WO1995013301A1

WO1995013301A1 - Cross-linked factor viii

Info

Publication number: WO1995013301A1
Application number: PCT/DK1994/000424
Authority: WO
Inventors: Egon Persson
Original assignee: Novo Nordisk A/S
Priority date: 1993-11-12
Filing date: 1994-11-10
Publication date: 1995-05-18
Also published as: AU1062895A; DK128193D0

Abstract

A new cross-linked Factor VIII polypeptide showing a coagulant activity of Factor VIII wherein the cross-linking is an inter-chain cross-linking. A cross-linked Factor VIIIa polypeptide showing a coagulant activity of Factor VIII wherein the cross-linking is an inter-chain cross-linking is stable at 4°C for an extended period. A new cross-linked Factor VIII polypeptide showing a coagulant activity of Factor VIII may be prepared by incubating the Factor VIII polypeptide with a cross-linking agent in a buffer having in the presence of a non-ionic surfactant whereafter lysine is added for quenching excess cross-linking agent optionally followed by isolation of the cross-linked Factor VIII polypeptide.

Description

TITLE

Crosslinked Factor VIII

FIELD OF THE INVENTION

The present invention relates to crosslinked Factor VIII showing a coagulant activity of Factor VIII, activated crosslinked Factor VIII (Factor Villa) as well as a method for the preparation of crosslinked Factor VIII and activated Factor VIII. Furthermore, the invention relates to pharmaceutical preparations comprising a crosslinked Factor VIII and the use of crosslinked Factor VIII for the preparation of a pharmaceutical preparation for the treatment of diseases caused by an absence or deficiency of the Factor VIII of a subject.

BACKGROUND OF THE INVENTION

Haemophilia A is an X-chromosome-linked inherited disease which afflicts 1-2 males per 10,000. The disease is caused by an absence or deficiency of Factor VIII. Factor VIII is a large glycoprotein (native M_r 330000 - 360000), which is present in plasma at low concentrations (0.1 nM (Rapaport, West.J.Med. (1993) 158:153-161)). It is an essential element in the proteolytic cascade which converts soluble fibrinogen to insoluble fibrin, forming a clot to prevent blood loss from traumatized tissue. In the bloodstream, it is found in noncovalent association with von Willebrand factor (vWF) which acts as a stabilizing carrier protein. Factor VIII is susceptible to cleavage by thrombin, activated protein C, plasmin, and other serine proteases. It is generally isolated from plasma or plasma products as a series of related polypeptides ranging from M_r 160000-40000 with predominant species of M_r 92000 and M_r 80000-77000. This complex pattern has made the analysis of the structure of active Factor VIII very difficult. Factor VIII and the related polypeptides have been described by F. Rotblat et al, Biochemistry (1985) 24:4294-4300; G.A. Vehar et al, Nature (1984) 312:337-342; J.J. Toole et al, Nature (1984) 312:342-347; and M.A. Truett et al, DNA (1985) 4:333-349. The sequence has been reported by J.J. Toole et al, supra; W.I. Wood et al, Nature (1984) 312:330-336; and M.A. Truett et al, supra.

The full-length protein contains three repeats of the A-domain and two repeats of the C-domain together with a heavily glycosylated B-domain, ordered A1-A2-B- A3-C1-C2. The B-domain is not required for the function of Factor VIII (Burke et al. (1986) J.Biol.Chem. 261 :12574-12578).

By thrombin activation, the heavy chain is cleaved between the A1 and the A2- domains and 41 amino acids are cleaved off from the N-terminus of the light chain.

Factor VIII has historically been isolated from blood in a concentrated form for therapeutic treatment of haemophilia. However, Factor VIII is only present in the blood in extremely small amounts and a vast number of donors have to be involved. Moreover, the purification process is laborious and expensive. Concerns regarding transmission of HIV and other blood-borne diseases as well as shortage of supplies have especially stimulated activity to provide alternative supplies of Factor VIII, thus leading to the development of recombinant techni- ques.

The preparation of proteins having Factor VIII activity by recombinant techniques has inter alia been disclosed in a number of patent publications. Thus, European Patent Application No. 160 457 and International Patent Application No. WO 86/01961 disclose recombinant production of full length Factor VIII, European Patent Application No. EP 150 735 discoses a complex of subunits of Factor VIII having coagulant activity and recombinant production of subunits of Factor VIII, European Patent Application No. EP 232 112 and International Patent Application No. WO 91/07490 disclose co-expression of subunits for the production of com¬ plexes showing coagulant activity, and International Patent Application No. WO 86/06101 , International Patent Application No. WO 87/04187, International Patent Application No. WO 87/07144, International Patent Application No. WO 88/00381 , European Patent Application No. EP 251 843, European Patent Application No. EP 253 455, European Patent Application No. EP 254 076, U.S. Patent No. 4.980.456, European Patent Application No. EP 294 910, European Patent Application No. EP 265 778, European Patent Application No. EP 303 540, and In¬ ternational Patent Application No. WO 91/09122 disclose recombinant expression of Factor VIII having one or more deletions in the molecule, or binding to an¬ tibodies inhibiting Factor VIII.

The development of the recombinant techniques has provided a means for ensuring sufficient supplies of Factor VIII.

The lack of stability of Factor VIII still imposes difficulties on the storing and handling of Factor VIII giving rise to vast losses of Factor VIII proteins.

Fay Biochimica et Biophvsica Acta(1987) 952:181-190 discloses a chemical crosslinking of Factor Villa giving a Factor Villa dimer composed of the 73000 and 51000 subunits comprising chemical "intra-chain crosslinks" and retaining more than 60% of its initial clotting activity. The resulting stabilized Factor Villa still had "a significant level of activity to allow the determination of the sedimentation coefficient" after 16 hours. However, there is no indication that the Factor Villa dimer prepared by Fay should retain coagulant activity for extended periods of time.

Thus, there is still a need for a method of stabilizing Factor VIII in order to ensure that as few units of Factor VIII as possible are lost during activation as well as storing and handling of Factor VIII and pharmaceutical preparations comprising the same. BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a crosslinked Factor VIII polypeptide. Such crosslinked Factor VIII polypeptides have surprisingly been found to be activatable using thrombin to form an activated crosslinked Factor Villa polypeptide exhibiting a very stable coagulant activity.

The invention also relates to a crosslinked Factor Villa polypeptide exhibiting a coagulant activity of Factor VIII. Such crosslinked Factor VIII polypeptides has surprisingly been found to be very stable and to retain a high activity over extended periods of time after activation by thrombin.

Furthermore, the invention relates to a method for preparing a crosslinked Factor VIII polypeptide having an inter-chain crosslinks and a method for preparing a crosslinked Factor Villa polypeptide having an inter-chain crosslinking.

The invention also relates to a pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide or a crosslinked Factor Villa polypeptide (activated Factor VIII).

In a further aspect, the invention relates to the use of a crosslinked Factor VIII polypeptide or a crosslinked Factor Villa polypeptide for the preparation of a pharmaceutical preparation.

In a still further aspect, the invention relates to methods for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject.

Still further the invention relates to a method for preparing a pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide or a crosslinked Factor Villa polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further explained with reference to the drawings on which

Fig. 1 shows the bands on SDS-PAGE of peak activity fraction of crosslinked Factor Villa as compared to Factor Villa.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a crosslinked Factor VIII polypeptide wherein the crosslinking is an inter-chain crosslinking. Such crosslinked Factor VIII polypeptides have surprisingly been found to be very stable and to give rise to coagulant activity upon activation using thrombin.

The Factor VIII polypeptide may be full length Factor VIII or a Factor VIII having one or more deletions in the molecule, subunits of Factor VIII or complexes of subunits of Factor VIII bound by ionic bridges provided that thrombin activation imparts the Factor VIII polypeptide coagulant activity.

The Factor VIII polypeptide may be isolated from plasma by methods known per se, e.g. as described in EP patent No. 83483, EP patent No. 150735 or EP patent No. 197901 or produced by recombinant techniques, e.g. as described in the patent applications listed above. The Factor VIII polypeptide may be purified by standard techniques.

The ionic bridge may be a divalent metal bridge comprising e.g. calcium, cobalt or manganese ions.

A preferred Factor VIII polypeptide is a calcium-bound complex of the M_r 92000 and M_r 77/80000 doublet subunits of Factor VIII. In a second aspect, the invention relates to a crosslinked Factor Villa polypeptide having inter-chain crosslinking and exhibiting a coagulant activity of Factor VIII. Such crosslinked Factor VIII polypeptides have surprisingly been found to be very stable and to retain a high activity over periods of several weeks after activation by thrombin.

Factor Villa is composed of three subunits; the A1 and A2 domains derived from the heavy chain of Factor VIII, and the light chain (A3-C1-C2) from which a 41 - amino-acid peptide has been released. A1 and A3-C1-C2 are held together by a high affinity divalent metal ion binding site. A2 is weakly bound to this complex, and dissociation of A2 and A1/A3-C1-C2 causes loss of Factor Villa activity.

The crosslinked Factor VIII polypeptides of the invention preferably comprise the M_r 92000 fragment calcium-bound to the M_r 80000 fragment and comprising a further chemical crosslinking. The crosslinking may be interchain or intrachain crosslinking or a combination thereof which after activation using thrombin gives rise to an activated Factor VIII comprising the M_r 50000 fragment, the M_r 40000 fragment of the M_r 92000 fragment and the M_r 73000 fragment of the M_r 80000 fragment bound by a ionic bridge and a chemical crosslink.

Such chemical crosslinking may connect the M_r 50000 and M_r 40000 fragments or the M_r 40000 and M_r 73000 of activated Factor VIII.

Thus, the invention relates to Factor VIII polypeptides comprising an ionic bridge and a chemical crosslinking and comprising the following fragments of Factor VIII:

1) M_r 92000 and M_r 80000 fragments

2) M_r 92000 and M_r 73000 fragments

3) M_r 50000, M_r 40000, and M_r 73000 fragments. In a further aspect, the invention relates to a method for preparing a crosslinked Factor VIII polypeptide having inter-chain crosslinking and exhibiting a coagulant activity of Factor VIII wherein the Factor VIII polypeptide is incubated with a crosslinking agent at a tempereture from -1 to +15 °C for a period of 5 to 60 minutes in a buffer having a pH from 6 to 8 in the presence of a non-ionic surfactant whereafter lysine is added for quenching excess crosslinking agent optionally followed by isolation of the crosslinked Factor VIII polypeptide.

The crosslinking agent to be used in accordance with the invention may be any crosslinking agent crosslinking primary amino groups and having no adverse effect on the coagulant activity of the crosslinked Factor VIII or ability for activation by thrombin, e.g. N-hydroxysuccinimide ester-based crosslinking agent such as disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl)suberate (BS³).

In a preferred aspect of the invention the Factor VIII polypeptide is incubated with from 0.1 to 10 mM bis(sulfosuccinimidyl) suberate or disuccinimidyl suberate as crosslinking agent, preferably from 0.1 to 5 mM.

The non-ionic surfactant may be a polysorbate, e.g. polysorbate 20, 80, etc. or a poloxamer, e.g. poloxamer 188.

A preferred non-ionic surfactant to be used in the method of the invention is polysorbate 80 which is inter alia sold under the trade mark Tween 80.

The invention also relates to a method for preparing an activated, crosslinked Factor VIII polypeptide (crosslinked Factor Villa) having an inter-chain crosslinking in which a crosslinked Factor VIII polypeptide showing a coagulant activity of Factor VIII and having an inter-chain crosslinking is activated using thrombin. The invention furthermore relates to a pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide and optionally pharmaceutically acceptable vehicle and/or excipient.

A pharmaceutical preparation comprising crosslinked Factor Villa and optionaly pharmaceutically acceptable carrier and/or diluent is also considered an aspect of the present invention.

In a further aspect, the invention relates to the use of a crosslinked Factor VIII polypeptide for the preparation of a pharmaceutical preparation.

A crosslinked Factor VIII polypeptide is preferably used for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.

In another aspect, the invention relates to the use of a crosslinked Factor Villa polypeptide for the preparation of a pharmaceutical preparation.

A crosslinked Factor Villa polypeptide is preferably used for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.

In another aspect, the invention relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject comprising administering to the subject a pharmaceutically active amount of a crosslinked Factor VIII polypeptide.

In yet another aspect, the invention relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject comprising administering to the subject a pharmaceutically active amount of a crosslinked Factor Villa polypeptide. Such diseases may e.g. be haemophilia A, both patients suffering from lack of Factor VIII due to lack of production or induction of Factor VIII being inactive, and inhibitor patients developing antibodies to Factor VIII. The chemical crosslinking itself may modify the epitope of Factor VIII recognized by the antibodies of an inhibitor patient and thus be used directly for treating the haemophilia and bypassing the inhibitor activity without having to take any special measures to neutralize or "by-pass" the antibodies.

In yet another aspect, the invention relates to a method of preparing a pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide and optionally pharmaceutically acceptable vehicle or excipient comprising combining the crosslinked Factor VIII polypeptide with pharmaceutically acceptable vehicle and/or excipient and forming a suitable dosis form of the pharmaceutical preparation.

In still another aspect the invention relates to a method of preparing a pharmaceutical preparation comprising a crosslinked Factor Villa polypeptide and optionally pharmaceutically acceptable vehicle or excipient comprising combining the crosslinked Factor Villa polypeptide with pharmaceutically acceptable vehicle and/or excipient and forming a suitable dosis form of the pharmaceutical preparation.

A suitable dosis form may e.g. be a lyophilized powder to be reconstituted with water for injection. Such lyophilized powder may be presented in a vial or in a prefilled syringe or pen device, e.g. a dual chamber syringe. The crosslinked Factor VIII or Factor Villa may also be presented in the form of a solution to be used in e.g. a pen device.

A pharmaceutical preparation according to the invention may comprise further pharmaceutical excipients well known to those skilled in the art. These include, for example, various bulking agents, additional buffering agents, chelating agents, antioxidants, preservatives, stabilizers, and the like.

A stable Factor Villa heterotrimer was produced by crosslinking recombinant Factor VIII (lacking the entire B domain) using disuccinimidyl suberate (DDS) prior to activation by thrombin. Thrombin cleavage of DSS-treated Factor VIII resulted in an 8-15 fold increase in activity as measured by an APTT assay. The activity was stable at 4°C during a 15-day period. In contrast, the activity of native Factor Villa rapidly decreased to approximately 5% of that of the original Factor VIII. Crosslinking per se using 0.5 mM disuccinimidyl suberate did not affect the biological activity of Factor VIII as measured in an APTT assay, indicating normal thrombin-catalyzed activation kinetics and cofactor activity.

In order to characterize the crosslinked Factor Villa, the active molecules were purified by a combination of immunoaffinity and gel permeation chromatography. The Factor Villa activity could be adsorbed to immobilized monoclonal antibodies against the A2 and C2 domains, respectively. SDS-PAGE of reduced samples revealed the presence of several molecular species. Two bands around 150 kDa gave positive results with both antibodies in Western blotting analysis. The molecular masses, which are similar to that of Factor VIII lacking the B domain, suggested that these bands represent A1/A2/A3-C1-C2 heterotrimers stabilized by covalent interdomain bonds. Bands corresponding to free A1 , A2, and A3-C1- C2 were also observed indicating the presence of non-covaleπtly stabilized heterotrimers.

As judged by the adsorption to F25-Sepharose, Factor Villa activity required the presence of the A2 domain. Crosslinked Factor Villa was then separated from free A2 by gel filtration and the material in the fraction with peak activity contained bands corresponding to a molecular weight about 150,000. These findings are new as compared with the results presented in Biochimica et Biophysica Acta (Supra) where no evidence for interchain crosslinks was obtained and hence no molecules with a molecular weight of more than 80,000 were detected. In the light of the present knowledge it is to be expected that all domains of Factor Villa need to be present to ensure coagulant activity of Factor VIII and thus that the active Factor Villa molecules must have a molecular weight of at least 150,000 (under denaturing conditions).

A species of Factor Villa supported by covalent interchain bonds with a long lifespan has obvious advantages over the native molecule with evanescent activity that vanishes only minutes after activation by thrombin. Due to the stability, crosslinked Factor Villa might be used for preparation of pharmaceutical preparations and could also be immobilized and used in the study of interactions with other proteins using solid-phase techniques such as the recently introduced biosensor technique disclosed in J. Chrom. (1992) 597:397-410.

MATERIALS AND METHODS

EΞXPERIMENTAL PART

Materials and methods

As source of Factor VIII was used a complex of the 92.5 kd and 80 kd subunits of Factor VIII joined by a calcium bridge and prepared as disclosed in WO 91/07490.

Monoclonal antibody F-25 was prepared by purifying Factor VIII HC from plasma as described in WO 88/00210. Using this isolated Factor VIII HC the monoclonal antibody was prepared using the procedure disclosed in Thromb. Haemostas. 1985:54, 586-590. Purification of Factor VIII

Conditioned medium comprising recombinant Factor VIII in the form of a complex of the M_r 90000 and M_r 80000 subunits of Factor VIII joined by a calcium bridge prepared as disclosed in WO 91/07490 containing 20 U Factor VIII per ml was filtrated. The filtrate was applied to a cation-exchange S-F (Pharmacia LKB) column and eluted using a salt gradient (increasing the ionic strength).

The eluate from the column was loaded on an immunoaffinity column consisting of an antibody (F25-lgG) directed against the C-terminal part of the heavy chain coupled to CnBr activated Sepharose 4B (Pharmacia LKB) using standard techniques as disclosed in Affinity Chromatography, Principles and Methods (Pharmacia 1983, p 15), equilibrated with 50 mM TrisCI pH 7.3 containing 150 mM NaCI, 10 mM CaCI₂, 10% (v/v) glycerol and 0.02% (v/v) Tween 80 at room temperature. The column was washed with 6 volumes of starting buffer. The column was washed further with 4 volumes 50 mM TrisCI pH 7.3 containing 0.65 M NaCI before eluting with 2.5 volumes of 20 mM TrisCI pH 7.3 containing 2.5 M NaCI, 50% (v/v) ethylenglycol, 10 mM NaCI and 0.02% Tween 80. The eluate containing FVIII was desalted on a Sephadex G25 column (5.3 x 32 cm, Phar¬ macia).

Bis(sulfosuccinimidyl)suberate (BS³) and disuccinimidyl suberate (DSS) were obtained from Pierce (Rockford, IL).

The automated APTT reagent and Factor Vlll-deficient plasma were obtained from Organon Teknika and the APTT assay was performed according to the manufacturer's instructions using an ACL 300 (Instrumentation Laboratory). SDS- PAGE on 4-15% gradient gels and silver staining were performed using the Phast system (Pharmacia). Alternatively, 7.5% gels were run and silver stained as described in Nature (1970), 227:680-685 and Anal.Biochem. (1981) 117:307-310. respectively. The. silver staining background was reduced by the use of Farmer's reducer (Tetenal Photowerk).

Crosslinking and activation of Factor VIII. Crosslinking of Factor VIII (50 units/ml) in 20 mM Hepes, pH 7.2, 0.1 M NaCI, 5 mM CaCI₂, 10% (v/v) glycerol, 0.02% (v/v) Tween 80 was carried out by adding DSS (25 mM in DMSO) to a final concentration of 0.5 mM and incubating at 0°C for 10 min. Lysine was then added to a final concentration of 10 mM to quench excess DSS, followed by incubation for 30 min. Activation of Factor VIII was achieved with thrombin (0.2 units/ml) in the above buffer at 37°C for 30 min.

Purification of crosslinked Factor Villa activity. 500 units of crosslinked Factor Villa was applied to a 0.5 ml column of F25-Sepharose equilibrated with 20 mM Tris- HCI, pH 7.3, 0.15 M NaCI, 10 mM CaCI₂, 0.02% (v/v) Tween 80 (buffer A). The column was washed with 12 ml of buffer A and the flow-through was collected. Bound protein was eluted with 50 mM imidazol, pH 7.3, 2.5 M NaCI, 10 mM CaCI₂, 50% (v/v) ethylene glycol, 0.02% (v/v) Tween 80. The bound fraction was desalted on a NAP-25 column (Pharmacia) equilibrated with buffer A and concentrated to a volume of 300 μl using a Centricon-10 (Amicon). 100 μl of the concentrated F25-Sepharose eluate was gel-filtered on a Superose 12 HR10/30 column equilibrated with 20 mM Hepes, pH 7.2, 1 M NaCI, 0.1 M lysine, 2.5 mM CaCI₂, 10% (v/v) glycerol, 0.01% (w/v) insulin, 0.02% (v/v) Tween 80 at a flow rate of 0.2 ml/min.

Results

The presence of chemical crosslinks in Factor Villa increased the stability of Factor Villa (activity). One hour after thrombin activation, the crosslinked Factor Villa was 200-400-fold more active than native Factor Villa. The difference was approximately the same after 24 hours. Crosslinked Factor Villa had about 10-fold higher activity than that of Factor VIII prior to thrombin activation, whereas non- crossliπked Factor Villa had an activity of approximately 5% of that of Factor VIII as appears from the below Table I showing the coagulant activity of activated Factor VIII and crosslinked activated Factor VIII as a function of time.

Table I

Activity in % of activity of original Factor VIII

Time (h) after thrombin Factor Villa Crosslinked Factor Villa activation

0 100 100

0.5 11 not determined 1.5 4 1300

It is noteworthy that treatment of Factor VIII with DSS affected neither the biological activity, i.e. the difference was only observed when comparing the thrombin-activated proteins nor the stability of Factor VIII over a 7-day period adversely.

In an attempt to further increase the activity of crosslinked Factor Villa, the incubation time with DSS was prolonged or the water-soluble analog of DSS, BS³, was used to increase the concentration of crosslinker to 5 mM. The increased incubation time had no effect, whereas an increased concentration of crosslinker had detrimental effects on Factor Villa activity. It is to be noted that DSS and BS³ were equally effective at 0.5 mM. After passing the crosslinked Factor Villa through a column of F25-Sepharose, more than 90% of the remaining FVIIIa activity could be eluted with ethylene glycol. The non-bound activity may be ascribed to Factor Villa molecules where the epitope for F25 had been modified beyond recognition by the crosslinker. When the same separation was repeated with native Factor VIII, no activity could be detected neither in the flow through nor in the eluate. The fraction of crosslinked Factor Villa which bound to F25-Sepharose was further chromatographed on a column of Superose 12. The Factor Villa activity in the fractions were monitored in an APTT assay and peak activity was found to elute after about 0.55 column volumes. Several bands corresponding to molecular weights of > 150,000 were visible upon SDS-PAGE of the material in the peak fraction of crosslinked activated Factor VIII as appears from Fig. 1 , see lane 1 , lane 2 showing Factor Villa as a reference showing no bands above about 70 kd.

The coagulant activity of activated crosslinked Factor VIII was determined as a function of time after activation.

The results are stated in the below Table II wherein the value at day 0 was determined after elution from the Superose 12 column.

Table II

Day No. Total Activity Activity in % of Activity ± SEM at Day 0

0 18950 ± 550 100

1 15950 ± 1455 84

4 23150 ± 4100 122

8 14700 ± 950 78

15 14400 ± 3700 76 The results demonstrate that the crosslinking of Factor VIII before activation stabilizes the resulting Factor Villa retaining about 3/4 of the initial activity after two weeks whereas the activity of activated Factor VIII normally is retained for less than 30 minutes.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Nordisk A/S

(B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : 2880

(G) TELEPHONE: 44448888 (H) TELEFAX: 44493256 (I) TELEX: 37304

(ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(EPO)

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER: DK

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 372 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Human Factor VIII

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15

Ivfet Gin Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn I le Ala Lys Pro 50 55 60

Arg Pro Pro Trp IVfet Gly Leu Leu Gly Pro Thr I le Gin Ala Glu Val 65 70 75 80

Tyr Asp Thr Val Val lie Thr Leu Lys Asn IVfet Ala Ser His Pro Val 85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys Val 115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140

Gly Pro N/fet Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu I le Gly Ala Leu 165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190

His Lys Phe I le Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205

His Ser Glu Thr Lys Asn Ser Leu IVfet Gin Asp Arg Asp Ala Ala Ser 210 215 220

Ala Arg Ala Trp Pro Lys Ivfet His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240

Ser Leu Pro Gly Leu I le Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255

Val I le Gly Ivfet Gly Thr Thr Pro Glu Val His Ser I le Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gin Ala Ser Leu Glu I le 275 280 285

Ser Pro I le Thr Phe Leu Thr Ala Gin Thr Leu Leu Ivfet Asp Leu Gly 290 295 300

Gin Phe Leu Leu Phe Cys His I le Ser Ser His Gin His Asp Gly Ivfet 305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg 325 330 335

Ivfet Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350

Ser Glu IVfet Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365

I le Gin le Arg 370

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 368 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal amino acid residues 373-740 of human Factor VIII

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr I le Ala Ala 1 5 10 15

Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp 20 25 30

Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro Gin Arg I le Gly 35 40 45 Arg Lys Tyr Lys Lys Val Arg Phe Ivfet Ala Tyr Thr Asp Glu Thr Phe 50 55 60

Lys Thr Arg Glu Ala I le Gin His Glu Ser Gly I le Leu Gly Pro Leu 65 70 75 80

Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu He He Phe Lys Asn Gin 85 90 95

Ala Ser Arg Pro Tyr Asn I le Tyr Pro His Gly I le Thr Asp Val Arg 100 105 110

Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp 115 120 125

Phe Pro I le Leu Pro Gly Glu I le Phe Lys Tyr Lys Trp Thr Val Thr 130 135 140

Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr 145 150 155 160

Tyr Ser Ser Phe Val Asn IVfet Glu Arg Asp Leu Ala Ser Gly Leu I le 165 170 175

Gly Pro Leu Leu lie Cys Tyr Lys Glu Ser Val Asp Gin Arg Gly Asn 180 185 190

Gin I le IVfet Ser Asp Lys Arg Asn Val I le Leu Phe Ser Val Phe Asp 195 200 205

Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn I le Gin Arg Phe Leu Pro 210 215 220

Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe Gin Ala Ser Asn 225 230 235 240

I le IVfet His Ser I le Asn Gly Tyr Val Phe Asp Ser Leu Gin Leu Ser 245 250 255

Val Cys Leu His Glu Val Ala Tyr Trp Tyr I le Leu Ser I le Gly Ala 260 265 270

Gin Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His 275 280 285

Lys IVfet Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu 290 295 300

Thr Val Phe IVfet Ser IVfet Glu Asn Pro Gly Leu Trp I le Leu Gly Cys 305 310 315 320

His Asn Ser Asp Phe Arg Asn Arg Gly

Thr Ala Leu Leu Lys Val 325 330 335 Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu 340 345 350

Asp I le Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala I le Glu Pro Arg 355 360 365

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 740 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: human Factor VIII

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15

IVfet Gin Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn I le Ala Lys Pro 50 55 60

Arg Pro Pro Trp IVfet Gly Leu Leu Gly Pro Thr I le Gin Ala Glu Val 65 70 75 80

Tyr Asp Thr Val Val I le Thr Leu Lys Asn IVfet Ala Ser His Pro Val 85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140

Gly Pro IVfet Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu I le Gly Ala Leu 165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190

His Lys Phe I le Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205

His Ser Glu Thr Lys Asn Ser Leu IVfet Gin Asp Arg Asp Ala Ala Ser 210 215 220

Ser Leu Pro Gly Leu I le Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255

Val I le Gly IVfet Gly Thr Thr Pro Glu Val His Ser I le Phe Leu Glu 260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gin Ala Ser Leu Glu I le 275 280 285

Ser Pro Me Thr Phe Leu Thr Ala Gin Thr Leu Leu IVfet Asp Leu Gly 290 295 300

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg 325 330 335

IVfet Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350

Ser Glu IVfet Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365

I le Gin I le Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380

Tyr I le Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu.Asn Asn Gly Pro 405 410 415 Gin Arg I le Gly Arg Lys Tyr Lys Lys Val Arg Phe fet Ala Tyr Thr 420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala I le Gin His Glu Ser Gly I le 435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Me Me 450 455 460

Phe Lys Asn Gin Ala Ser Arg Pro Tyr Asn Me Tyr Pro His Gly Me 465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495

His Leu Lys Asp Phe Pro Me Leu Pro Gly Glu Me Phe Lys Tyr Lys 500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn IVfet Glu Arg Asp Leu Ala 530 535 540

Ser Gly Leu I le Gly Pro Leu Leu I le Cys Tyr Lys Glu Ser Val Asp 545 550 555 560

Gin Arg Gly Asn Gin I le IVfet Ser Asp Lys Arg Asn Val I le Leu Phe 565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn I le Gin 580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe 595 600 605

Gin Ala Ser Asn I le IVfet His Ser I le Asn Gly Tyr Val Phe Asp Ser 610 615 620

Leu Gin Leu Ser Val O/s Leu His Glu Val Ala Tyr Trp Tyr I le Leu 625 630 635 640

Ser I le Gly Ala Gin Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655

Thr Phe Lys His Lys /fet Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670

Phe Ser Gly Glu Thr Val Phe IVfet Ser IVfet Glu Asn Pro Gly Leu Trp 675 680 685

I le Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly IVfet Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720

Asp Ser Tyr Glu Asp I le Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735

Me Glu Pro Arg 740

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 684 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: amino acid residues 1649-2332 of human Factor VIII

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: C-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Glu I le Thr Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu I le Asp Tyr 1 5 10 15

Asp Asp Thr I le Ser Val Glu IVfet Lys Lys Glu Asp Phe Asp I le Tyr 20 25 30

Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 35 40 45

His Tyr Phe I le Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly IVfet Ser 50 55 60

Ser Ser Pro His Val Leu Arg Asn Arg Ala Gin Ser Gly Ser Val Pro 65 70 75 80

Gin Phe Lys Lys Val Val Phe Gin Glu Phe Thr Asp Gly Ser Phe Thr 85 90 95

Gin Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 100 105 110

Pro Tyr I le Arg Ala Glu Val Glu Asp Asn I le IVfet Val Thr Phe Arg 115 120 125 Asn Gin Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Me Ser Tyr 130 135 140

Glu Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 145 150 155 160

Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gin His His IVfet Ala 165 170 175

Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 180 185 190

Val Asp Leu Glu Lys Asp Val His Ser Gly Leu I le Gly Pro Leu Leu 195 200 205

Val Oys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gin Val Thr 210 215 220

Val Gin Glu Phe Ala Leu Phe Phe Thr I le Phe Asp Glu Thr Lys Ser 225 230 235 240

Trp Tyr Phe Thr Glu Asn IVfet Glu Arg Asn Cys Arg Ala Pro Cys Asn 245 250 255

Me Gin IVfet Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 260 265 270

I le Asn Gly Tyr I le IVfet Asp Thr Leu Pro Gly Leu Val IVfet Ala Gin 275 280 285

Asp Gin Arg I le Arg Trp Tyr Leu Leu Ser IVfet Gly Ser Asn Glu Asn 290 295 300

I le His Ser I le His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 305 310 315 320

Glu Glu Tyr Lys IVfet Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 325 330 335

Thr Val Glu IVfet Leu Pro Ser Lys Ala Gly I le Trp Arg Val Glu Cys 340 345 350

Leu I le Gly Glu His Leu His Ala Gly IVfet Ser Thr Leu Phe Leu Val 355 360 365

Tyr Ser Asn Lys Cys Gin Thr Pro Leu Gly IVfet Ala Ser Gly His I le 370 375 380

Arg Asp Phe Gin I le Thr Ala Ser Gly Gin Tyr Gly Gin Trp Ala Pro 385 390 395 400

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Me Asn Ala Trp Ser Thr 405 410 415 Lys Glu Pro Phe Ser Trp I le Lys Val Asp Leu Leu Ala Pro IVfet I le 420 425 430

I le His Gly I le Lys Thr Gin Gly Ala Arg Gin Lys Phe Ser Ser Leu 435 440 445

Tyr I le Ser Gin Phe Me I le IVfet Tyr Ser Leu Asp Gly Lys Lys Trp 450 455 460

Gin Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu IVfet Val Phe Phe Gly 465 470 475 480

Asn Val Asp Ser Ser Gly I le Lys His Asn I le Phe Asn Pro Pro I le 485 490 495

I le Ala Arg Tyr Me Arg Leu His Pro Thr His Tyr Ser Me Arg Ser 500 505 510

Thr Leu Arg IVfet Glu Leu IVfet Gly Oys Asp Leu Asn Ser Cys Ser IVfet 515 520 525

Pro Leu Gly IVfet Glu Ser Lys Ala I le Ser Asp Ala Gin I le Thr Ala 530 535 540

Ser Ser Tyr Phe Thr Asn IVfet Phe Ala Thr Trp Ser Pro Ser Lys Ala 545 550 555 560

Arg Leu His Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val Asn 565 570 575

Asn Pro Lys Glu Trp Leu Gin Val Asp Phe Gin Lys Thr IVfet Lys Val 580 585 590

Thr Gly Val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr Ser IVfet Tyr 595 600 605

Val Lys Glu Phe Leu I le Ser Ser Ser Gin Asp Gly His Gin Trp Thr 610 615 620

Leu Phe Phe Gin Asn Gly Lys Val Lys Val Phe Gin Gly Asn Gin Asp 625 630 635 640

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 645 650 655

Tyr Leu Arg I le His Pro Gin Ser Trp Val His Gin I le Ala Leu Arg 660 665 670

IVfet Glu Val Leu Gly Cys Glu Ala Gin Asp Leu Tyr 675 680

Claims

CLAIMSWhat is claimed is:

1. A crosslinked Factor VIII polypeptide showing a coagulant activity of Factor VIII wherein the crosslinking is an inter-chain crosslinking.

2. A crosslinked Factor Villa polypeptide showing a coagulant activity of Factor VIII wherein the crosslinking is an inter-chain crosslinking.

3. A method for preparing a crosslinked Factor VIII polypeptide showing a coagulant activity of Factor VIII having an inter-chain crosslinking wherein the Factor VIII polypeptide is incubated with a crosslinking agent at a tempereture from -1 to + 15°C for a period of from 5 to 15 minutes in a buffer having pH from 6 to 8 in the presence of a non-ionic surfactant whereafter lysine is added for quenching excess crosslinking agent optionally followed by isolation of the crosslinked Factor VIII polypeptide.

4. A method as claimed in claim 3 wherein the Factor VIII polypeptide is incubated with from 0.1 to 10 mM bis(sulfosuccinimidyl) suberate or disuccinimidyl suberate as crosslinking agent.

5. A method as claimed in claim 3 or 4 wherein the non-ionic surfactant is polysorbate 80.

6. A method for preparing a crosslinked Factor Villa polypeptide having an inter- chain crosslinking wherein a crosslinked Factor VIII polypeptide showing a coagulant activity of Factor VIII and having an inter-chain crosslinking is activated using thrombin.

7. A pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide and optionally pharmaceutically acceptable vehicle and/or excipient.

8. A pharmaceutical preparation comprising a crosslinked Factor Villa polypeptide and optionally pharmaceutical acceptable vehicle or excipient.

5 9. Use of a crosslinked Factor VIII polypeptide for the preparation of a pharmaceutical preparation.

10. Use as claimed in Claim 8 wherein a crosslinked Factor VIII polypeptide is used for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.

10 11. Use of a crosslinked Factor Villa polypeptide for the preparation of a pharmaceutical preparation.

12. Use as claimed in claim 11 wherein a crosslinked Factor Villa polypeptide is used for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.

15 13. A method for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject comprising administering to the subject a pharmaceutically active amount of a crosslinked Factor VIII polypeptide.

14. A method for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject comprising administering to the subject a 0 pharmaceutically active amount of a crosslinked Factor Villa polypeptide.

15. A method of preparing a pharmaceutical preparation comprising a crosslinked Factor VIII polypeptide and optionally pharmaceutically acceptable vehicle or excipient comprising combining the crosslinked Factor VIII polypeptide ^* with pharmaceutically acceptable vehicle and/or excipient and forming a suitable dosis form of the pharmaceutical preparation.

16. A method of preparing a pharmaceutical preparation comprising a crosslinked Factor Villa polypeptide and optionally pharmaceutically acceptable vehicle or excipient comprising combining the crosslinked Factor Villa polypeptide with pharmaceutically acceptable vehicle and/or excipient and forming a suitable dosis form of the pharmaceutical preparation.