%, 70% or 75% of the Factor VIII binding
activity of wild type von Willebrand Factor. A Factor VIII binding
domain is located at the N-terminus of the mature VWF, for example
in the D'D3 domain or fragments thereof.
[0045] The gene encoding wild type von Willebrand Factor is
transcribed into a 9 kb mRNA which is translated into a
pre-propolypeptide of 2813 amino acids with an estimated molecular
weight of 310,000 Da. The pre-propolypeptide contains a 22 amino
acid long signal peptide, a 741 amino acid pro-polypeptide and the
mature subunit. Cleavage of the 741 amino acid long propolypeptide
from the N-terminus results in mature VWF consisting of 2050 amino
acids. The amino acid sequence of the VWF pre-propolypeptide is
shown in SEQ ID NO: 2, and several variants are published, for
example NCBI reference sequence NP_000543.2. Unless indicated
otherwise, the amino acid numbering of VWF residues in this
application refers to SEQ ID NO:2, even though the VWF molecule
does not need to comprise all residues of SEQ ID NO:2. The amino
acid sequence of mature wildtype VWF corresponds to residues 764 to
2813 of SEQ ID NO: 2. The term "VWF" as used herein refers to any
form of VWF or variant thereof that shows at least a partial VWF
activity of any one VWF function mentioned above.
[0046] The propolypeptide of wild type VWF comprises multiple
domains which are arranged in the following order (domain structure
of pro-VWF domains D1 to D4 according to Schneppenheim and Budde
(2011) J Thrombosis Haemostasis 9 (Suppl. 1) 209-215, domain
structure and nomenclature of C-domains according to Zhou et al
(2012) Blood 120, 449-458): [0047]
D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK
[0048] The D1 and D2 domains represent the propeptide which is
cleaved off to yield the mature VWF. The D' domain encompasses
amino acids 764 to 865 of SEQ ID NO:2; the D'D3 domain encompasses
amino acids 764 to 1241, 764 to 1242, 764 to 1247, or 764 to 1270,
or any sequence between 764 and 1241 to 1270, respectively, of SEQ
ID NO: 2. The carboxy-terminal 90 residues comprise the "CK" domain
that is homologous to the "cystine knot" superfamily of proteins.
These family members have a tendency to dimerize through disulfide
bonds. The C-terminal domains C1 to C6 as defined by Zhou et al
correspond to residues 2255 to 2333 (C1), 2334 to about 2402 (C2),
2429 to 2496 (C3), 2497 to 2577 (C4), 2578 to 2646 (C5), and 2647
to 2722 (C6) in SEQ ID NO: 2.
[0049] Wild type von Willebrand Factor comprises the amino acid
sequence of mature von Willebrand Factor as shown in SEQ ID NO: 2,
residues 764 to 2813. Also encompassed are additions, insertions,
N-terminal, C-terminal or internal deletions of VWF as long as a
biological activity of VWF is retained. The biological activity is
retained in the sense of the invention if the VWF with deletions
and/or other modifications retains at least 10%, preferably 15%,
20%, 25%, or 30%, more preferably at least 40% or 50%, even more
preferably at least 60%, 70% or 75% of any activity measured for
the wild-type VWF. The biological activity of wild-type VWF can be
determined by the skilled person, for example, using methods for
measuring ristocetin co-factor activity (Federici A B et al. 2004.
Haematologica 89:77-85), binding of VWF to GP Ib.sub..alpha. of the
platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin
Appl Thromb Hemost. 12:305-310), or a collagen binding assay
(Kallas & Talpsep. 2001. Annals of Hematology 80:466-471), or
measuring collagen binding, e.g. by surface plasmon resonance.
Other methods of determining biological activity of VWF that may be
used comprise the determination of FVIII binding capacity
(Veyradier et al., Haemophilia 2011).
[0050] The terms "blood coagulation Factor VIII", "Factor VIII" and
"FVIII" are used interchangeably herein. "Blood coagulation Factor
VIII" or "Factor VIII" includes wild-type blood coagulation FVIII
as well as derivatives or variants of wild-type blood coagulation
FVIII where the procoagulant activity of wild-type blood
coagulation FVIII is at least partially retained. Derivatives may
have deletions like that of the B domain or parts of the B domain,
insertions and/or additions compared with the amino acid sequence
of wild-type FVIII. The term Factor VIII includes proteolytically
processed forms of FVIII, e.g. the two-chain form before
activation, comprising heavy chain and light chain, as well as
uncleaved, single-chain Factor VIII.
[0051] The term "Factor VIII" includes any FVIII variants or
mutants that retain at least 10%, preferably at least 15%, 20% or
25%, more preferably at least 30%, 40% or 50%, most preferably at
least 60%, 70% or even 75% of the biological activity of wild-type
FVIII.
[0052] FVIII is synthesized as a single polypeptide chain with a
molecular weight of about 280 kDa. The amino-terminal signal
peptide is removed upon translocation of FVIII into the
endoplasmatic reticulum, and the mature (i.e. after the cleavage of
the signal peptide) native FVIII molecule is then proteolytically
cleaved in the course of its secretion between the B and a3 domain
or within the B-domain. This results in the release of a
heterodimer which consists of a C-terminal light chain of about 80
kDa in a metal ion-dependent association with an N-terminal heavy
chain fragment of about 90-200 kDa (see also review by Kaufman,
Transfusion Med. Revs. 6:235 (1992)).
[0053] Physiological activation of the heterodimer occurs through
proteolytic cleavage of the protein chains by thrombin. Thrombin
cleaves the heavy chain to a 90 kDa protein, and then to 54 kDa and
44 kDa fragments. Thrombin also cleaves the 80 kDa light chain to a
72 kDa protein. It is the latter protein, and the two heavy chain
fragments (54 kDa and 44 kDa above), held together by calcium ions,
that constitute active FVIII. Inactivation occurs when the 44 kDa
A2 heavy chain fragment dissociates from the molecule or when the
72 kDa and 54 kDa proteins are further cleaved by thrombin,
activated protein C or FXa. In plasma, FVIII is stabilized by
association with an about 50-fold molar excess of von Willebrand
Factor protein ("VWF"), which appears to inhibit proteolytic
degradation of FVIII as described above.
[0054] The amino acid sequence of FVIII is organized into three
structural domains: a triplicated A domain of 330 amino acids each,
a single B domain of 980 amino acids, and a duplicated C domain of
150 amino acids each. The B domain has no homology to other
proteins and provides 18 of the 25 potential asparagine(N)-linked
glycosylation sites of this protein. The B domain has apparently no
function in coagulation and can be deleted, with the B-domain
deleted FVIII molecule still having procoagulatory activity.
[0055] As non-limiting examples, Factor VIII as used herein
includes FVIII mutants providing reduced or prevented APC cleavage
(Amano 1998. Thromb. Haemost. 79:557-563), FVIII mutants with a
further stabilized A2 domain (WO 97/40145), FVIII mutants resulting
in increased expression (Swaroop et al. 1997. JBC 272:24121-24124),
FVIII mutants with reduced immunogenicity (Lollar 1999. Thromb.
Haemost. 82:505-508), FVIII reconstituted from independently
expressed heavy and light chains (Oh et al. 1999. Exp. Mol. Med.
31:95-100), FVIII mutants with reduced binding to receptors leading
to catabolism of FVIII like HSPG (heparan sulfate proteoglycans)
and/or LRP (low density lipoprotein receptor related protein)
(Ananyeva et al. 2001. TCM, 11:251-257), disulfide bond-stabilized
FVIII variants (Gale et al., 2006. J. Thromb. Hemost. 4:1315-1322),
FVIII mutants with improved secretion properties (Miao et al.,
2004. Blood 103:3412-3419), FVIII mutants with increased cofactor
specific activity (Wakabayashi et al., 2005. Biochemistry
44:10298-304), FVIII mutants with improved biosynthesis and
secretion, reduced ER chaperone interaction, improved ER-Golgi
transport, increased activation or resistance to inactivation and
improved half-life (summarized by Pipe 2004. Sem. Thromb. Hemost.
30:227-237), and single-chain FVIII mutants not cleavable by furin.
All of these FVIII mutants and variants are incorporated herein by
reference in their entirety.
[0056] Preferably Factor VIII comprises the full length sequence of
FVIII as shown in SEQ ID NO: 6, more preferably, the Factor VIII is
a variant of FVIII with a partial or full deletion of the B-domain.
Also encompassed are additions, insertions, substitutions,
N-terminal, C-terminal or internal deletions of FVIII as long as
the biological activity of FVIII is at least partially retained.
The biological activity is retained in the sense of the invention
if the FVIII with modifications retains at least 10%, preferably at
least 15%, 20% or 25%, more preferably at least 30%, 40% or 50%,
most preferably at least 60%, 70% or even 75% of the biological
activity of wild-type FVIII. The biological activity of Factor VIII
can be determined by the artisan as described below.
[0057] A suitable test to determine the biological activity of
Factor VIII is for example the one stage coagulation assay (Rizza
et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom ed. The
Hemophilias. NY Churchchill Livingston 1992) or the chromogenic
(two-stage) substrate FVIII activity assay (S. Rosen, 1984. Scand J
Haematol 33: 139-145, suppl.). The content of these references is
incorporated herein by reference.
[0058] The amino acid sequence of the mature wild-type form of
human blood coagulation FVIII is shown in SEQ ID NO: 6. The
reference to an amino acid position of a specific sequence means
the position of said amino acid in the FVIII wild-type protein and
does not exclude the presence of mutations, e.g. deletions,
insertions and/or substitutions at other positions in the sequence
referred to. For example, a mutation of residue 2004 referring to
SEQ ID NO: 6 does not exclude that in the modified homologue one or
more amino acids at positions 1 through 2332 of SEQ ID NO: 6 are
missing.
[0059] "Factor VIII" and/or "VWF" within the above definition also
include natural allelic variations that may exist and occur from
one individual to another and FVIII from other mammalian species,
e.g. porcine FVIII. "Factor VIII" and/or "VWF" within the above
definition further includes variants of FVIII and or VWF. Such
variants differ in one or more amino acid residues from the
wild-type sequence. Examples of such differences may include
conservative amino acid substitutions, i.e. substitutions within
groups of amino acids with similar characteristics, e.g. (1) small
amino acids, (2) acidic amino acids, (3) polar amino acids, (4)
basic amino acids, (5) hydrophobic amino acids, and (6) aromatic
amino acids. Examples of such conservative substitutions are shown
in the following table 1.
TABLE-US-00001 TABLE 1 (1) Alanine Glycine (2) Aspartic acid
Glutamic acid (3) Asparagine Glutamine Serine Threonine (4)
Arginine Histidine Lysine (5) Isoleucine Leucine Methionine Valine
(6) Phenylalanine Tyrosine Tryptophane
[0060] The term "conserved residue" in FVIII or VWF relates to an
evolutionarily conserved residue, i.e. where at the respective
position an identical residue or conservative substitution is found
in at least two, preferably at least three mammalian sequences.
[0061] The term "variant" of FVIII, VWF or domains thereof refers
to proteins or domains with at least 50% sequence identity,
preferably at least 55%, 60%, 65%, 70%, 75% or 80% sequence
identity, more preferably at least 82%, 84%, 85%, 86%, or 88%
sequence identity, even more preferably at least 90%, 92%, 94%, 95%
sequence identity to the sequence or relevant part of the sequence
shown in SEQ ID NO 6 or 2 respectively, provided that the variant
retains at least 10%, preferably 15%, 20%, 25%, or 30%, more
preferably at least 40% or 50%, even more preferably at least 60%,
70% or 75% of the biological activity of the protein or domain
thereof respectively. It is recognized that certain positions may
be more suitable to variation than others. For example, variants of
the CK domain of VWF will need to retain the cysteine at position
2773 (or equivalent thereof), which appears to be essential for the
formation of dimers. Other cysteine residues in the CK domain (Zhou
et al (2012) Blood 120, 449-458), and other domains of VWF and also
of FVIII, may also be essential.
[0062] To determine % sequence identity, the sequences are aligned
using a suitable sequence alignment program, such as the GAP
program of the GCG suite, using default parameters (Devereux et al
(1984) Nucl Acids Res 12, 387). Other programs that can be used to
align sequences include FASTA (Lipman & Pearson (1985) Science
227, 1436-1441), BLAST (Altschul et al (1990) J Mol Biol 215,
403-410), and ClustalW (Thompson et al (1994) Nucl Acids Res 22,
4673-4680).
[0063] In one embodiment of the invention, the covalent linkage is
achieved by a disulphide bridge between a cysteine residue in
FVIII, which is introduced into FVIII by genetic engineering, and a
cysteine residue in VWF, which can be either a cysteine found in
the wild-type VWF sequence, or it can also be introduced at an
appropriate location in the VWF sequence by genetic
engineering.
[0064] One preferred embodiment of the present invention relates to
a covalent complex comprising half-life extended VWF and Factor
VIII, wherein Factor VIII is modified by substitution of at least
one naturally occurring amino acid with a cysteine residue or
insertion of at least one cysteine residue at an appropriate
location in the FVIII which forms a disulphide bridge with a
cysteine residue in VWF (FIG. 3).
[0065] Therefore, according to the invention, the amino acid
sequence of the Factor VIII component of the complex differs from
that of wild-type FVIII as shown in SEQ ID NO: 6. The modified
Factor VIII has at least one mutation, for example a substitution
of a naturally occurring amino acid with a cysteine, or an
insertion of a cysteine residue at an appropriate position, for
example in the a3 domain or the C-terminal domain. Thus there may
be one or more, e.g. two, three, four, five or more additional
cysteine residues in the Factor VIII of the complex of the
invention; more preferably, only one or two additional cysteine
residues are introduced, most preferably one additional cysteine
residue is introduced.
[0066] More preferably, the naturally occurring amino acid that is
substituted in Factor VIII is an amino acid in the a3 domain. More
preferably, the naturally occurring amino acid that is substituted
in Factor VIII is located within amino acids 1653 to 1660 or within
amino acids 1667 to 1674 or within amino acids 1675 to 1688 of the
FVIII a3 domain. More preferably, the naturally occurring amino
acid in the a3 domain is an acidic residue, preferably a conserved
acidic residue, or a residue involved in a haemophilic phenotype,
or a Tyr residue which may be sulphated in the FVIII a3 domain.
Even more preferably, the naturally occurring amino acid in the a3
domain that is substituted with cysteine is selected from E1649,
D1658, E1660, D1663, Y1664, D1665, D1666, E1671, E1675, D1676,
D1678, I1679, Y1680, E1682, D1683, E1684, even more preferably from
T1653, L1655, D1658, E1660, S1669, V1670, N1672, K1673, K1674,
E1675, D1676 and/or N1685 in SEQ ID NO: 6 or equivalent position,
e.g. in a genetically engineered form of Factor VIII. Most
preferably, the naturally occurring amino acid in the a3 domain
that is substituted with cysteine is selected from T1654, Q1656,
F1677, D1678, I1679, Y1680, D1681, E1682, D1683, E1684, Q1686,
S1687 and/or P1688 in SEQ ID NO: 6 or equivalent position, e.g. in
a genetically engineered form of FVIII.
[0067] Preferably, the naturally occurring amino acid in VWF is
selected by a relative ratio higher than 0.5 of covalently bound
Factor VIII to VWF as experimentally assessed shown in example 6
and activity of Factor VIII as experimentally assessed shown in
example 9. Most preferably, the naturally occurring amino acid in
VWF is the selected by a said ratio of higher than 1.0.
[0068] In another preferred embodiment of the first aspect of the
invention, the naturally occurring amino acid that is substituted
with cysteine is in the C-terminal domain of FVIII, preferably an
amino acid in the FVIII region between amino acids 2051 and 2270,
more preferably the residue is selected from I2098, S2119, N2129,
R2150, P2153, W2229, Q2246 in SEQ ID NO: 6 or equivalent position,
e.g. in an engineered form of FVIII.
[0069] In a further, preferred embodiment of the first aspect of
the invention, VWF is also modified by substitution of a naturally
occurring amino acid with a cysteine residue, or insertion of a
cysteine residue, which forms a disulphide bridge with a cysteine
residue introduced into Factor VIII. The naturally occurring amino
acid in VWF is a residue within the D' or D3 region, preferably a
basic residue in the D' or D3 region or a highly conserved residue
in the D' or D3 region or a residue involved in type N-VWD or an
amino acid exposed on the surface of the VWF molecule. In a
preferred embodiment of the invention a cysteine residue is
inserted into the TIL' domain, the E' domain, the VWD3 domain, the
C8-3 domain, the TIL-3 domain or the E-3 domain or the naturally
occurring amino acid in VWF that is substituted with a cysteine
residue is a residue in the TIL' domain, the E' domain, the VWD3
domain, the C8-3 domain, the TIL-3 domain or the E-3 domain
(domains as defined by Zhou et al (2012) Blood 120(2) 449-458). For
example, the naturally occurring amino acid in VWF is selected from
R768, R782, R816, R820, R826, R852, R854, R906, R924, R945, R960,
R976, R1035, H817, H831, H861, H874, H916, H952, H977, H1047, K773,
K790, K834, K843, K855, K882, K912, K920, K922, K923, K940, K948,
K968, K985, K991, K1026, K1036, K1052, K1073, G785, M800, D879,
Q1053, E1078, E787, A789, T789, T791, Q793, N794, Y795, P828, F830,
E835, P838, D853, W856, L884. Preferably, the naturally occurring
amino acid in VWF is selected from T795, R816, D879, D951, E1161,
and/or R1204 in SEQ ID NO: 2 or equivalent position, e.g. in an
engineered form of VWF. More preferably, the naturally occurring
amino acid in VWF is selected from R768, R782, H817, D853, E933,
L984, E1015, D1076, E1078, P1079, K1116 and/or N1134 in SEQ ID NO:
2 or equivalent position, e.g. in an engineered form of VWF.
[0070] Preferably, the naturally occurring amino acid in VWF is
selected by a relative ratio higher than 0.5 of covalently bound
FVIII to VWF as experimentally assessed shown in example 6 and
activity of Factor VIII as experimentally assessed shown in example
9. Most preferably, the naturally occurring amino acid in VWF is
selected by a said ratio higher than 1.0.
[0071] Preferably, one or more of the following combinations of
substitutions of naturally occurring amino acid residues in FVIII
and VWF are introduced:
A/T789C:D1658C, M800C:D1658C, P828C:D1658C, F830C:D1658C,
P838C:D1658C, D853C:D1658C, R924C:D1658C, E1078C:D1658C,
F830C:D1663C, P838C:D1663C, D853C:D1663C, E1078C:D1663C,
E1078C:Y1664C, P838C:D1665C, R816C:D1666C, F830C:D1666C,
E835C:D1666C, T791C:E1671C, F830C:E1671C, E835C:E1671C,
D879C:E1671C, A/T789C:E1675C, T791C:E1675C, N794C:E1675C,
P828C:E1675C, F830C:E1675C, E835C:E1675C, P838C:E1675C,
D879CE1675C, R924C:E1675C, E1078C:E1675C, A/T789C:D1676C,
T791C:D1676C, N794C:D1676C, F830C:D1676C, E835C:D1676C,
A/T789C:D1678C, F830C:D1678C, E835C:D1678C, A/T789C:I1679C,
M800C:I1679C, F830C:I1679C, E835C:I1679C, R854C:I1679C,
D879C:I1679C, A/T789C:Y1680C, T791C:Y1680C, Y795C:Y1680C,
M800C:Y1680C, R816C:Y1680C, F830C:Y1680C, E835C:Y1680C,
R854C:Y1680C, D879C:Y1680C, A/T789C:E1682C, Y795C:E1682C,
R816C:E1682C, P828C:E1682C, E835C:E1682C, P838C:E1682C,
R854C:E1682C, D879C:E1682C, Q1053C:E1682C.
[0072] More preferably, one or more of the following combinations
of substitutions of naturally occurring amino acid residues in
FVIII and VWF are introduced:
F1677C:R768C, I1679C:R768C, Y1680C:R768C, N1685C:R768C,
T1654C:R782C, E1675C:R782C, N1685C:R782C, Q1686C:Y795C,
S1687C:Y795C, P1688C:Y795C, P1688C:Y795C, E1675C:H816C,
D1676C:R816C, Y1680C:R816C, E1682C:R816C, P1688C:R816C,
Y1680C:H817C, N1685C:H817C, Q1686C:H817C, S1687C:H817C,
I1679C:P828C, Y1680C:D853C, N1685C:D853C, T1654C:D879C,
P1688C:E933C, P1688:T951C, T1653C:L984C, T1654C:L984C,
L1655C:L984C, S1669C:L984C, K1673C:L984C, D1683C:L984C,
T1653C:E1015C, L1655C:E1015C, S1669C:E1015C, V1670C:E1015C,
N1672C:E1015C, K1673C:E1015C, D1678C:E1015C, I1679C:E1015C,
E1684C:E1015C, S1687C:E1015C, F1677C:V1027C, I1679C:V1027C,
P1688C:V1027C, S1657C:D1076C, K1673C:D1076C, D1676C:D1076C,
F1677C:D1076C, I1679C:D1076C, E1682C:D1076C, D1683C:D1076C,
Q1686C:D1076C, D1676C:E1078C, I1679C:E1078C, Y1680C:E1078C,
T1653C:P1079C, L1655C:P1079C, S1657C:P1079C, D1658C:P1079C,
E1682C:P1079C, V1670C:K1116C, K1673C:K1116C, D1676C:K1116C,
D1678C:K1116C, D1681C:K1116C, Q1686C:K1116C, P1688C:K1116C,
T1653C:N1134C, L1655C:N1134C, E1660C:N1134C, D1678C:N1134C,
D1683C:N1134C, E1684C:N1134C, Q1686C:N1134C, T1653C:E1161C,
L1655C:E1161C, K1674C:E1161C, D1676C:E1161C, E1684C:E1161C,
S1687C:E1161C, P1688C:R1204C.
[0073] Most preferably, one or more of the following combinations
of substitutions of naturally occurring amino acid residues in
FVIII and VWF are introduced:
T1654:P1079, T1654:N1134, Q1656:D1076, F1677:K1116, D1678:R782,
I1679:K1116, Y1680:H817, Y1680:D853, Y1680:E1078, D1681:R768,
E1682:R768, D1683:R768, E1684:R768, Q1686:R768, Q1686:E1015,
S1687:R768, S1687:N1134, P1688:R768, P1688:H817, P1688:E933,
P1688:L984, P1688:E1015, P1688:D1076 and P1688:N1134.
[0074] Preferably, the naturally occurring amino acids of the
combination of one or more inserted cysteine residues in VWF and
Factor VIII are selected by a relative ratio higher than 0.5 of
covalently bound Factor VIII to VWF as experimentally assessed
shown in example 6 and activity of Factor VIII as experimentally
assessed shown in example 9. Most preferably, the naturally
occurring amino acids of the combination of one or more inserted
cysteine residues in VWF and Factor VIII are selected by a said
ratio of higher than 1.0.
[0075] Preferably, the Factor VIII in the complex of the invention
is a genetically engineered Factor VIII. The engineered Factor VIII
may contain a partial or complete B-domain deletion, it may be a
mutated Factor VIII comprising one or more amino acid
substitutions, insertions, deletions or combinations thereof, it
may be a single chain version of Factor VIII, or it may be a fusion
polypeptide with a half-life extending moiety, e.g. a half-life
extending polypeptide (HLEP). It may also be a chemically modified
Factor VIII, e.g. modified by attachment of a half-life extending
moiety such as polyethylene glycol (PEGylation), glycosylated PEG,
hydroxyl ethyl starch (HESylation), polysialic acids, elastin-like
polypeptides, heparosan polymers or hyaluronic acid. It may also be
a Factor VIII from another species, e.g. another mammalian species,
e.g. porcine Factor VIII.
[0076] Preferably, the VWF in the complex of the invention is a
half-life extended form of VWF.
[0077] As used herein, the term "half-life" indicates the
functional half-life of the respective protein, i.e. the time it
takes for half the activity to be lost in vivo, i.e. in blood.
[0078] In a preferred embodiment, the half-life extended form of
VWF in the complex of the invention is a genetically engineered
form of VWF. More preferably, the genetically engineered VWF is a
fusion protein of VWF with a half-life extending moiety such as a
half-life extending polypeptide (HLEP).
[0079] A "half-life enhancing polypeptide" or "half-life extending
polypeptide" (HLEP) as used herein is a moiety that is fused to the
protein of interest, in particular to VWF, in order to extend its
half-life. Preferred HLEPs are selected from the group consisting
of albumin, a member of the albumin-family, the constant region of
immunoglobulin G and fragments thereof, polypeptides or lipids
capable of binding under physiological conditions to albumin, to
members of the albumin family as well as to portions of an
immunoglobulin constant region. It may be a full-length
half-life-enhancing protein described herein (e.g. albumin, a
member of the albumin-family or the constant region of
immunoglobulin G) or one or more domains or fragments thereof that
are capable of stabilizing or prolonging the therapeutic activity
or the biological activity of the coagulation factor. Such
fragments may be composed of 10 or more amino acids in length or
may include at least about 15, at least about 20, at least about
25, at least about 30, at least about 50, at least about 100, or
more contiguous amino acids from the HLEP sequence or may include
part or all of specific domains of the respective HLEP, as long as
the HLEP fragment provides a functional half-life extension of at
least 25% compared to a wild-type VWF or Factor VIII.
[0080] The HLEP may be a variant of a HLEP. The term "variants"
includes insertions, deletions and substitutions, either
conservative or non-conservative, where such changes allow the
half-life extending properties of the HLEP to be at least partially
maintained.
[0081] In particular, the proposed VWF HLEP fusion constructs of
the invention may include naturally occurring polymorphic variants
of HLEPs and fragments of HLEPs. The HLEP may be derived from any
vertebrate, especially any mammal, for example human, monkey, cow,
sheep, or pig. Non-mammalian HLEPs include, but are not limited to,
hen and salmon.
[0082] Preferably, the half-life extending moiety is selected from
albumin or variants or fragments thereof, immunoglobulin constant
region or variants and portions thereof, e.g. the Fc fragment,
solvated random chains with large hydrodynamic volume (e.g. XTEN,
homo-amino acid repeats (HAP) or proline-alanine-serine repeats
(PAS)), afamin or variants thereof, alpha-fetoprotein or variants
thereof, Vitamin D binding protein or variants thereof, transferrin
or variants thereof, carboxyl-terminal peptide (CTP) of human
chorionic gonadotropin-.beta. subunit, polypeptides or lipids
capable of binding under physiological conditions to albumin or
immunoglobulin constant regions. Most preferably, the HLEP is human
serum albumin.
[0083] The terms, "human serum albumin" (HSA) and "human albumin"
(HA) are used interchangeably in this application. The terms
"albumin" and "serum albumin" are broader, and encompass human
serum albumin (and fragments and variants thereof) as well as
albumin from other species (and fragments and variants
thereof).
[0084] As used herein, "albumin" refers collectively to albumin
polypeptide or amino acid sequences, or an albumin fragment or
variant, having one or more functional activities (e.g., biological
activities) of albumin such as binding of Ca.sup.2+, Na.sup.+,
K.sup.+, Zn.sup.2+ ions, fatty acids, hormones, bilirubin or
binding to FcRn. In particular, "albumin" refers to human albumin
or fragments thereof, especially the mature form of human albumin
as shown in SEQ ID NO: 7 herein or albumin from other vertebrates
or fragments thereof, or analogs or variants of these molecules or
fragments thereof.
[0085] In particular, the proposed VWF fusion constructs of the
invention may include naturally occurring polymorphic variants of
human albumin and fragments of human albumin. Generally speaking,
an albumin fragment or variant will be at least 30, most preferably
more than 70 amino acids long. The albumin variant may
preferentially consist of or alternatively comprise at least one
whole domain of albumin or fragments of said domains, for example
domains 1 (amino acids 1-194 of SEQ ID NO:7), 2 (amino acids
195-387 of SEQ ID NO: 7), 3 (amino acids 388-585 of SEQ ID NO: 7),
1+2 (1-387 of SEQ ID NO: 7), 2+3 (195-585 of SEQ ID NO: 7) or 1+3
(amino acids 1-194 of SEQ ID NO: 3+ amino acids 388-585 of SEQ ID
NO: 7). Each domain is itself made up of two homologous subdomains
namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with
flexible inter-subdomain linker regions comprising residues Lys106
to Glu119, Glu292 to Val315 and Glu492 to Ala511.
[0086] The albumin portion of the VWF fusion constructs within the
complex of the invention may comprise at least one subdomain or
domain of HA or conservative modifications thereof.
[0087] In a preferred embodiment the N-terminus of albumin is fused
to the C-terminus of the amino acid sequence of the modified VWF.
That is, the complex of the present invention may comprise the
structure: [0088] mVWF-L-A wherein mVWF is the modified VWF as
described hereinabove, L is an optional peptidic linker sequence
and A is albumin as defined hereinabove.
[0089] The modified VWF or the complex of the FVIII with the
modified VWF of the invention may comprise more than one HLEP
sequence, e.g. two or three HLEP sequences.
[0090] These multiple HLEP sequences may be fused to the C-terminal
part of VWF in tandem, e.g. as successive repeats.
[0091] The HLEP may also be coupled to VWF by a peptide linker. The
linker should be non-immunogenic and may be a non-cleavable or
cleavable linker. Non-cleavable linkers may be comprised, for
example, of alternating glycine and serine residues as exemplified
in WO2007/090584.
[0092] A possible peptidic linker between the VWF moiety and the
HLEP moiety may also consist of peptide sequences, which serve as
natural interdomain linkers in human proteins. Preferably such
peptide sequences in their natural environment are located close to
the protein surface and are accessible to the immune system so that
one can assume a natural tolerance against this sequence. Examples
are given in WO2007/090584. Preferably, the linker region comprises
a sequence of VWF, which should result in a decreased risk of
neoantigenic properties of the expressed fusion protein.
[0093] Cleavable linkers should be flexible enough to allow
cleavage by proteases. The linker peptides are preferably cleavable
by the proteases of the coagulation system, for example FIIa, FIXa,
FXa, FXIa, FXIIa and/or FVIIa.
[0094] The HLEP may also be a peptide that can non-covalently bind
a half-life extending moiety such as a protein naturally occurring
in human plasma (e.g. albumin, immunoglobulins). In this case, VWF
would be modified in a way that it bears, preferably C-terminally
or N-terminally to the D'D3 domain, a peptide binding the half-life
extending moiety.
[0095] In another embodiment of the invention, the half-life of VWF
is extended by chemical modification, e.g. attachment of a
half-life extending moiety such as polyethylene glycol
(PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation),
polysialic acids, elastin-like polypeptides, heparosan polymers or
hyaluronic acid.
[0096] Another embodiment of the invention is a covalent complex of
Factor VIII and half-life extended VWF, where Factor VIII is
connected to VWF via an additional peptide or polypeptide sequence
added to Factor VIII. Preferably the added sequence comprises one
or more VWF domains.
[0097] As mentioned above, during biosynthesis in the endoplasmatic
reticulum, the VWF propeptide monomers are assembled into dimers
via a C-terminal disulphide bridge formed between the C-terminal
cystine knot domains (CK). The inventors have now surprisingly
found that this CK domain, when fused with Factor VIII, leads to a
covalent, disulphide linkage between the CK domains introduced into
Factor VIII, and that naturally present in VWF. Thus, this presents
another novel way of achieving the covalent complex between Factor
VIII and VWF of the present invention. The efficiency with which
the covalent linkage is formed can be enhanced if additional C
domains are included in the portion of the VWF that is fused to
Factor VIII. These may be for example the C5 to C6 domains, the C3
to C6 domains or the C1 to C6 domains as defined by Zhou et al
(2012, Blood 120, 449-458), optionally extended by additional VWF
domains.
[0098] Therefore, another embodiment of the invention is a covalent
complex comprising VWF and Factor VIII, wherein the VWF is a
half-life extended form of VWF, wherein Factor VIII is modified to
comprise the C-terminal domain CK of VWF, optionally containing
additional VWF domains. Preferably, the Factor VIII is so modified
at its C-terminus. More preferably, the Factor VIII comprises,
preferably at its C-terminus, residues 2723 to 2813, 2722-2813,
2724-2813, 2580-2813, 2578-2813, 2497-2813, 2429-2813, 2400-2813,
2334-2813, 2255-2813, 1873-2813, 1683-2813, 1277-2813, 1264-2813 or
764-2813 of SEQ ID NO: 2 or a variant thereof, provided that
cysteine residue 2773 (or equivalent thereof) is preserved.
Preferably, the modified Factor VIII in addition to the CK domain
comprises C6, C5 to C6, C4 to C6, C3 to C6, C2 to C6, or C1 to C6
domains of VWF as defined by Zhou et al (2012, Blood 120, 449-458)
or variants thereof. Optionally, the CK and C domains may be
extended by additional domains of VWF.
[0099] In another embodiment of the invention, the Factor VIII is
N-terminally fused with one or more of the C-terminal domains of
VWF (see FIGS. 6A-6C). Such C-terminal domains may be derived from
the C-terminal cystine knot (CK) domain of VWF and may additionally
comprise one or more of the C-domains, D-domains, or A-domains of
VWF up to the whole VWF sequence (see FIGS. 2A and 2B for the
structure of VWF). More preferably, the Factor VIII comprises,
preferably at its N-terminus, residues 2723 to 2813, 2722-2813
2724-2813, 2580-2813, 2578-2813, 2497-2813, 2429-2813, 2400-2813,
2334-2813, 2255-2813, 1873-2813, 1683-2813, 1277-2813 or 1264-2813
or 764-2813 of SEQ ID NO: 2 or variants thereof, provided that
cysteine residue 2773 (or equivalent thereof) is preserved. In this
embodiment, a signal peptide is added to the N-terminus of the VWF
domains, and the VWF domains are fused to the N-terminus of mature
Factor VIII (without signal peptide) either directly or via a
polypeptide linker.
[0100] Preferably, the C-terminal CK domain, optionally extended by
additional domains, of VWF is attached to Factor VIII by a
cleavable linker. A linker sequence may consist of one or more
amino acids, e.g. of 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to
30, 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3 (e.g. 1, 2 or 3)
amino acids and which may be equal or different from each other.
Usually, the linker sequences are not present at the corresponding
position in the wild-type coagulation factor. Preferably, the
linker is a cleavable linker, i.e. it comprises a cleavage site for
a protease, preferably it comprises a cleavage site that is
cleavable by a protease related to blood coagulation, more
preferably, the cleavable linker comprises a thrombin cleavage
site, even more preferably it comprises one of the thrombin
cleavage sites of FVIII.
[0101] Examples of cleavable linkers are
EDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN (aa1675-1720 of SEQ
ID NO: 6 (FVIII)) or
NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHRSTRQKQFNATTIPEN (aa714-764 of
SEQ ID NO: 6) or WRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV
(aa357-399 of SEQ ID NO: 6) or
WRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYA (aa357-396 of SEQ ID NO: 6)
or WRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWD (aa357-394 of SEQ ID NO:
6)
[0102] Including deletions, insertions and/or substitutions
thereof, given that cleavability is retained.
[0103] Optionally, the linker comprises additional amino acid
residues, which are preferably introduced between the domain(s)
derived from VWF and the cleavable part of the linker. Preferably,
the additional residues provide a peptide of sufficient length to
permit the interaction of Factor VIII and VWF, in particular via
the a3 and D'D3 regions, respectively. The additional amino acid
residues may be more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
120 or 150 amino acids. Preferably the additional amino acid
residues form a flexible, "non-structural" peptide, and more
preferably comprise or even consist of glycine-serine repeats,
proline-alanine-serine repeats, homo-amino acid repeats, or
sequences of the FVIII B-domain.
[0104] Another embodiment of the invention is a covalent complex
comprising VWF and Factor VIII, wherein the VWF is a half-life
extended form of VWF, and wherein Factor VIII is modified to
comprise the D'D3 region of VWF, and optionally additional domains
of VWF (FIGS. 5A-5E). Preferably, the Factor VIII is so modified
that its partial or complete B-domain is replaced by the VWF D'D3
region or fragments thereof (FIGS. 5D and 5E). More preferably, the
Factor VIII comprises, preferably instead of its (or part of its) B
domain, residues 764 to 1241, 764 to 1242, 764 to 1247 or 764 to
1270 or any sequence between 764 and 1241 to 1270, respectively, of
SEQ ID NO: 2 or a variant or a fragment thereof.
[0105] Preferably, the D'D3 domain of VWF is attached to Factor
VIII such that a two-chain molecule is generated upon secretion of
the molecule into the cell culture medium and that the D'D3 domain
is located at the N-terminus of the Factor VIII light chain. This
can be achieved by introducing a cleavable linker, comprising, for
example, a cleavage site for PACE/furin, between the Factor VIII a2
domain and the VWF D'D3 domain (FIGS. 5D and 5E). Optionally, the
linker comprises additional residues between the D'D3 domain of VWF
and the Factor VIII a3 domain (FIG. 5E). The additional residues
comprise a peptide of sufficient length to permit the
intramolecular interaction of Factor VIII and VWF via the a3 and
D'D3 regions, respectively. Preferably, the additional residues are
less than 300, 250, 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30,
25, 20, 15, or 10 amino acids. The additional amino acid residues
may comprise a flexible, "non-structural" peptide, preferably they
comprise or even consist of glycine-serine repeats,
proline-alanine-serin repeats or homo-amino acid repeats, or
sequences derived from the FVIII B-domain.
[0106] The embodiments described above are the mature form; the
skilled person will be able to construct a protein to be expressed
in order to obtain the mature form, e.g. by including additional
sequences, e.g. a signal sequence at the N-terminus.
[0107] Alternatively, the N-terminus of Factor VIII is connected to
the C-terminus of the VWF D'D3 domains or fragments thereof,
optionally containing further domains of VWF (e.g. the D1 and D2
domains), which is preferably N-terminally extended by a signal
peptide. This will aid in the expression and intracellular
formation of covalent bonds with half-life extended VWF. More
preferably, the VWF portion comprises N-terminally residues 1 to
1241 or residues 764 to 1241 (after cleavage of the propeptide) of
SEQ ID NO: 2 or a variant or a fragment thereof.
[0108] Preferably, the D'D3 or D1D2D'D3 domains of VWF,
respectively, are attached to the N-terminus of Factor VIII by a
cleavable linker. More preferably, the cleavable linker comprises a
protease cleavage site, more preferably a cleavage site for one of
the proteases of the coagulation system, even more preferably a
thrombin cleavage site, preferably one of the thrombin cleavage
sites of FVIII. Optionally, the linker comprises additional
residues between the D'D3 or D1D2D'D3 domains of VWF and the Factor
VIII molecule, the additional residues comprising a peptide of
sufficient length to permit the intramolecular interaction of
Factor VIII and VWF via the a3 and D'D3 regions, respectively.
Preferably, more than 20, 30, 40, 50, 70, 100 or 150 additional
amino acids are added. Preferably the additional amino acids
comprise a flexible, non-structural peptide, more preferably they
comprise or even consist of glycine-serine repeats,
proline-alanine-serine repeats, homo-amino acid repeats, or
sequences of the FVIII B-domain.
[0109] As a further alternative, the C-terminus of Factor VIII is
connected to the N-terminus of the VWF D'D3 domains or fragments
thereof. This will aid in the expression and intracellular
formation of covalent bonds with coexpressed half-life extended
VWF. More preferably, the VWF comprises amino acids 764 to 1241,
764 to 1242, 764 to 1247 or 764 to 1270 or any sequence between 764
and 1241 to 1270, respectively, of SEQ ID NO: 2 or a variant or a
fragment thereof.
[0110] Preferably, the D'D3 domains of VWF (or the D1D2D'D3
domains) are attached to the N-terminus of Factor VIII by a
cleavable linker; inclusion of a signal peptide N-terminal to the
VWF domains would lead to secretion upon expression of the protein
in mammalian cells. More preferably, the cleavable linker comprises
a thrombin cleavage site, preferably one of the thrombin cleavage
sites of FVIII which comprise of sequences encompassing the
thrombin cleavage sites at amino acid positions 372, 740 and/or
1689 of SEQ ID NO. 6, respectively.
[0111] Optionally, the linker comprises additional residues between
the D'D3 domains of VWF and the Factor VIII molecule, the
additional residues comprising a peptide of sufficient length to
permit the interaction of Factor VIII and VWF via the a3 and D'D3
regions, respectively. Preferably, more than 20, 30, 40, 50, 70,
100, 120 or 150 additional amino acids are added. Preferably the
additional amino acids comprise a flexible, non-structural peptide,
more preferably they comprise or even consist of glycine-serine
repeats, proline-alanine-serine repeats, homo-amino acid repeats,
or sequences of the FVIII B-domain.
[0112] Examples of such fusion proteins with various linkers are
shown in SEQ ID NOs: 144-177; each sequence shown, i.e. the DNA and
its translation product (the fusion protein), as well as DNA
sequences encoding the same translation product by virtue of the
redundancy of the genetic code (e.g. codon-optimized versions of
those DNA sequences) are specific embodiments of the invention.
However, the skilled person will be able to design many more
examples of such fusion proteins that also fall within the present
invention.
[0113] Preferably the VWF portion of the complex of the invention
forms multimers as it does in nature. For particular reasons it may
be desirable for the VWF portion of the complex to form not more
than a dimer. This can be achieved by deleting the propeptide
sequence of the VWF and fusing the VWF signal peptide directly to
the N-terminus of D', thereby allowing for the expression of a
propeptide depleted VWF molecule. Due to the absence of the
propeptide the multimerization via the D'D3 domain will be blocked.
For other particular reasons it may be desirable for the VWF
portion of the complex to form not more than a monomer. This can be
achieved by deleting the propeptide sequence of the VWF and fusing
the VWF signal peptide directly to D' allowing for the expression
of a propeptide depleted VWF molecule and in addition by
introducing a mutation of Cys2773 into another suitable amino acid,
e.g. alanine.
[0114] Another embodiment of the invention is the combination of
any of the embodiments described above to form a Factor VIII/VWF
complex where one or more covalent bond(s) exist(s) directly
between the Factor VIII and VWF binding sites (preferably a
disulphide bond), and where another covalent bond exists between
the Factor VIII and the VWF part of the molecule and where one or
more HLEPs is connected to Factor VIII, to VWF, or to both (FIGS.
12A-12N). Such Factor VIII/VWF complexes may be advantageous
because they may be producible with higher yields than complexes
with only a disulphide bond between the Factor VIII and the VWF
moiety.
[0115] A second aspect of the invention is a method of producing
the covalent complexes of Factor VIII and VWF described above,
comprising co-expressing the Factor VIII and VWF in a eukaryotic
cell line. Therefore, the invention also relates to polynucleotides
encoding the proteins forming the complex of the invention.
[0116] The term "polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide that may be
unmodified RNA or DNA or modified RNA or DNA. The polynucleotide
may be single- or double-stranded DNA, single or double-stranded
RNA. As used herein, the term "polynucleotide(s)" also includes
DNAs or RNAs that comprise one or more modified bases and/or
unusual bases, such as inosine. It will be appreciated that a
variety of modifications may be made to DNA and RNA that serve many
useful purposes known to those of skill in the art. The term
"polynucleotide(s)" as it is employed herein embraces such
chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including, for example, simple
and complex cells.
[0117] The skilled person will understand that, due to the
degeneracy of the genetic code, a given polypeptide can be encoded
by different polynucleotides. These "variants" are encompassed by
this invention.
[0118] Preferably, the polynucleotide of the invention is an
isolated polynucleotide. The term "isolated" polynucleotide refers
to a polynucleotide that is substantially free from other nucleic
acid sequences, such as and not limited to other chromosomal and
extrachromosomal DNA and RNA. Isolated polynucleotides may be
purified from a host cell. Conventional nucleic acid purification
methods known to the skilled person may be used to obtain isolated
polynucleotides. The term also includes recombinant polynucleotides
and chemically synthesized polynucleotides.
[0119] The invention further relates to a group of polynucleotides
which together encode the modified VWF and/or the modified Factor
VIII of the invention, or the polypeptide of the invention
comprising the modified VWF and/or the modified Factor VIII. For
example, a first polynucleotide in the group may encode the heavy
chain of a modified Factor VIII, and a second polynucleotide may
encode the light chain of a modified Factor VIII, and a third
polynucleotide may encode the modified VWF.
[0120] Yet another aspect of the invention is a plasmid or vector
comprising a polynucleotide according to the invention. Preferably,
the plasmid or vector is an expression vector. In a particular
embodiment, the vector is a transfer vector for use in human gene
therapy.
[0121] The invention also relates to a group of plasmids or vectors
that comprise the above group of polynucleotides. A first plasmid
or vector may contain said first polynucleotide, and a second
plasmid or vector may contain said second polynucleotide.
Alternatively, two or more coding sequences are cloned into one
expression vector either using separate promoter sequences or one
promoter and an internal ribosome entry site (IRES) element to
direct the expression of more than one protein that is part of the
complex of the invention.
[0122] Still another aspect of the invention is a host cell
comprising a polynucleotide, a plasmid or vector of the invention,
or a group of polynucleotides or a group of plasmids or vectors as
described herein.
[0123] The host cells of the invention may be employed in a method
of producing the covalent complex of the invention. The method
comprises: [0124] (a) culturing host cells of the invention under
conditions such that the desired protein complex is expressed; and
[0125] (b) optionally recovering the desired protein complex from
the host cells or from the culture medium.
[0126] The production of recombinant mutant proteins at high levels
in suitable host cells requires the assembly of the above-mentioned
modified cDNAs into efficient transcriptional units together with
suitable regulatory elements in a recombinant expression vector
that can be propagated in various expression systems according to
methods known to those skilled in the art. Efficient
transcriptional regulatory elements could be derived from viruses
having animal cells as their natural hosts or from the chromosomal
DNA of animal cells. Preferably, promoter-enhancer combinations
derived from the Simian Virus 40, adenovirus, BK polyoma virus,
human cytomegalovirus, or the long terminal repeat of Rous sarcoma
virus, or promoter-enhancer combinations including strongly
constitutively transcribed genes in animal cells like beta-actin or
GRP78 can be used. In order to achieve stable high levels of mRNA
transcribed from the cDNAs, the transcriptional unit should contain
in its 3'-proximal part a DNA region encoding a transcriptional
termination-polyadenylation sequence. Preferably, this sequence is
derived from the Simian Virus 40 early transcriptional region, the
rabbit beta-globin gene, or the human tissue plasminogen activator
gene.
[0127] The cDNAs may then be integrated into the genome of a
suitable host cell line for expression of the modified Factor VIII
and/or VWF proteins, which then assemble into the covalent complex
of the invention. Alternatively, stable episomal vectors can also
be used that remain in the cell as stable extrachromosomal
elements. Preferably this cell line should be an animal cell-line
of vertebrate origin in order to ensure correct folding, disulfide
bond formation, asparagine-linked glycosylation and other
post-translational modifications as well as secretion into the
cultivation medium. Examples of other post-translational
modifications are tyrosine O-sulfation and proteolytic processing
of the nascent polypeptide chain. Examples of cell lines that can
be used are monkey COS-cells, mouse L-cells, mouse C127-cells,
hamster BHK-21 cells, human HEK-293 cells, and hamster
CHO-cells.
[0128] The recombinant expression vector encoding the corresponding
cDNAs can be introduced into an animal or human cell line in
several different ways. For instance, recombinant expression
vectors can be created from vectors based on different animal
viruses. Examples of these are vectors based on baculovirus,
vaccinia virus, adenovirus, and preferably bovine papilloma
virus.
[0129] The transcription units encoding the corresponding DNAs can
also be introduced into animal cells together with another
recombinant gene which may function as a dominant selectable marker
in these cells in order to facilitate the isolation of specific
cell clones which have integrated the recombinant DNA into their
genome. Examples of this type of dominant selectable marker genes
are Tn5 amino glycoside phosphotransferase, conferring resistance
to geneticin (G418), hygromycin phosphotransferase, conferring
resistance to hygromycin, and puromycin acetyl transferase,
conferring resistance to puromycin. The recombinant expression
vector encoding such a selectable marker can reside either on the
same vector as the one encoding the cDNA of the desired protein, or
it can be encoded on a separate vector which is simultaneously
introduced and integrated to the genome of the host cell,
frequently resulting in a tight physical linkage between the
different transcription units.
[0130] Other types of selectable marker genes which can be used
together with the cDNA of the desired proteins are based on various
transcription units encoding dihydrofolate reductase (dhfr). After
introduction of this type of gene into cells lacking endogenous
dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44), it will
enable these to grow in media lacking nucleosides. An example of
such a medium is Ham's F12 without hypoxanthine, thymidin, and
glycine. These dhfr-genes can be introduced together with the cDNA
transcriptional units into CHO-cells of the above type, either
linked on the same vector or on different vectors, thus creating
dhfr-positive cell lines producing recombinant protein.
[0131] If the above cell lines are grown in the presence of the
cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to
methotrexate will emerge. These cell lines may produce recombinant
protein at an increased rate due to the amplified number of linked
dhfr and the desired protein's transcriptional units. When
propagating these cell lines in increasing concentrations of
methotrexate (1-10000 nM), new cell lines can be obtained which
produce the desired protein at very high rate.
[0132] The above cell lines producing the desired protein can be
grown on a large scale, either in suspension culture or on various
solid supports. Examples of these supports are micro carriers based
on dextran or collagen matrices, or solid supports in the form of
hollow fibres or various ceramic materials. When grown in cell
suspension culture or on micro carriers the culture of the above
cell lines can be performed either as a batch culture or as a
perfusion culture with continuous production of conditioned medium
over extended periods of time. Thus, according to the present
invention, the above cell lines are well suited for the development
of an industrial process for the production of the desired
recombinant mutant proteins
[0133] It is preferred to purify the complex of the invention to
80% purity, more preferably 95% purity, and particularly preferred
is a pharmaceutically pure state that is greater than 99.9% pure
with respect to contaminating macromolecules from the cell culture,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, an isolated or
purified modified covalent complex of the invention is
substantially free of other, non-related polypeptides.
[0134] The covalent complex of the invention, which accumulates in
the medium of secreting cells of the above types, can be
concentrated and purified by a variety of biochemical and
chromatographic methods, including methods utilizing differences in
size, charge, hydrophobicity, solubility, specific affinity, etc.
between the desired protein and other substances in the cell
cultivation medium.
[0135] An example of such purification is the adsorption of the
recombinant mutant protein to a monoclonal antibody, directed to
e.g. a HLEP, preferably human albumin, or directed to the
respective coagulation factor, which is immobilised on a solid
support. After adsorption of the complex to the support, washing
and desorption, the protein can be further purified by a variety of
chromatographic techniques based on the above properties. The order
of the purification steps is chosen e.g. according to capacity and
selectivity of the steps, stability of the support or other
aspects. Preferred purification steps e.g. are but are not limited
to ion exchange chromatography steps, immunoaffinity chromatography
steps, affinity chromatography steps, hydrophobic interaction
chromatography steps, dye chromatography steps, hydroxyapatite
chromatography steps, multimodal chromatography steps, and size
exclusion chromatography steps.
[0136] In order to minimize the theoretical risk of virus
contaminations, additional steps may be included in the process
that allow effective inactivation and/or elimination of viruses.
Such steps e.g. are heat treatment in the liquid or solid state,
treatment with solvents and/or detergents, radiation in the visible
or UV spectrum, gamma-irradiation or nanofiltration.
[0137] The modified polynucleotides (e.g. DNA) of this invention
may also be integrated into a transfer vector for use in the human
gene therapy.
[0138] In another embodiment of this aspect of the invention, the
(modified) Factor VIII and the (modified) VWF are covalently
connected by chemical cross-linking.
[0139] The various products of the invention are useful as
medicaments. Accordingly, a third aspect of the invention is a
covalent complex as described above for use in medicine, preferably
for use in the treatment or prophylaxis of a bleeding disorder.
Preferably, the bleeding disorder is hemophilia A or VWD.
[0140] A fourth aspect of the invention is a pharmaceutical
composition comprising the covalent complex described above. The
covalent complex as described in this invention can be formulated
into pharmaceutical preparations for therapeutic use. The purified
protein may be dissolved in conventional physiologically compatible
aqueous buffer solutions to which there may be added, optionally,
pharmaceutical excipients to provide pharmaceutical
preparations.
[0141] Such pharmaceutical carriers and excipients as well as
suitable pharmaceutical formulations are well known in the art (see
for example "Pharmaceutical Formulation Development of Peptides and
Proteins", Frokjaer et al., Taylor & Francis (2000) or
"Handbook of Pharmaceutical Excipients", 3.sup.rd edition, Kibbe et
al., Pharmaceutical Press (2000)). Standard pharmaceutical
formulation techniques are well known to persons skilled in the art
(see, e.g., 2005 Physicians' Desk Reference.RTM., Thomson
Healthcare:Montvale, N.J., 2004; Remington: The Science and
Practice of Pharmacy, 20th ed., Gennaro et al., Eds. Lippincott
Williams & Wilkins: Philadelphia, Pa., 2000). In particular,
the pharmaceutical composition comprising the covalent complex of
the invention may be formulated in lyophilized or stable liquid
form. The polypeptide variant may be lyophilized by a variety of
procedures known in the art. Lyophilized formulations are
reconstituted prior to use by the addition of one or more
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0142] Formulations of the composition are delivered to the
individual by any pharmaceutically suitable means of
administration. Various delivery systems are known and can be used
to administer the composition by any convenient route.
Preferentially, the compositions of the invention are administered
systemically. For systemic use, the complex of the invention is
formulated for parenteral (e.g. intravenous, subcutaneous,
intramuscular, intraperitoneal, intracerebral, intrapulmonar,
intranasal or transdermal) or enteral (e.g., oral, vaginal or
rectal) delivery according to conventional methods. The most
preferential routes of administration are intravenous and
subcutaneous administration. The formulations can be administered
continuously by infusion or by bolus injection. Some formulations
encompass slow release systems.
[0143] The covalent complex of the present invention is
administered to patients in a therapeutically effective dose,
meaning a dose that is sufficient to produce the desired effects,
preventing or lessening the severity or spread of the condition or
indication being treated without reaching a dose which produces
intolerable adverse side effects. The exact dose depends on many
factors as e.g. the indication, formulation, mode of administration
and has to be determined in preclinical and clinical trials for
each respective indication.
[0144] The pharmaceutical composition of the invention may be
administered alone or in conjunction with other therapeutic agents.
These agents may be incorporated as part of the same pharmaceutical
preparation.
[0145] A further aspect of the invention is a method of treating or
preventing a bleeding disorder by administering an effective amount
of a complex described above to a subject in need thereof. In
another embodiment, the method comprises administering to the
individual an efficient amount of a polynucleotide of the invention
or of a plasmid or vector of the invention. Alternatively, the
method may comprise administering to the individual an efficient
amount of the host cells of the invention described herein.
[0146] The present invention will be further described in the
following, non-limiting examples. This description of specific
embodiments of the invention will be made in conjunction with the
appended figures.
[0147] FIGS. 1A-1C: FIG. 1A, domain structure of mature FVIII
protein; FIG. 1B, domain structure of a B-domain deleted mature
FVIII protein; FIG. 1C, domain structure of a B-domain deleted
single-chain mature FVIII protein. The arrows show the PACE/Furin
cleavage sites, the triangles the thrombin cleavage sites for
activation.
[0148] FIGS. 2A and 2B: Domain structure of pro-VWF (FIG. 2A) and
mature VWF (FIG. 2B) according to Zhou et al., 2012.
VWF-dimerization and multimerization are not shown.
[0149] FIG. 3: Example of a covalent complex where Factor VIII and
VWF are linked via a disulphide bridge. VWF domains are shown in
grey, Factor VIII in white.
[0150] FIGS. 4A-4J: Examples of modified FVIII with VWF domains
including the VWF CK domain. FVIII domains are shown in white, VWF
domains are shown in grey. Black triangles show thrombin cleavage
sites, open triangles show protease cleavage sites introduced into
the linker.
[0151] FIGS. 5A-5E: Examples of modified FVIII with VWF domains
including D'D3 domains. Arrows show PACE/Furin cleavage sites,
black triangles show thrombin cleavage sites, open triangles show
protease cleavage sites introduced into the linker.
[0152] FIGS. 6A-6C: FVIII modified by additional VWF domains.
Symbols as explained above.
[0153] FIG. 7: Example of a covalent complex linked by chemical
crosslinking.
[0154] FIGS. 8A and 8B: Western Blot of covalently linked
FVIII-SC/VWF-FP molecules. M, molecular size marker. FIG. 8A,
anti-FVIII, FIG. 8B, anti-VWF antibody blot.
[0155] FIG. 9: Separation of covalently linked FVIII-SC/VWF-FP
molecules on a reducing SDS-PAGE after purification (lane 1) and
subsequent thrombin cleavage (lane 2).
[0156] FIGS. 10A and 10B: Multimer gel analysis of a covalently
linked FVIII-SC/VWF-FP multimer molecule by anti-VWF (FIG. 10A) and
anti-FVIII (FIG. 10B) antibodies. Lane 1, plasma.derived VWF; lane
2 and 3, supernatant of two clones expressing covalently linked
FVIII-SC/VWF-FP multimers; lane 4, rVWF-FP.
[0157] FIGS. 11A and 11B: Pharmacokinetic analysis of a covalently
linked FVIII-SC/VWF-FP multimer (circles) in rats. FIG. 11A, FVIII
data, FIG. 11B, VWF data.
[0158] FIGS. 12A-12N: Examples of constructs with both disulphide
bridge and fusion of VWF to Factor VIII, optionally via a peptide
linker.
SEQUENCE LISTING
[0159] SEQ ID NO: 1: cDNA sequence of human VWF
[0160] SEQ ID NO: 2: Protein sequence of human VWF
[0161] SEQ ID NO: 3: PCR primer VWF+
[0162] SEQ ID NO: 4: PCR primer VWF-
[0163] SEQ ID NO: 5: cDNA sequence of human FVIII
[0164] SEQ ID NO: 6: Protein sequence of mature human FVIII
[0165] SEQ ID NO: 7: Protein sequence of mature human serum
albumin
[0166] SEQ ID NOs: 8-143: Various primers and oligonucleotides for
mutagenesis as listed in the examples.
[0167] SEQ ID NOs: 144-177: Fusion protein sequences (DNA and
protein) of human single chain FVIII with various VWF-CK comprising
sequences, connected through various linkers.
EXAMPLES
Example 1: Generation of VWF Mutants with Cysteine Residues in the
D'D3 Region
[0168] An expression plasmid (pIRESpuro3; BD Biosciences, Franklin
Lakes, N.J., USA) containing a full length VWF cDNA sequence in its
multiple cloning site had been generated previously (pVWF-2448).
The VWF cDNA sequence contained in this vector is displayed as SEQ
ID No.1, its corresponding protein sequence as SEQ ID No. 2.
[0169] For generating such expression vectors, the VWF cDNA may be
amplified by polymerase chain reaction (PCR) using primer set VWF+
and VWF- (SEQ ID NOs. 3 and 4) under standard conditions known to
those skilled in the art (and as described e.g. in Current
Protocols in Molecular Biology, Ausubel F M et al. (eds.) John
Wiley & Sons, Inc.; http://www.currentprotocols.com/WileyCDA/)
from a plasmid containing VWF cDNA (as obtainable commercially,
e.g. pMT2-VWF from ATCC, No. 67122). The resulting PCR fragment may
be digested by restriction endonuclease EcoRI and ligated into
expression vector pIRESpuro3 which had been linearized by EcoRI.
The resulting expression plasmid, screened for correct orientation
of the insert, will contain a wild-type cDNA of VWF downstream of
the CMV promoter suitable for VWF expression.
[0170] In order to introduce mutations into the VWF sequence site
directed mutagenesis (QuickChange XL Site Directed Mutagenesis Kit,
Agilent Technologies, La Jolla, Calif., USA) was applied on plasmid
pVWF-2448 according to the following protocol as suggested by the
kit manufacturer. Per mutagenesis reaction 5 .mu.l of 10.times.
reaction buffer, 1 .mu.l of plasmid DNA pVWF-2448 (50 ng), 1 .mu.l
(10 pmol/.mu.l) each of the respective two mutagenesis
oligonucleotides, 1 .mu.l dNTP Mix, 3 .mu.l Quick-Solution, 1 .mu.l
Turbo Polymerase (2.5 U/.mu.l) and 37 .mu.l H.sub.2O were mixed and
subjected to a polymerase chain reaction with an initial
denaturation for 2 min at 95.degree. C., 18 cycles of a)
denaturation for 50 sec. at 95.degree. C., b) annealing for 50 sec
at 60.degree. C. and c) elongation for 14 min at 68.degree. C.,
followed by a single terminal elongation phase of 7 min at
68.degree. C. Subsequently 1 .mu.l of DpnI enzyme from the kit was
added and the reaction incubated for another 60 min at 37.degree.
C. After that 3 .mu.l of the mutagenesis reaction were transformed
into E. coli competent cells (e.g. XL10 Gold, Agilent
Technologies). Clones were isolated, plasmid DNA extracted and the
mutations in the VWF sequences were verified by DNA sequencing.
[0171] The following table lists oligonucleotides used for
mutagenesis of the VWF cDNA sequence and the respective mutations
introduced.
TABLE-US-00002 SEQ VWF Mutagenesis oligonucleotide sequence ID
mutation Designation (5'.fwdarw.3') NO R768C We4674
GTGTTCCCTGAGCTGCTGCCCTCCTATGGTCAAACTGG 89 We4675
CCAGTTTGACCATAGGAGGGCAGCAGCTCAGGGAACAC 90 R782C We4218
CCCGCTGACAACCTGTGCGCTGAAGGGCTCGAGTG 8 We4219
CACTCGAGCCCTTCAGCGCACAGGTTGTCAGCGGG 9 G785C We4226
CAACCTGCGGGCTGAATGCCTCGAGTGTACCAAAACG 10 We4227
CGTTTTGGTACACTCGAGGCATTCAGCCCGCAGGTTG 11 E787C We4236
GGGCTGAAGGGCTCTGCTGTACCAAAACGTGCCAG 12 We4237
CTGGCACGTTTTGGTACAGCAGAGCCCTTCAGCCC 13 A789C We4238
GGGCTCGAGTGTTGCAAAACGTGCCAGAACTATGAC 14 We4239
GTCATAGTTCTGGCACGTTTTGCAACACTCGAGCCC 15 T789C We4238
GGGCTCGAGTGTTGCAAAACGTGCCAGAACTATGAC 14 We4239
GTCATAGTTCTGGCACGTTTTGCAACACTCGAGCCC 15 T791C We4240
GGGCTCGAGTGTACCAAATGCTGCCAGAACTATGACCTG 16 We4241
CAGGTCATAGTTCTGGCAGCATTTGGTACACTCGAGCCC 17 Q793C We4242
GAGTGTACCAAAACGTGCTGCAACTATGACCTGGAGTGC 18 We4243
GCACTCCAGGTCATAGTTGCAGCACGTTTTGGTACACTC 19 N794C We4244
GTACCAAAACGTGCCAGTGCTATGACCTGGAGTGCATGAGC 20 We4245
GCTCATGCACTCCAGGTCATAGCACTGGCACGTTTTGGTAC 21 Y795C We4246
GTACCAAAACGTGCCAGAACTGTGACCTGGAGTGCATGAGC 22 We4247
GCTCATGCACTCCAGGTCACAGTTCTGGCACGTTTTGGTAC 23 M800C We4228
CTATGACCTGGAGTGCTGCAGCATGGGCTGTGTCTC 24 We4229
GAGACACAGCCCATGCTGCAGCACTCCAGGTCATAG 25 R816C We4220
CCCCGGGCATGGTCTGCCATGAGAACAGATGTGTG 26 We4221
CACACATCTGTTCTCATGGCAGACCATGCCCGGGG 27 H817C We4248
GGGCATGGTCCGGTGTGAGAACAGATGTGTGGCC 28 We4249
GGCCACACATCTGTTCTCACACCGGACCATGCCC 29 P828C We4250
TGGCCCTGGAAAGGTGTTGCTGCTTCCATCAGGGC 30 We4251
GCCCTGATGGAAGCAGCAACACCTTTCCAGGGCCA 31 F830C We4252
GAAAGGTGTCCCTGCTGCCATCAGGGCAAGGAG 32 We4253
CTCCTTGCCCTGATGGCAGCAGGGACACCTTTC 33 E835C We4254
CTTCCATCAGGGCAAGTGCTATGCCCCTGGAGAAAC 34 We4255
GTTTCTCCAGGGGCATAGCACTTGCCCTGATGGAAG 35 P838C We4256
GGGCAAGGAGTATGCCTGTGGAGAAACAGTGAAGATT 36 We4257
AATCTTCACTGTTTCTCCACAGGCATACTCCTTGCCC 37 D853C We4258
CACTTGTGTCTGTCGGTGCCGGAAGTGGAACTGCAC 38 We4259
GTGCAGTTCCACTTCCGGCACCGACAGACACAAGTG 39 R854C We4222
CTTGTGTCTGTCGGGACTGCAAGTGGAACTGCACAG 40 We4223
CTGTGCAGTTCCACTTGCAGTCCCGACAGACACAAG 41 K855C We4260
CTGTCGGGACCGGTGCTGGAACTGCACAGACCATG 42 We4261
CATGGTCTGTGCAGTTCCAGCACCGGTCCCGACAG 43 W856C We4262
CTGTCGGGACCGGAAGTGCAACTGCACAGACCATG 44 We4263
CATGGTCTGTGCAGTTGCACTTCCGGTCCCGACAG 45 D879C We4230
CCACTACCTCACCTTCTGCGGGCTCAAATACCTGTTCC 46 We4231
GGAACAGGTATTTGAGCCCGCAGAAGGTGAGGTAGTGG 47 R924C We4224
CCTCAGTGAAATGCAAGAAATGCGTCACCATCCTGGTGG 48 We4225
CCACCAGGATGGTGACGCATTTCTTGCATTTCACTGAGG 49 E933C We4435
GTCGAGGGCGGCTGCATCGAACTGTTCGACGGC 143 We4436
GCCGTCGAACAGTTCGATGCAGCCGCCCTCGAC 91 T951C We4447
GGCCTATGAAGGACGAATGCCATTTCGAGGTGGTCGAG 92 We4448
CTCGACCACCTCGAAATGGCATTCGTCCTTCATAGGCC 93 L984C We4469
CCTGTCCATTAGTGTGGTGTGCAAACAGACCTATCAGGAAAAAGTCTG 94 We4470
CAGACTTTTTCCTGATAGGTCTGTTTGCACACCACACTAATGGACAGG 95 E1015C We4485
CTAGCAACCTGCAGGTCTGCGAGGACCCCGTGG 96 We4486
CCACGGGGTCCTCGCAGACCTGCAGGTTGCTAG 97 Q1053C We4232
CTGCCATAACAACATCATGAAGTGCACGATGGTGGATTCCTCCTG 50 We4233
CAGGAGGAATCCACCATCGTGCACTTCATGATGTTGTTATGGCAG 51 D1076 We4600
GGATTGCAACAAACTGGTCTGCCCTGAACCTTACCTGGACG 98 We4601
CGTCCAGGTAAGGTTCAGGGCAGACCAGTTTGTTGCAATCC 99 E1078C We4234
CAACAAGCTGGTGGACCCCTGCCCATATCTGGATGTCTGC 52 We4235
GCAGACATCCAGATATGGGCAGGGGTCCACCAGCTTGTTG 53 P1079C We4604
CAACAAACTGGTCGATCCTGAATGCTACCTGGACGTGTGTATCTAC 100 We4605
GTAGATACACACGTCCAGGTAGCATTCAGGATCGACCAGTTTGTTG 101 K1116C We4519
GCGCTCAGCACGGATGCGTCGTGACATGGCGC 102 We4520
GCGCCATGTCACGACGCATCCGTGCTGAGCGC 103 N1134C We4525
CCTGCGAGGAACGGTGCCTGCGCGAGAATGGC 104 We4526
GCCATTCTCGCGCAGGCACCGTTCCTCGCAGG 105 E1161C We4531
CACATGCCAGCATCCCTGCCCCCTGGCTTGTCC 106 We4532
GGACAAGCCAGGGGGCAGGGATGCTGGCATGTG 107 R1204C We4539
CGAAGTGGCCGGCTGCAGATTCGCCTCCGGC 108 We4540
GCCGGAGGCGAATCTGCAGCCGGCCACTTCG 109
[0172] Using the protocols and plasmids described above and by
applying molecular biology techniques known to those skilled in the
art (and as described e.g. in Current Protocols in Molecular
Biology, ibid) other constructs can be made by the artisan for
mutation of any amino acid residue within SEQ ID No. 2.
[0173] As the half-life extending principle in these examples
albumin fusion to VWF has been chosen. This is indicated by the
suffix -FP.
[0174] To generate albumin fusions of VWF and VWF mutants,
insertion of linker and albumin cDNA sequences was performed in
analogy to the examples described in WO 2009/156137.
[0175] For generation of an expression cassette containing a VWF
mutant which does not contain the propeptide sequence a mutagenesis
as described above is performed using primers with SEQ ID 54 and
55.
[0176] This will result in a VWF sequence wherein the signal
peptide (amino acids 1 to 22 of SEQ ID no. 2) is fused directly to
the D' region (amino acid 764 of SEQ ID no. 2).
[0177] The following table lists residues that were interchanged
with cysteine in VWF D'D3 domain in a scattered approach:
TABLE-US-00003 Cystein residues in human von Willebrand Factor S
764 D 853 E 957 T 1064 R 768 R 854 R 960 D 1067 P 770 K 855 I 963 V
1075 K 773 W 856 L 966 D 1076 N 780 I 870 A 969 P 1077 R 782 A 873
V 972 E 1078 G 785 L 876 D 975 P 1079 E 787 D 879 L 978 Y 1080 T
789 K 882 S 981 L 1081 T 791 F 885 L 984 I 1094 Q 793 V 892 T 987 A
1105 N 794 Q 895 E 990 A 1108 Y 795 P 902 D 1000 K 1116 M 800 F 905
Q 1003 W 1120 S 801 L 908 D 1006 A 1123 M 802 N 911 S 1009 N 1134 G
813 S 918 L 1012 N 1138 R 816 R 924 E 1015 R 1145 H 817 I 927 P
1018 E 1161 E 818 E 930 F 1021 K 1181 L 824 E 933 S 1024 E 1185 P
828 L 936 V 1027 P 1193 F 830 G 939 R 1035 R 1204 Q 832 N 942 L
1039 S 1208 E 835 R 945 A 1042 T 1213 P 838 K 948 I 1050 S 1217 T
841 T 951 Q 1053 V 1230 K 843 E 954 V 1056 G 1241
Example 2: Generation of FVIII Mutants with Cysteine Residues in
the a3 Domain
[0178] Any FVIII cDNA sequence cloned in an expression plasmid can
be used to introduce Cys mutations into the a3 domain. Preferably a
single-chain FVIII construct with partial B domain depletion is
used (see examples in WO 2004/067566).
[0179] For generating FVIII expression vectors, the FVIII cDNA may
be amplified by polymerase chain reaction (PCR) using primer set of
SEQ ID NO 56 and 57 under standard conditions known to those
skilled in the art (and as described e.g. in Current Protocols in
Molecular Biology, Ausubel F M et al. (eds.) John Wiley & Sons,
Inc.; http:/www.currentprotocols.com/WileyCDA/) from a plasmid
containing FVIII cDNA. The resulting PCR fragment is digested by
restriction endonucleases NheI and NotI and ligated into expression
vector pIRESpuro3 (BD Biosciences, Franklin Lakes, N.J., USA) which
had been linearized by NheI and NotI. The resulting expression
plasmid will contain a cDNA of FVIII downstream of the CMV promoter
and is suitable for FVIII expression in animal cell culture.
[0180] In order to introduce mutations into the FVIII sequence site
directed mutagenesis (QuickChange XL Site Directed Mutagenesis Kit,
Agilent Technologies, La Jolla, Calif., USA) is applied on the
FVIII expression plasmid as suggested by the kit manufacturer.
[0181] The following table lists the oligonucleotides used for
mutagenesis of the FVIII cDNA sequence and the respective mutations
introduced.
TABLE-US-00004 SEQ FVIII Mutagenesis oligonucleotide sequence ID
mutation Designation (5'.fwdarw.3') NO T1654C We4630
GCCACCACAATTCCAGAAAATACTTGCCTTCAGTCAGATCAAGAGG 110 We4631
CCTCTTGATCTGACTGAAGGCAAGTATTTTCTGGAATTGTGGTGGC 111 Q1656C We4634
CACAATTCCAGAAAATACTACTCTTTGCTCAGATCAAGAGGAAATTGAC 112 We4635
GTCAATTTCCTCTTGATCTGAGCAAAGAGTAGTATTTTCTGGAATTGTG 113 D1658C We4196
CTACTCTTCAGTCATGTCAAGAGGAAATTGACTATGATGATACC 58 We4197
GGTATCATCATAGTCAATTTCCTCTTGACATGACTGAAGAGTAG 59 E1660C We4640
CTACTCTTCAGTCAGATCAATGCGAAATTGACTATGATGATACCATATC 114 We4641
GATATGGTATCATCATAGTCAATTTCGCATTGATCTGACTGAAGAGTAG 115 D1663C We4198
CAGTCAGATCAAGAGGAAATTTGCTATGATGATACCATATCAGTTG 60 We4199
CAACTGATATGGTATCATCATAGCAAATTTCCTCTTGATCTGACTG 61 Y1664C We4200
GATCAAGAGGAAATTGACTGTGATGATACCATATCAGTTGAAATG 62 We4201
CATTTCAACTGATATGGTATCATCACAGTCAATTTCCTCTTGATC 63 D1665C We4202
GATCAAGAGGAAATTGACTATTGTGATACCATATCAGTTGAAATGAAGAAGG 64 We4203
CCTTCTTCATTTCAACTGATATGGTATCACAATAGTCAATTTCCTCTTGATC 65 D1666C
We4204 GATCAAGAGGAAATTGACTATGATTGTACCATATCAGTTGAAATGAAGAAGG 66
We4205 CCTTCTTCATTTCAACTGATATGGTACAATCATAGTCAATTTCCTCTTGATC 67
S1669C We4650 GAAATTGACTATGATGATACCATATGCGTTGAAATGAAGAAGGAAGATTTTG
116 We4651 CAAAATCTTCCTTCTTCATTTCAACGCATATGGTATCATCATAGTCAATTTC 117
V1670C We4652 GACTATGATGATACCATATCATGCGAAATGAAGAAGGAAGATTTTGAC 118
We4653 GTCAAAATCTTCCTTCTTCATTTCGCATGATATGGTATCATCATAGTC 119 E1671C
We4206 TGACTATGATGATACCATATCAGTTTGCATGAAGAAGGAAGATTTTGACATTTATG 68
We4207 CATAAATGTCAAAATCTTCCTTCTTCATGCAAACTGATATGGTATCATCATAGTCA 69
M1672C We4608 GATGATACCATATCAGTTGAATGCAAGAAGGAAGATTTTGACATTTATG 120
We4609 CATAAATGTCAAAATCTTCCTTCTTGCATTCAACTGATATGGTATCATC 121 K1673C
We4610 GATACCATATCAGTTGAAATGTGCAAGGAAGATTTTGACATTTATGATG 122 We4611
CATCATAAATGTCAAAATCTTCCTTGCACATTTCAACTGATATGGTATC 123 K1674C We4612
CCATATCAGTTGAAATGAAGTGCGAAGATTTTGACATTTATGATGAGGATG 124 We4613
CATCCTCATCATAAATGTCAAAATCTTCGCACTTCATTTCAACTGATATGG 125 E1675C
We4208 GATGATACCATATCAGTTGAAATGAAGAAGTGCGATTTTGACATTTATGATGAGG 70
We4209 CCTCATCATAAATGTCAAAATCGCACTTCTTCATTTCAACTGATATGGTATCATC 71
D1676C We4210
GATACCATATCAGTTGAAATGAAGAAGGAATGTTTTGACATTTATGATGAGGATG 72 We4211
CATCCTCATCATAAATGTCAAAACATTCCTTCTTCATTTCAACTGATATGGTATC 73 F1677C
We4614 CAGTTGAAATGAAGAAGGAAGATTGCGACATTTATGATGAGGATGAAAATCAG 126
We4615 CTGATTTTCATCCTCATCATAAATGTCGCAATCTTCCTTCTTCATTTCAACTG 127
D1678C We4212 GAAATGAAGAAGGAAGATTTTTGCATTTATGATGAGGATGAAAATCAGAGCCC
74 We4213 GGGCTCTGATTTTCATCCTCATCATAAATGCAAAAATCTTCCTTCTTCATTTC 75
I1679C We4294 GAAATGAAGAAGGAAGATTTTGACTGTTATGATGAGGATGAAAATCAGAGCCC
76 We4295 GGGCTCTGATTTTCATCCTCATCATAACAGTCAAAATCTTCCTTCTTCATTTC 77
Y1680C We4214 GAAGAAGGAAGATTTTGACATTTGCGATGAGGATGAAAATCAGAGCC 78
We4215 GGCTCTGATTTTCATCCTCATCGCAAATGTCAAAATCTTCCTTCTTC 79 D1681C
We4616 GAAGAAGGAAGATTTTGACATTTATTGCGAGGATGAAAATCAGAGCCCCC 128
We4617 GGGGGCTCTGATTTTCATCCTCGCAATAAATGTCAAAATCTTCCTTCTTC 129
E1682C We4216 GGAAGATTTTGACATTTATGATTGCGATGAAAATCAGAGCCCCCGCAG 80
We4217 CTGCGGGGGCTCTGATTTTCATCGCAATCATAAATGTCAAAATCTTCC 81 D1683C
We4618 GGAAGATTTTGACATTTATGATGAGTGCGAAAATCAGAGCCCCCGCAG 130 We4619
CTGCGGGGGCTCTGATTTTCGCACTCATCATAAATGTCAAAATCTTCC 131 E1684C We4620
GGAAGATTTTGACATTTATGATGAGTGCGAAAATCAGAGCCCCCGCAG 132 We4621
CTGCGGGGGCTCTGATTTTCGCACTCATCATAAATGTCAAAATCTTCC 133 N1685C We4622
GGAAGATTTTGACATTTATGATGAGGATGAATGCCAGAGCCCCCGCAG 134 We4623
CTGCGGGGGCTCTGGCATTCATCCTCATCATAAATGTCAAAATCTTCC 135 Q1686C We4624
GAAGATTTTGACATTTATGATGAGGATGAAAATTGCAGCCCCCGCAGC 136 We4625
GCTGCGGGGGCTGCAATTTTCATCCTCATCATAAATGTCAAAATCTTC 137 S1687C We4654
CATTTATGATGAGGATGAAAATCAGTGCCCCCGCAGCTTTCAAAAG 138 We4655
CTTTTGAAAGCTGCGGGGGCACTGATTTTCATCCTCATCATAAATG 139 P1688C We4656
TGATGAGGATGAAAATCAGAGCTGCCGCAGCTTTCAAAAGAAAACACG 140 We4657
CGTGTTTTCTTTTGAAAGCTGCGGCAGCTCTGATTTTCATCCTCATCA 141
[0182] Using the protocols and plasmids described above and in WO
2004/067566 by applying molecular biology techniques known to those
skilled in the art (and as described e.g. in Current Protocols in
Molecular Biology, ibid) any other constructs can be made by the
artisan for mutation of any other amino acid residue within the a3
domain of FVIII.
[0183] The following table lists residues interchanged with
cysteine in Factor VIII a3, C1 and C2 domains.
TABLE-US-00005 Cystein residues in Factor VIII SingleChain T 1653 T
1654 L 1655 Q 1656 S 1657 D 1658 Q 1659 E 1660 E 1661 I 1662 D 1663
Y 1664 D 1665 D 1666 T 1667 I 1668 S 1669 V 1670 E 1671 M 1672 K
1673 K 1674 E 1675 D 1676 F 1677 D 1678 I 1679 Y 1680 D 1681 E 1682
D 1683 E 1684 N 1685 Q 1686 S 1687 P 1688 R 1689 I 2098 S 2119 N
2129 R 2150 P 2153 W 2229 Q 2246
Example 3: Generation of Expression Vectors for FVIII Molecules
with VWF-Derived C-Terminal Extensions
[0184] FVIII molecules with VWF domains or fragments added to its
carboxyterminus were generated by molecular biology methods known
to those skilled in the art. These were used to cotransfect with
VWF-FP to generate heterodimers containing modified FVIII and
VWF-FP which were covalently linked via the CK domains at the
C-terminus of both proteins.
[0185] For that FVIII cDNA was amplified by primers
TABLE-US-00006 (SEQ ID NO: 82) We4323
GTGGCTAGCGCATGGAAATAGAGCTCTCCAC (SEQ ID NO: 83) We4324
CACGCGGCCGCGTTACCGGTGTAGAGGTCCTGTGCCTCGC
and the resulting PCR fragment was inserted into a suitable
expression vector, e.g. pIRESpuro3 (ibid) opened by NheI and NotI.
Through the resulting AgeI and NotI sites the coding sequence of
the VWF-derived C-terminal domains C3-C4-C5-C6-CK (VWF amino acids
2400 to 2813), C5-C6-CK (VWF amino acids 2544 to 2813) or of the CK
domain alone (VWF amino acids 2724 to 2813) that had been amplified
by PCR using primer pairs
TABLE-US-00007 (SEQ ID NO: 84) We4264
GTGACCGGTAACTCCACAGTGAGCTGTCCC (SEQ ID NO: 85) We4267
ACAGCGGCCGCTATCACTTGCTGCACTTCCTGG and (SEQ ID NO: 142) We4265
GTGACCGGTCAAAGGAACGTCTCCTGCCC (SEQ ID NO: 85) We4267
ACAGCGGCCGCTATCACTTGCTGCACTTCCTGG and (SEQ ID NO: 86) We4266
GTGACCGGTTGCAACGACATCACTGCCAG (SEQ ID NO: 85) We4267
ACAGCGGCCGCTATCACTTGCTGCACTTCCTGG,
respectively, were inserted. This resulted in expression vectors
containing FVIII cDNA with C-terminal extensions by VWF C-terminal
domains C3-C4-C5-C6-CK, C5-C6-CK or CK, respectively.
[0186] Into the AgeI restriction site cleavable linker sequences
were introduced which would release the FVIII from the VWF-FP
during FVIII activation. The linker sequences were chosen from
sequences surrounding one of the thrombin cleavage sites of FVIII,
but any other thrombin cleavage site could be used as well (e.g. as
described in WO 03/035861). As an example thrombin cleavage sites
372 and 1689 are represented by the following cDNA sequences:
TABLE-US-00008 CS372 (SEQ ID NO: 87):
.sup.5'ACCGGTGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCC
AAGAAGCATCCTAAAACTTGGACCGGT.sup.3' CS1689 (SEQ ID NO: 88):
.sup.5'ACCGGTGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAA
ACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGACCGGT.sup.3'
[0187] These sequences can be amplified by suitable PCR primers
containing AgeI restriction sites at their termini. PCR fragments
are then cleaved by AgeI and inserted into AgeI opened expression
vectors as described above.
[0188] Similar approaches can be used by the artisan to construct
expression plasmids containing FVIII cDNA molecules where its B
domain or parts of it have been replaced by the VWF D'D3 region or
where the VWF D'D3 region is connected directly or via a linker to
the N-terminus or C-terminus of FVIII.
Example 4: Transfection of Plasmids for Stable Expression of VWF
Mutants in CHO Cells
[0189] Expression plasmids based on pIRESneo3 were grown up in XL10
Gold (Agilent Technologies) and purified using standard protocols
(Qiagen, Hilden, Germany).
[0190] CHO cells, preferably CHO-K1, were transfected using
standard methods, for example nucleofection or lipofection, and
single clones expressing the desired VWF-FP mutant were
selected.
[0191] For proper VWF propeptide cleavage an expression plasmid
encoding protease furin (NM002569.2) is cotransfected together with
the VWF plasmid in a molar ratio of 1:4 (furin:VWF mutant).
Example 5: Transfection of CHO Cells Expressing VWF-FP Mutants and
Transient Expression of FVIII Mutants
[0192] FVIII mutant expression plasmids were purified as described
above. Transient transfections into the stable VWF-FP mutant CHO
clones (example 4) were conducted according to standard
methods.
[0193] Harvest of transient transfections was performed by
centrifugation to separate the cells from supernatant. Aliquots of
the supernatant were generated and the recombinant product was
characterized.
[0194] The following table describes representative results from
transient transfections of FVIII mutant expression plasmids (column
1) into CHO cells stably expressing VWF-FP mutants (column 2). The
results have been selected so that the ratio of covalently linked
FVIII antigen to total FVIII activity (column 7) or the ratio of
covalently linked FVIII antigen to total FVIII antigen (column 8)
are equal to or greater than 1.0, which we have used as the
selection criteria for the most preferred mutant combinations.
[0195] The amount of covalently linked FVIII antigen has been
determined by the assay described in example 6, FVIII and VWF
activity and antigen by assays as described in examples 9 and
10.
TABLE-US-00009 ratio ratio ratio Cys Cys covalent covalent FVIII
residue at residue at covalent FVIII FVIII VWF FVIII/ FVIII/Total
activity/ FVIII aa VWF aa FVIII activity antigen antigen Total
FVIII FVIII FVIII position position [amU/ml].sup.1 [mU/ml] [mU/ml]
[mU/ml] activity antigen antigen 1654 1079 409 257 421 12 1.59 0.97
0.61 1654 1134 424 371 495 56 1.14 0.85 0.75 1656 1134 435 981 436
66 0.44 1.00 2.25 1677 1116 1076 649 1361 1208 1.66 0.79 0.48 1679
1079 398 316 349 12 1.26 1.14 0.91 1679 1116 992 889 1171 1400 1.12
0.85 0.76 1681 768 87 58 56 71 1.50 1.55 1.04 1681 1116 1440 2414
646 1405 0.60 2.23 3.74 1682 768 328 156 147 93 2.11 2.23 1.06 1682
1116 488 1424 397 1575 0.34 1.23 3.59 1683 768 2209 543 721 86 4.07
3.07 0.75 1683 1116 1346 3190 767 1475 0.42 1.76 4.16 1684 768 491
467 711 63 1.05 0.69 0.66 1684 1116 1150 2996 752 1548 0.38 1.53
3.98 1684 1134 741 844 236 63 0.88 3.15 3.58 1686 768 506 490 997
75 1.03 0.51 0.49 1686 1015 1639 1360 2568 331 1.21 0.64 0.53 1686
1116 2744 3180 523 1484 0.86 5.24 6.08 1686 1134 693 914 128 71
0.76 5.43 7.16 1687 768 390 271 797 77 1.44 0.49 0.34 1687 1134 843
804 411 65 1.05 2.05 1.95 1688 768 2058 110 968 69 18.75 2.13 0.11
1688 817 367 194 1279 1636 1.89 0.29 0.15 1688 984 1904 777 1974
438 2.45 0.96 0.39 1688 1015 1083 680 2053 390 1.59 0.53 0.33 1688
1116 666 1083 353 1654 0.61 1.89 3.07 1688 1134 650 348 106 62 1.87
6.11 3.27 2129 817 292 285 898 1789 1.02 0.33 0.32 .sup.1arbitrary
milli-Unit per ml
Example 6: Detection of FVIII Mutants Covalently Attached to VWF
Mutants by Elisa
[0196] Cell culture supernatant samples (10 ml) from transient
transfections were concentrated with Amicon Ultracell-30K
(Millipore UFC903024; 3000 g centrifugation). FVIII covalently
attached to VWF-FP in culture supernatant (concentrates) was
determined by a standard ELISA. Briefly, microplates were incubated
with 100 .mu.L per well of the capture antibody (rabbit anti human
VWF-IgG, Dako A0082 [Dako, Hamburg, Germany], diluted 1:2000 in
buffer A [Sigma C3041, Sigma-Aldrich, Munich, Germany]) overnight
at ambient temperature. After washing plates three times with
buffer B (Sigma T9039), each well was incubated with 200 .mu.L
buffer C (Sigma T6789) for 1.5 hours at ambient temperature
(blocking). After another three wash steps with buffer B, serial
dilutions of the test sample in buffer B as well as serial
dilutions of a control preparation of covalently linked
FVIII-VWF-FP, (2.0-0.03 arbitrary U/ml in buffer B (we call these
"arbitrary units" as they may not correspond to the standard FVIII
units as determined using Standard Human Plasma); volumes per well:
100 .mu.L) were incubated for 1.5 hours at ambient temperature.
After three wash steps with buffer B, 200 .mu.L of 350 mM
CaCl.sub.2 were added to each well and incubated for 1 hour at
ambient temperature. CaCl.sub.2 was removed (without washing) and
additional 200 .mu.l were added to each well and incubated further
for 1 hour. After three wash steps with buffer B 100 .mu.L of a 1:2
dilution in buffer B of the detection antibody (Detecting Antibody
for FVIII:C, peroxidase labelled, Cedarlane CL20035K-D) were added
to each well and incubated for 1 hour at ambient temperature. After
three wash steps with buffer B, 100 .mu.L of substrate solution
(OUVF, Siemens Healthcare Diagnostics) were added per well and
incubated for 15 minutes at ambient temperature in the dark.
Addition of 100 .mu.L undiluted stop dilution (OSFA, Siemens
Healthcare Diagnostics) prepared the samples for reading in a
suitable microplate reader at 450 nm wavelength. Concentrations of
the test samples were then calculated using the standard curve with
the control preparation.
Example 7: Detection of FVIII Mutants Covalently Attached to VWF
Mutants by Western Blot and Coomassie Stain
[0197] Alternatively covalent complexes were detected by staining
or Western blotting. Samples were examined with denaturing SDS-PAGE
under reducing or non-reducing conditions and subsequent Western
blot. For the detection of FVIII, an in house murine anti-FVIII
monoclonal antibody mix followed by an alkaline phosphatase coupled
secondary anti-mouse antibody (Invitrogen) and for VWF detection an
HRP labeled polyclonal rabbit anti-human VWF (Fa. Dako P0226)
antibody were used.
[0198] FIGS. 8A and 8B show the Western blot analysis of FVIII
(FVIII-SingleChain) covalently linked to rVWF-FP dimers by the two
principles described above from a non-reduced SDS-PAGE. FIG. 8A has
been detected using anti-FVIII antibodies, FIG. 8B using anti-VWF
antibodies. Lane 1 represents material linked where the FVIII
moiety is linked to a VWF-FP dimer by a VWF derived C3-C4-C5-C6-CK
sequence added to its C-terminus and an additional thrombin
cleavage site between FVIII and the C3-C4-C5-C6-CK sequence. It had
a ratio of covalent FVIII as measured by the specific Elisa
described in example 8 to total FVIII activity of 5.78 and to total
FVIII antigen of 7.47. High molecular weight bands above 460 kDa
are visualized by both anti-FVIII and anti-VWF antibodies and
demonstrate the presence of covalently linked FVIII-VWF-FP
complexes. M denotes the molecular size marker. Lanes 2 and 3
represent control preparations of FVIII-SC and VWF-FP,
respectively.
[0199] Lanes 4 and 5 represent covalent complexes linked through
disulfide bridges between FVIII a3 and VWF-FP D3 domains by
respective Cys mutations. Lane 4 represents FVIII-SC I1679C mutant
on VWF-FP E1078C mutant. Lane 5 represents FVIII-SC I1675C mutant
on VWF-FP E1078C mutant. Lane 6 is a control preparation only
containing VWF-FP mutant E1078C. The blot demonstrates the presence
of high molecular weight FVIII-VWF-FP complexes covalently linked
to each other besides free FVIII molecules.
[0200] FIG. 9 shows a reduced SDS-PAGE stained with Gelcode Blue
Stain reagent. Lane 1 contains a purified FVIII (FVIII-SingleChain)
covalently linked to a rVWF-FP dimer, lane 2 shows the same
preparation after thrombin digest releasing the covalently linked
FVIII moiety in the linker sequence while in parallel activating
FVIII. The bands in lane 1 represent the covalent complex and FVIII
dimers, the prominent band in lane 2 between the 268 and 460 kDa
markers represents the VWF-FP moiety, while the bands in the below
71 KDa range represent FVIII fragments.
Example 8: Chemical Crosslinking of FVIII to VWF
[0201] FVIII is reacted in an aqueous buffer solution containing
preferably physiological NaCl and CaCl.sub.2 concentrations at a
constant temperature of preferably between 4.degree. C. and
37.degree. C. with a bi-specific bis-succinimide ester (PEG)n with
a molecular weight of between 500 Da and 100 kDa in a molar ratio
of FVIII and cross-linker of 2:1 to 1:1000 (preferred about 1:1).
The FVIII concentration is preferably low to minimize cross-linking
of FVIII with itself. After a period of 1 min to 60 min a half-life
extended VWF is added to the FVIII solution in a molar excess of
2:1 to 200:1 based on the monomer building units of VWF. After an
incubation period of 1 to 300 min at the temperature given above
the residual reagent is quenched using preferably a low molecular
weight compound containing a primary amino group and the covalent
complex of FVIII and VWF is purified by methods known to the expert
in the field, removing non-reacted FVIII or oligomers of FVIII and
non-reacted VWF. The reaction times and temperatures for the
different incubation steps are optimized by methods known to the
expert in the field, e.g. by using SDS-PAGE/Western blot analysis
with anti-FVIII or anti-VWF antibodies with the goal to maximize
the content of the desired covalent complex and to minimize the
content of side products.
[0202] Different reagents can be used for the chemical
cross-linking of modified FVIII and VWF molecules. They are based
on the cross-linking of different reactive groups of FVIII and VWF:
[0203] a) Amine-to-Amine cross-linkers (e.g. bis-Imidoester(PEG)n
or bis-succinimide ester(PEG)n) [0204] b) Carboxyl-to-Carboxyl
cross-linkers [0205] c) Sufhydryl-to-Sulfhydryl cross-linkers (e.g.
bis-maleimide(PEG)n) [0206] d) Carbohydrate-to-Carbohydrate
cross-linkers [0207] e) Amine-to-Sulfhydryl cross-linkers [0208] f)
Sulfhydryl-to-Carbohydrate cross-linkers [0209] g)
Sulfhydryl-to-Hydroxyl cross-linkers [0210] h) Carboxyl-to-Amine
cross-linkers
Example 9: Analysis of Factor VIII Activity and Antigen
[0211] For activity determination of FVIII:C in vitro either a
clotting assay (e.g. Pathromtin SL reagent and FVIII deficient
plasma delivered by Dade Behring, Germany) or a chromogenic assay
(e.g. Coamatic FVIII:C assay delivered by Haemochrom) are used. The
assays are performed according to the manufacturers'
instructions.
[0212] FVIII antigen (FVIII:Ag) is determined by a standard ELISA.
Briefly, microplates are incubated with 100 .mu.L per well of the
capture antibody (sheep anti-human FVIII IgG, Cedarlane CL20035K-C,
diluted 1:200 in Buffer A [Sigma C3041]) for 2 hours at ambient
temperature. After washing plates three times with buffer B (Sigma
P3563), serial dilutions of the test sample in sample diluent
buffer (Cedarlane) as well as serial dilutions of a FVIII
preparation (CSL Behring; 200-2 mU/mL) in sample diluent buffer
(volumes per well: 100 .mu.L) are incubated for two hours at
ambient temperature. After three wash steps with buffer B, 100
.mu.L of a 1:2 dilution in buffer B of the detection antibody
(sheep anti-human FVIII IgG, Cedarlane CL20035K-D, peroxidase
labelled) are added to each well and incubated for another hour at
ambient temperature. After three wash steps with buffer B, 100
.mu.L of substrate solution (1:10 (v/v) TMB OUVF:TMB Buffer OUVG,
Dade Behring) are added per well and incubated for 30 minutes at
ambient temperature in the dark. Addition of 100 .mu.L stop
solution (Dade Behring, OSFA) prepares the samples for reading in a
suitable microplate reader at 450 nm wavelength. Concentrations of
test samples are then calculated using the standard curve with the
FVIII preparation as reference.
Example 10: Analysis of VWF Activity and Antigen
[0213] Samples are analysed by immunoturbidimetric determination of
VWF:Ag (OPAB03, Siemens Healthcare Diagnostics, Marburg, Germany)
and for collagen binding (Technozym VWF:CBA ELISA, Ref. 5450301
with calibrator set 5450310 and control set 5450312, Technoclone,
Vienna, Austria) as described by the manufacturer.
[0214] VWF:RCo testing is done using the BC VWF reagent of Siemens
Healthcare Diagnostics, Marburg, Germany according to the
manufacturer's description. The International Concentrate Standard
is used as a primary standard preparation to calibrate an in-house
standard preparation for day to day use.
[0215] For pharmacokinetic analyses VWF antigen is determined by a
standard ELISA. Briefly, microplates are incubated with 100 .mu.L
per well of the capture antibody (rabbit anti human vWF-IgG, Dako
A0082 [Dako, Hamburg, Germany], diluted 1:2000 in buffer A [Sigma
C3041, Sigma-Aldrich, Munich, Germany]) overnight at ambient
temperature. After washing plates three times with buffer B (Sigma
P3563), each well is incubated with 200 .mu.L buffer C (Sigma
P3688) for 1.5 hours at ambient temperature (blocking). After
another three wash steps with buffer B, serial dilutions of the
test sample in buffer B as well as serial dilutions of standard
human plasma (ORKL21; 20-0.2 mU/mL; Siemens Healthcare Diagnostics,
Marburg, Germany) in buffer B (volumes per well: 100 .mu.L) are
incubated for 1.5 hours at ambient temperature. After three wash
steps with buffer B, 100 .mu.L of a 1:16000 dilution in buffer B of
the detection antibody (rabbit anti human vWF-IgG, Dako P0226,
peroxidase labelled) are added to each well and incubated for 1
hour at ambient temperature. After three wash steps with buffer B,
100 .mu.L of substrate solution (OUVF, Siemens Healthcare
Diagnostics) are added per well and incubated for 30 minutes at
ambient temperature in the dark. Addition of 100 .mu.L undiluted
stop dilution (OSFA, Siemens Healthcare Diagnostics) prepares the
samples for reading in a suitable microplate reader at 450 nm
wavelength. Concentrations of the test samples are then calculated
using the standard curve with standard human plasma as
reference.
Example 11: VWF Multimer Analysis
[0216] VWF multimer analysis was performed by SDS-agarose gel
electrophoresis as recently described (Tatewaki et al. Thromb. Res.
52: 23-32 (1988), and Metzner et al., Haemophilia 4 (Suppl. 3):
25-32 (1998)) with minor modifications. Briefly, after
equilibration in running buffer ready to use 1% agarose mini gels
(BioRad) were used to standardize the method as far as possible.
Comparable amounts of VWF antigen were subjected to electrophoresis
on the SDS-agarose gels. After Western blotting the protein bands
were detected using anti-VWF, anti-FVIII or anti-albumin antibodies
followed by alkaline phosphatase labelled anti-IgG antibodies
(SIGMA, prod. No. 1305) and colour reaction quantified by
densitometry.
[0217] Two preparations of covalent FVIII-SC/VWF-FP multimer
complexes were analysed by multimer gel analysis. Lanes 2 and 3 of
FIGS. 10A and 10B represent material wherein the FVIII moiety is
linked to a VWF-FP multimer by a VWF derived C3-C4-C5-C6-CK
sequence added to its C-terminus with an additional thrombin
cleavage site between FVIII and the C3-C4-C5-C6-CK sequence
(example 3). Lane 1 represents plasma-derived VWF, lane 4 VWF-FP.
The results demonstrate that covalent FVIII/VWF-FP complexes do
multimerize to an extent similar to VWF-FP or natural VWF.
Additional bands represent the addition of one or more covalent
FVIII molecules. Most multimer bands detected by anti-VWF
antibodies (FIG. 10A) can also be stained by anti-FVIII (FIG. 10B),
demonstrating covalent FVIII/VWF-FP multimers.
Example 12: Purification of Covalently Linked FVIII/VWF-FP
Complexes
[0218] Cell culture supernatants containing covalently linked
FVIII/VWF-FP dimer complexes are sterile-filtered through a 0.2
.mu.m filter and concentrated with a 30 kDa UF unit
(Centramate.TM., Pall) up to 20-fold. Cell culture supernatants
containing covalently linked FVIII/VWF-FP multimer complexes are
sterile-filtered through a 0.2 .mu.m filter and concentrated with a
Cadence.TM. Single-Use Inline Concentrator (30 kDa cut-off, Pall).
This material is then applied to a Human Albumin Capture Select
column (BAC) equilibrated with equilibration buffer (EB, 20 mM Tris
pH 7.0). The column is washed with EB and FVIII/VWF-FP complexes
are eluted with 2M MgCl.sub.2 in EB. The elution peak is pooled and
dialysed against running buffer of the SEC HiPrep Sephacryl S-500
High Resolution (GE Healthcare) containing 50 mM HEPES, 400 mM
CaCl.sub.2, 50 mM NaCl, pH 7, as described by McCue et al., 2009;
J. Chrom. A, 1216(45): 7824-30 with minor modification). This
material is then applied to a preequilibrated SEC HiPrep Sephacryl
S-500 High Resolution (GE Healthcare) and after separating by size
only the fractions containing the covalently linked FVIII/VWF-FP
were pooled and SEC HiPrep Sephacryl S-500 High Resolution (GE
Healthcare). This pool is dialysed against 1.7 mM CaCl.sub.2, 10 mM
L-His, 308 mM NaCl, 8.76 mM saccharose, 0.01% Tween 80, pH 7.
Finally the material is frozen in aliquots.
[0219] Alternatively for certain constructs the VIIISelect column
(GE Healthcare) may provide better purification results than the
Human Albumin Capture Select Column. In such case, the cell culture
supernatant concentrate is applied to a preequilibrated VIIISelect
column (GE Healthcare) and after washing with equilibration buffer
(10 mM HEPES, 5 mM CaCl.sub.2, 150 mM NaCl, 0.03% Tween80 pH 7), it
is followed by equilibration buffer with a high salt concentration
(1 M NaCl) and then again by equilibration buffer. The FVIII/VWF-FP
complexes are eluted with 20 mM L-His, 5 mM CaCl.sub.2, 150 mM
NaCl, 60% ethylene glycol, 0.03% Tween 80, pH 7. he elution peak is
pooled and dialysed against the running buffer of the subsequent
SEC column, containing 50 mM HEPES, 400 mM CaCl.sub.2, 50 mM NaCl,
pH 7. This material is then applied to a preequilibrated SEC HiPrep
Sephacryl S-500 High Resolution (GE Healthcare) column. Fractions
containing the covalently linked FVIII/VWF-FP are pooled. This pool
is dialysed against 1.7 mM CaCl.sub.2, 10 mM L-His, 308 mM NaCl,
8.76 mM saccharose, 0.01% Tween 80, pH 7. Finally the material is
frozen in aliquots.
Example 13: Pharmacokinetic Analysis of Covalently Linked FVIII/VWF
Complexes in FVIII Deficient Mice and in Rats
[0220] The FVIII/VWF complexes are administered intravenously to
FVIII deficient mice (12 mice per substance) with a dose of 100
IU(FVIII:Ag)/kg body weight. Blood samples are drawn at appropriate
intervals using an alternating sampling scheme, resulting in
samples from 3 animals/timepoint (t=0 min and 16 h for subset No 1,
5 min and 24 h for subset No 2, 2 h and 4 h for subset No 3, and 8
h and 32 h for subset No 4). The scheme is designed to minimize
potential effects of blood sampling on the plasma concentration to
be quantified. Blood is processed to plasma and stored deep frozen
until analysis. FVIII and VWF antigen content is subsequently
quantified by specific ELISA assays (see examples 7, 9 and 10). The
mean values of the treatment groups are used to calculate in vivo
recovery after 5 min. Half-lives are calculated using the time
points of the beta phase of elimination according to the formula
t1/2=In2/k, whereas k is the slope of the regression line. Antigen
is usually used as a measure in pharmacokinetic studies. It is
expected that antigen and functional activity will correlate.
[0221] The FVIII/VWF complexes are administered intravenously to
narcotized CD/Lewis rats (6 rats per substance) with a dose of 100
IU(VWF:Ag)/kg body weight. Blood samples are drawn at appropriate
intervals starting at 5 minutes after application of the test
substances using an alternating sampling scheme, resulting in
samples from 3 animals/timepoint (t=0, 5, 30, 90 min, 4 h, 1 d for
subset Nr. 1 and 0, 15 min, 1, 2, 8 h and 2 d for subset Nr. 2).
The scheme is designed to minimize potential effects of blood
sampling on the plasma concentration to be quantified. Blood is
processed to plasma and stored deep frozen until analysis. FVIII
and VWF antigen content is subsequently quantified by specific
ELISA assays (see above). The mean values of the treatment groups
are used to calculate in vivo recovery after 5 min. Half-lives are
calculated using the time points of the beta phase of elimination
according to the formula t.sub.1/2=In2/k, whereas k is the slope of
the regression line. Antigen is usually used as a measure in
pharmacokinetic studies in normal animals in order to eliminate the
background of the intrinsic FVIII activity in the animals from the
measurements. It is expected that antigen and functional activity
will correlate.
[0222] A covalent FVIII/VWF-FP preparation consisting of a
single-chain FVIII sequence with the VWF C3 to C6 and CK domains
attached to its carboxyterminus via a cleavable linker and an
albumin-fused VWF (as described in example 3) were tested for their
half-lives in a rat PK model. FIGS. 11A and 11B show the
elimination kinetics of the covalent complex (circles, named
FVIII-CK+VWF-FP in the figure legend) in comparison to a
recombinant FVIII (Advate, squares in FIG. 11A), a VWF-FP (squares
in FIG. 11B) and a plasma-derived FVIII-VWF complex (Haemate,
triangles). FIG. 11A shows the FVIII data (the covalent complex
being measured by the specific Elisa as described in example 6; all
other compounds were measured by FVIII Elisa), FIG. 11B shows the
data of a VWF Elisa. The elimination kinetics of the covalent
construct were similar when FVIII and VWF antigen were measured, as
expected when both moieties were covalently attached. The terminal
half-life for FVIII antigen was calculated to be 7.9 hours, that of
VWF 7.8 hours. Surprisingly the terminal half-life calculated for
the VWF-FP control (VWF antigen) was very similar, 8.1 hours.
Clearance rates were also similar with 9.8 IU/mL/h for the covalent
complex and 10.1 IU/mL/h for VWF-FP. The half-life of rFVIII
(Advate) was calculated with 2.5 hours, which would result in an
about 3-fold half-life extension of the covalent complex over free
FVIII.
[0223] These results indicate that the covalent attachment of a
FVIII sequence to a half-life extended VWF molecule does extend the
half-life of that FVIII molecule significantly and to an extent
that it resembles the half-life of the unfused half-life extended
VWF molecule.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180092966A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180092966A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References