WO1995013300A1

WO1995013300A1 - New factor viii polypeptides

Info

Publication number: WO1995013300A1
Application number: PCT/DK1994/000423
Authority: WO
Inventors: Marianne Kjalke; Mirella Ezban Rasmussen
Original assignee: Novo Nordisk A/S
Priority date: 1993-11-12
Filing date: 1994-11-10
Publication date: 1995-05-18
Also published as: DK128093D0; AU8140094A

Abstract

A factor VIII polypeptide comprising a heavy chain having an amino acid sequence corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domain of full length Factor VIII exhibits the coagulating effect of Factor VIII and may be used for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject.

Description

TITLE

New Factor VIII polypeptides.

FIELD OF THE INVENTION

The present invention relates to new Factor VIII polypeptides showing coagulant activity, a method for the preparation thereof, pharmaceutical preparations comprising the new Factor VIII polypeptides, the use of these polypeptides for the preparation of a pharmaceutical preparation especially 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 desease 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 (S.I. 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 as a series of related polypeptides ranging from M_r 160000-40000 with predominant species of M_r 92000 (the heavy chain) and M_r 80000 (the light chain). 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 (the heavy chain) and A3-C1-C2 (the light chain). 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 between the A2 and B domains, and 41 amino acids is 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 and the isolation and purification process which is, moreover, 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 techniques.

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 discloses 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 of Factor VIII for the production of complexes 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 International Patent Application No. WO 91/09122 disclose re- combinant expression of Factor VIII having one or more deletions in the molecule, or binding to antibodies inhibiting Factor VIII.

It is advantageous to express shortened forms of Factor VIII (as compared to the full length molecule) as it is difficult to reach an acceptable level of production due to low expression levels and instability of the expressed product during expression and purification.

Furthermore, it is desirable to identify the smallest fraction of full length Factor VIII showing coagulant activity as such a shortened form may also be used for treating inhibitor patients having developed antibodies against epitopes in the C- terminal part of the heavy chain.

WO 87/07144 discloses the preparation of deletion analogues of Factor VIII lacking 1-1317 amino acid residues from Ser-373 through Arg-1689. However, no examples discloses the preparation of analogues comprising a shortened A2 domain of Factor VIII, and no results are present showing coagulant activity for Factor VIII analogues comprising a shortened A2 domain.

British Journal of Haematology 1993 (85), 133-142 discloses the preparation of deletion analogues of Factor VIII lacking amino acid residues 713 through 1637. Such analogues show no activity in clotting assay but do show activity in a Factor Xa-generating assay.

BRIEF DESCRIPTION OF THE INVENTION

It has surprisingly been found that new shortened forms of Factor VIII lacking a part of the A2 domain of the heavy chain exhibits coagulant activity up to the same level as the complex of the full A1-A2 and A3-C1-C2 domains.

Thus, in a first aspect, the invention relates to a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain.

In a second aspect, the invention relates to a method for the preparation of a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain.

In a further aspect, the invention relates to a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain where a part of it or all is lacking a part of the C terminal part of the A2 domain.

In a still further aspect, the invention relates to the use of a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain 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 accordance with another aspect, the invention relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII.

In yet another aspect, the invention relates to a method of preparing a pharmaceutical preparation comprising a Factor VIII polypeptide of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 1 shows an SDS-PAGE of Factor VIII polypeptides of the invention as compared to Factor VIII polypeptides, containing the complete A2- domain,

Fig. 2 shows RP-HPLC profiles of LysC peptides from the heavy chain of Factor VIII polypeptides of the invention as compared to the heavy chain, containing the complete A2-domain,

Fig. 3 shows RP-HPLC profiles of AspN peptides from the heavy chain of Factor VIII polypeptides of the invention as compared to the heavy chain of Factor VIII polypeptides containing the complete A2-domain,

Fig. 4 shows the inhibition of Factor VIII activity by a monoclonal antibody for peptides of the invention as compared to Factor VIII polypeptides containing the complete A2-domain and plasma Factor VIII,

Fig. 5 shows a time-study of thrombin activation of FVIII polypeptides as measured by SDS-PAGE,

Fig. 6 shows a time-study of thrombin activation of FVIII polypeptides as measured by a clotting assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domain of full length Factor VIII. The heavy and light chains are preferably bridged via a metal ion bridge. Such a bridge is suitably formed via a divalent ion such as Mn²⁺, Ca²⁺ or Co ⁺. Preferably the bridge is a calcium bridge.

The Factor VIII polypeptides of the invention prefererably lacks a part of the C- terminal part of the A2 domain. Preferred Factor VIII polypeptides of the invention comprises a heavy chain comprising the amino acid residues 1-720 or 1 -729 of the heavy chain of full length Factor VIII.

It has surprisingly been found that the Factor VIII polypeptide of the invention comprising a heavy chain comprising amino acid residues 1-729 of the full length Factor VIII exhibits a coagulant activity of the same level as Factor VIII polypeptide comprising the full heavy chain.

It has also surprisingly been found that the Factor VIII polypeptide of the invention comprising a heavy chain comprising amino acid residues 1-720 of full length Factor VIII exhibits a specific activity as measured in a chromogenic assay of the same level as Factor VIII polypeptides comprising full heavy chain and a specific activity as measured in a clot assay of about 50%.

The Factor VIII polypeptides of the invention normally will comprise a light chain having an amino acid sequence corresponding to amino acids 1649-2332 of the C terminal of full length Factor VIII.

In the alternative, the Factor VIII polypeptides comprise a light chain having an amino acid sequence corresponding to amino acids 1690-2332 of the C terminus full length Factor VIII. The invention further relates to a method for preparing a Factor VIII polypeptide comprising a heavy chain having an amino acid sequence corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain shorter than the A1-A2 domain of full length Factor VIII wherein a Factor VIII polypeptide comprising the full A1-A2 domain of full length Factor VIII is treated with a protease cleaving off the C-terminal part of the A2 domain.

The Factor VIII polypeptides of the invention may be prepared starting from a Factor VIII polypeptide 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.

In a preferred embodiment of the invention the Factor VIII polypeptides of the invention are prepared by coexpession of the heavy and light chains of Factor VIII as disclosed in WO91/07490. Such Factor VIII polypeptides lack the B domain of full length Factor VIII and comprise a heavy chain metal ion-bridged to a light chain showing coagulant activity. The Factor VIII polypeptides of the invention may be generated by proteolytic digestion in the medium.

The Factor VIII polypeptides of the invention may be purified and isolated by methods known per se for purification and isolation of Factor VIII polypeptides.

The invention also relates to a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII in admixture with a parenterally acceptable vehicle or excipient.

Furthermore, the invention relates to the use of a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domains of full length Factor VIII for the preparation of a pharmaceutical preparation.

Preferably the Factor VIII polypeptides of the invention are 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.

The invention also 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 Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII in admixture with a pharmaceutically acceptable vehicle or excipient.

in a further aspect, the invention relates to a method of preparing a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII with pharmaceutically acceptable vehicle and/or excipients and forming a suitable dosis form of the pharmaceutical preparation.

As used herein the term "full length Factor VIII" designates the full molecule comprising the amino acid residues 1-2332 as disclosed in Nature (1984) 312:339.

As used herein the term "heavy chain" or "HC" designates the A1-A2 repeats of Factor VIII comprising the amino acid residues 1-740 of full length Factor VIII as disclosed in Nature (1984) 312:341.

The term "light chain" or "LC" as used herein designates the A3-C1-C2 repeats of Factor VIII comprising the amino acid residues 1649-2332 as disclosed in Nature (1984) 312:341.

EXPERIMENTAL PART

Summary

Recombinant Factor VIII lacking the B domain was purified by a procedure including an affinity chromatography step using a monoclonal antibody directed against the C-terminal part of the heavy chain. A part of the Factor VIII did not bind to the column, due to C-terminal truncation of the A2-domain of the heavy chains. Two Factor VIII forms were purified from the fractions not bound to the antibody column. By peptide mapping and isolation of C-terminal peptides by affinity chromatography followed by amino acid sequencing and mass spectro- metry, it is shown, that the two C-terminal truncated forms of Factor VIII contains heavy chains. consisting of amino acids 1-720 (FVIII(HC:1-720)) and 1-729

(FVIII(HC:1-729)), respectively, while the Factor VIII bound to the antibody column contains a heavy chain consisting of amino acid 1 -740 corresponding to the entire A1 and A2-domains. FVIII (HC: 1-729) have the same specific activity as FVIII(HC:1-740), and is activated by thrombin at a similar rate. FVI II (HC: 1-720) have the same specific activity when the activity is measured in a chromogenic assay, however, the specific activity is a factor two lower when the specific activity is measured in a clotting assay. Similary, FVIII (HC: 1-720) is activated by thrombin at a slower rate and to a lower level compared with FVIII(HC:1-740), FVIII(HC:1- 729), and plasma Factor VIII.

Materials and Methods

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 pr. 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) 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 flow-through containing FVIII (HC: 1-729) and FVIII (HC: 1-720) was collected. 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 CaCI₂ and 0.02% Tween 80. The eluate containing FVIII(HC: 1-740) were desalted on a Sephadex G25 column (5.3 x 32 cm, Pharmacia). The F25-lgG was prepared by purifying Factor VIII HC from plasma as described in WO 88/00210. Using this isolated Factor VIII HC the monoclonal antibody (F25- IgG) was prepared using the procedure disclosed in Thromb. Haemostas 1985:54, 586-590.

The Factor VIII forms were finally purified on a MonoQ PC 1.6/5 column using the SMART system (Pharmacia). Approximately 800 U FVIII(HC: 1-740) or FVIII(HC:1- 729) and FVIII(HC: 1-720) was loaded on the column equilibrated with 20 mM TrisCI pH 7.5 containng 150 mM NaCI, 10 mM CaCI₂, 10% glycerol (v/v) and 0.02% (v/v) Tween 80 at room temperature at a flow of 100 μl min^'1. After washing, a 30 min gradient of 150 to 500 mM NaCI in the buffer was applied.

The heavy chain forms were separated from the complex and isolated as described in Thromb. Haemostas. 1987:58, 1043-1048.

Enzymatic cleavage

Reduced and amidomethylated heavy chains from FVIII (HC: 1-740) (570 pmol), FVIII(HC:1-729) (460 pmol), and FVIII(HC: 1-720) (40 pmol) were incubated with LysC endopeptidase (Boehringer Mannheim) at a LysC endopeptidase to heavy chain ratio of 1 :50 (w/w) for four hours at 37°C in 0.2 M ammonium bicarbonate. The reaction was stopped by adding 10% triflouroacetic acid to pH 2.

For anhydrotrypsin affinity purification approximately 4 nmol heavy chain from FVIII (HC: 1-720) was reduced and alkylated, and cleaved with 3.1 μg LysC endoprotease as described. The reaction was stopped by adding PMSF (Phenylmethane sulfonyl-fluoride) to a final concentration of 1 mM.

AspN endoprotease digestions were made on heavy chains from FVIII(HC: 1-740) (approximately 1.6 nmol), FVIII (HC: 1-729) (approximately 0.9 nmol), and FVIII- (HC: 1-720) (approximately 0.2 nmol). After redissolvation in 8 M urea, 0.75 μg AspN endoprotease (Boehringer Mannheim) in 10 mM TrisCI pH 7.5 was added to each sample to a final concentration of urea of 1 M. After 1 hour at 22°C another 0.75 μg AspN endoprotease was added to each sample and the reactions were continued for 15 hours before stopping by adding 10% trifluoro- acetic acid to pH 2.

For time-study of thrombin cleavage of Factor VIII forms measured by SDS-PAGE plasma Factor VIII, FVIII(HC:1-740), FVIII(HC: 1 -729), and FVIII (HC: 1-720) were diluted into 1.25 ml of 20 mM TrisCI pH 7.5 containing 150 mM NaCI, 10 mM CaCI₂, 10% (v/v) glycerol and 0.02% (v/v) Tween 80 to a final concentration of 20 U/ml. The samples were preincubated 2 min at 37°C before a 100 μl sample was withdrawn an added to 20 μl icecold 50% (w/w) trichloracetic acid containing 0.2% (w/w) sodiumdeoxycolat. Human α-thrombin (Boehringer Mannheim) was added to the remaining Factor VIII to a final concentration of 0.1 U/ml, and 100 μl samples were withdrawn at the times indicated in Fig. 5 and the cleavage stopped as described above. The samples were incubated 30 min at 4°C before centrifugation 10 min at 18000 x g, and the precipitate analyzed by SDS-PAGE.

When the time-study is measured by a clotting assay, the Factor VIII concen¬ tration was 0.9 U/ml, and the final volume 0.8 ml. Samples of 50 μl were withdrawn at times indicated on Fig. 6 and analyzed by a clotting assay on Amelung Coagulometer as described below.

For thrombin cleavage of samples to SDS-PAGE (Fig. 1) 2 U Factor VIII were diluted to 100 μl in 20 mM TrisCI pH 7.5 containing 150 mM NaCI, 10 mM CaCI₂, 10% (v/v) glycerol and 0.02% (v/v) Tween 80 and incubated for one hour at 37°C with 0.5 U/ml human α-thrombin (Boehringer Mannheim). The Factor VIII was precipitated by adding 20 μl of icecold 50% (w/w) trichloracetic acid containing 0.2% (w/w) sodiumdeoxycolat as described above. Affinity purification of C-terminal peptides

The LysC endoprotease peptides from FVIII(HC:1-720) heavy chain was added to 1 ml anhydrotrypsin agarose (Clontec) packed in a BioRad column with a diametre of 0.9 cm equilibrated with 50 mM sodium acetate pH 5.0 containing 20 mM CaCI₂ at a flow of 0.18 ml min^"1. The column was washed with 20 volumes of starting buffer and fractions of 0.5 ml collected. Absorbance at 227 nm was detected. The bound peptides were eluted with 10 volumes 5 mM HCI pH 2.5. The pH of the pooled fractions containing the non-bounded peptides was adjusted to pH 2 by adding 10% trifluoroacetic acid before rechromatography on reverse phase-HPLC. A sample containing only LysC endoprotease and buffer was runned in parallel.

Reverse phase-HPLC

All digests and pooled fractions from the anhydrotrypsin agarose column were analyzed by reverse phase-HPLC (Applied Biosystems model 130A) using a Brownlee C18 column (2.1 x 220 mm, Applied Biosystems) and a 50 min linear gradient from 3.5 to 50.4% acetonitrile in 0.1% trifluoroacetic acid. The flow rate was 275 μl min^"1. Effluents were monitored at 214 nm, and fractions collected manually.

Sequence, mass and amino acid analyses

The peptides (10-100 pmol) were sequenced on an Applied Biosystem model 477A sequencer equipped with on-line model 120A HPLC using standard pro¬ grams as described by the manufacturer. Mass spectra were recorded with a time-of-flight plasma desorption mass spectrometre (Bio-Ion 20, Applied Biosystem) at 16 kV acceleration.

Samples for amino acid analyses were hydrolyzed for 20 hours under vacuum at 110°C in 6 M HCI containing 0.1% phenol and 0.1 % dithiodipropionic acid (Barkholt and Jensen (1989) Anal. Biochem. 177: 318-322). For determination of specific activity 2-3 hydrolyses of each sample were made. The samples were acetone precipitated and redissolved in H₂0 to approximately 0.1 μg μl^'1. Norleu- sine was added to each sample (1.5 nmol each) as internal standard. The samples were dryed in a speed-vac, and hydrolyses performed as decsribed above.

Activity determination

Chromogenic Assay

The activity of Factor VIII was measured in a chromogenic assay (Coatest, Cro- mogenix), as described by the manufacturers, except that all reactions were carried out at room temperature and that the incubation times were altered:

Phospholipid, Factor IXa + Factor X, CaCI₂ and the diluted sample was incubated 15 min before adding the substrate + the thrombin inhibitor, and the colour reaction was allowed to take place for 10 min.

Clotting Activity

Clotting activity of Factor VIII was measured as the ability to restore clotting activity of FVIII deficient plasma (ACL analyses, IL Laboratories, or Amelung coagulometer, Pharmacia). Clotting time analysis on the ACL instrument was carried out as described by the manufacturer. All reagens were from IL Laboratories except Factor VIII deficient plasma and APTT reagens that were from Organon Teknika. For clotting time analysis on the Amelung instrument, 50 μl Factor VIII deficient plasma, 20 μl APTT reagens (both from Organon Teknika) and 80 μl 20 mM TrisCI pH 7.5 containing 150 mM NaCI was incubated 5 to 10 min at 37°C before the sample was added and clotting-time measured. In all cases activities were calculated by comparing with the activity of a plasma standard calibrated against a WHO standard or by using a partially purified Factor VIII standard with known activity.

Electrophoresis

SDS-PAGE was performed on reduced samples in 7.5% polyacrylamide gels as described in Biochemistry (1991) 30:1533-1537 using the BioRad Mini-Protean system. The gels were silver stained.

Results

Purification of recombinant Factor VIII forms

FVIII(HC: 1-740), FVIII(HC: 1-729) and FVIII(HC:1-720) together with heavy chains from the three Factor VIII forms were purified as described in Methods. Fig. 1 shows SDS-PAGE of the three Factor VIII forms and thrombin generated fragments from theese compared with plasma Factor VIII containing no B-domain. FVIII(HC: 1-740) have similar Mr as the plasma Factor VIII, while FVIII(HC: 1-729) and FVIII(HC: 1-720) had slightly lower Mr of the heavy chains. The Mr differences is located in the A2-domains as seen by the Mr of the fragments generated by thrombin cleavage (see arrows on Fig. 1). Peptide mapping of Factor VIII forms

Identification of the differences of the Factor VIII forms was done by LysC and AspN peptide mapping of the isolated heavy chains on reverse phase-HPLC. Fig. 2 shows the LysC peptide maps of reduced and alkylated heavy chain from the recombinant Factor VIII forms. Peaks not seen in all three maps were analyzed by amino acid sequencing and mass spectrometry (see Table I below).

Table I. Amino acid sequences and masses of LysC peptides from the heavy chains marked on Fig. 2. Observed M_r (Obs.) are the masses determined by mass spectrometry. Calculated M_r (Cal.) are deduced from the cDNA sequence. The amino acid numbering (AA no.) corresponds to the mature Factor VIII heavy chain. Small letters indicate tentative sequence assignments. ND = not determined.

M_r

Pep¬

Sample tide AA no. Sequence Obs. Cal.

FVIII(HC: 1-740) A 734-740 NNAIEPR 814.4 813.9 FVIII(HC: 1-740) B 714-733 NTGDYYEDSY- ND 2347.4 EDISaYLLSK 214-230 NSLMQDRDAA- ND 1918.1 SARAWPK

FVIII(HC:1-740) C 714-733 NTGDYYEDSY- 2348.3 2347.4 EDISAYLLSK

FVIII (HC: 1-729) D 714-729 NTGDYYEDSY- - 1905.9 EDISAY

FVIII(HC: 1-729) E 714-720? ntgDYyx - 861.8

FVIII(HC:1-720) E 714-720 NTGDYYE - 861.8

The peptide marked A, corresponding to amino acid 734-740, was only present in FVIII(HC:1-740), showing that only this form contains the full-length A2-domain. Only FVIII(HC: 1-729) contained the peak marked D, which corresponds to amino acid 714-729. Because LysC is not cleaving peptides C-terminal to Tyr, this indi¬ cates that FVIII(HC:1-729) have C-terminus at Tyr729. Both FVIII(HC:1-729) and FVIII(HC: 1-720) contains the peaks marked E corresponding to amino acid 714- 720. This peak is not seen in FVIII(HC: 1-740) indicating that some Factor VIII have C-terminus at Glu720. The prescence of peak E in FVIII(HC: 1-729) could be due to copurification of some FVIII(HC: 1-720) in the FVIII(HC: 1-729) preparation. FVIII- (HC: 1-740) contains the peaks marked B and C both containing amino acid 714- 733. The prescence of this peptide in two peaks could be due to partial sulfata- tion of one or more of the expected sulfate groups at Tyr 118, Tyr 119 and 723 as seen in the full-length FVIII molecule expressed in Chinese Hamster ovary cells (Mikkelsen et al., Biochemistry (1991), 30, 1533-1537.

AspN peptide mapping of the unreduced heavy chains from FVIII(HC: 1-740), FVIII (HC: 1-729), and FVIII(HC: 1-720) is shown in Fig. 3. Table II shows amino acid sequences and mass spectrometry data of the peaks deviating in retention time among the three maps.

Table II. Amino acid sequences and masses of AspN peptides from the heavy chains marked on Fig. 3. Observed M_r (Obs.) are the masses determined by mass spectrometry. Calculated M_r (Cal.) are deduced from the cDNA sequence. The amino acid numbering (AA no.) corresponds to the mature Factor VIII heavy chain. Small letters indicate tentative sequence assighnments. ND = not detected.

M,

Source Pep¬ AA no. Sequence Obs. Cal. tide

FVIII(HC: 1-740) A 725-740 DISAYLLSKNNA- 1804.9 1805.0 IEPR

FVIII (HC: 1-740) B 717-720 DYYE - 589.6

FVIII(HC: 1-740) D 519-524 DGPTKS (604.1) 604.6 717-720 DYYE 589.6 721-724 DSYE 513.5 FVIII(HC: 1-729) C 725-729 DISAY - 568.6

FVIII(HC: 1-729) D 717-720 DYyE ND 589.6

FVIII(HC: 1-729) E 519-524 DGPTKS ND 604.6

FVIII(HC: 1-729) F 721-724 DSYE ND 513.5

The peak marked A in the map of FVIII(HC: 1-740) corresponds to the C-terminal AspN peptide. Sequencing of the "shoulder" (retention time 27.5 min, Fig. 3) of the peak from FVIII (HC: 1-729) eluting just before peptide A, did not show the se¬ quence of the C-terminal peptide (not shown). In accordance with the results of the LysC peptide maps, this shows that only FVIII(HC: 1-740) contains the full- length A2-domain. The peptide from FVIII(HC: 1-729) marked C on Fig. 3 showed the sequence corresponding to amino acid 725-729, showing that FVIII(HC:1-729) have C-terminus at Tyr729. The peaks marked E, D and F in the map of FVIII(HC: 1-729) corresponds to amino acid 717-720, 519-524, and 721-724, respectively. All peptides was also seen in the broad pead marked D from FVIII- (HC: 1-740). The corresponding broad peak from FVIII(HC1-720) was not sequen- ced. FVIII(HC:1-740) did also contain amino acid 717-720 in the peak marked B. Like in the LysC peptide mapping, this is probably due to partial sulfation of Tyr 718 or Tyr 719. Because AspN cleaves between amino acid 720 and 721 , a specific C-terminal peptide is not observed for FVIII (HC: 1-720).

Affinity purification of the C-terminal peptide from FVHI(HC:1-720) heavy chain

The C-terminal LysC peptides from the heavy chain of FVIII(HC:1-720) were purifi- ed by anhydrotrypsin affinity chromatography. Anhydrotrypsin is a catalytical inac¬ tive derivative of trypsin with the ability of binding peptides with C-terminal Lys or Arg (Ishii and Kumazaki, (1988) in Methods in Protein Seguence Analysis. (B. Witman-Liebold, ed.) pp. 156-163, Springer Verlag, Berlin). Because the results of the peptide maps indicated, that the FVIII(HC: 1-720) did not have Arg as the C- terminal amino acid of the heavy chain, the C-terminal peptide was recovered from the flow-through fraction from a LysC digest of reduced and alkylated heavy chain from FVIII(HC: 1-720) loaded on an anhydrotrypsin column. Table III shows the amino acid sequence of this C-terminal peptide, clearly confirming that FVIII(HC: 1-720) have C-terminus at Glu720.

Table III. Sequence analysis of the peptide from a LysC digest of heavy chain from FVIII (HC: 1-740) not bound to anhydrotrypsin agarose. It was not possible to obtain a mass by mass spectrometry. The amino acid numbering (AA no.) corresponds to the mature Factor VIII heavy chain.

M_r

Source AA no. Sequence Obs. Cal. FVIII(HC: 1-720) 714-720 NTGDYYE - 813.9 Specific activity

The specific activity of FVIII(HC:1-740), FVIII(HC: 1-729), and FVIII (HC: 1-720) was measured by both a chromogenic assay and a one-stage clotting assay (see Table IV, below).

Table IV. Specific activity of the FVIII(HC:1-740), FVIII (HC: 1-729) and FVIII(HC:1- 720) determined in a clot-assay (ACL analysis) and a chromogenic assay (Coatest). For each sample by 8-12 activity analyses were made. The protein concentration was measured by 2-3 amino acid analysis of each sample. The values are ± standard deviation.

Specific activity Sample Clot-assay Chromogenic assay (10³ U/mg) (10³ U/mg

Unmodified FVIII 10.0 ± 2.5 8.5 ± 1.3

Modified "middle" FVIII 9.6 ± 3.0 8.2 ± 1.2

Modified "lower" FVIII 5.1 ± 1.2 10.0 ± 1.4

The specific activity of all the Factor VIII forms was approximately 1.0 x 10⁴ U/mg as determined by the chromogenic assay. FVIII (HC: 1-740) and FVIII (HC: 1-729) also have the same specific activity within the experimental error as determined by the clotting assay. However, FVIII (HC: 1-720) have a specific activity a factor two lower as determined in the clotting assay.

Inhibition of Factor VIII activity by a monoclonal antibody

The monoclonal antibody (F25-lgG) used for separation of FVIII(HC: 1-740) from FVIII(HC:1-729) and FVIII(HC: 1-720) was used for inhibition of activity of plasma Factor VIII and the three recombinant Factor VIII forms as measured by a chromogenic assay and a one-stage clotting assay (Fig. 4). The antibody inhibits the activity of FVIII(HC: 1-740) and plasma Factor VIII at a similar degree as measured in the clotting assay, but was not able to inhibit in the chromogenic assay. In the clotting assay the time of clotting of Factor VIII deficient plasma is measured. In the chromogenic assay, the Factor VIII form is incubated with Factor IXa, Factor X and phospholipid for 15 minutes before the Factor Xa substrate assay is added. This means, that any differences in affinity of for example thrombin to Factor VIII would be masked by the long incubation time in the chromogenic assay but not in the clotting assay. As expected, the inhibitory effect of the antibody was not seen for FVIII(HC:1-729) and FVIII(HC:1-720).

Time-study of thrombin activation of Factor VIII forms

Thrombin activation of plasma Factor VIII and the three recombinant Factor VIII forms was evaluated in a time-study by SDS-PAGE (Fig. 5) and a clotting assay (Fig. 6). Plasma Factor VIII, FVIII(HC:1-740) and FVIII(HC:1-729) is activated at similar rates, while FVIII(HC: 1-720) is activated more slowly. At SDS-PAGE (Fig. 5) it is seen, that the heavy chain bands dissapeares within 5 to 10 min after thrombin is added for FVIII(HC:1-740), FVIII (HC: 1-729), and plasma Factor VIII, while thrombin cleavage of FVIII (HC: 1-720) requires at least 15 min before the heavy chain band dissapeares. Similary, the bands corresponding to the A1 and A2-domains from the activated heavy chain and the activated light chain (marked LC on Fig. 5) is observed later for FVIII(HC: 1-720) compared with the other Factor VIII forms. It is difficult to compare the dissapearance of the light chain band due to the prescence of free light chain in the plasma Factor VIII preparation. At Fig. 6 it is seen that plasma Factor VIII, FVIII(HC: 1-740) and FVIII- (HC: 1-729) all reaches maximum activity after 1 to 2 min of incubation with thrombin, and have similar profiles of the activity curves. FVIII(HC: 1-720) is activated more slowly, ie. the activity curve shows a broad maximum after 3 to 5 min. As expected from the differences in specific activity, FVIII(HC: 1-720) is less active in the clotting assay compared with the other Factor VIII forms.

SEQUENCE LISTING

( 1) GENERAL INFORMATION :

(i) APPLICANT:

(A) NAME: Novo Nordis 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

(ϋ) 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: 720 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

Mat 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

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 Oys His Arg Lys Ser Val Tyr Trp His 245 250 255

Val I le Gly M3t 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 lie Thr Phe Leu Thr Ala Gin Thr Leu Leu

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 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 IVfet 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 lie 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 I le 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 I fet 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 Cys 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 IVfet 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 e 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

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 729 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:2:

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

M3t 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

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

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro 405 410 415

Gin Arg Me Gly Arg Lys Tyr Lys Lys Val Arg Phe IVfet 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 I le 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 Gys 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 IVfet 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

Me 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 725

(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

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

Ser Pro I le 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 Giu 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 Giu 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 IVfet 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 I le Tyr Pro His Gly I le 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 I le 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 Oys 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

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 IVfet 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

Me 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

I le 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: 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 Giu 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 Cys 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

I le 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 /fet 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 I le 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 Me Ser Gin Phe Me Me 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

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

Thr Leu Arg IVfet Glu Leu IVfet Gly Cys 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

What is claimed is:

1. A Factor VIII polypeptide comprising a heavy chain having an amino acid sequence corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domain of full length Factor VIII.

2. A Factor VIII polypeptide as claimed in claim 1 wherein the heavy chain comprises the amino acids 1-729 of the heavy chain of full length Factor VIII.

3. A Factor VIII polypeptide as claimed in claim 1 wherein the heavy chain comprises the amino acids 1-720 of the heavy chain of full length Factor VIII.

4. A Factor VIII polypeptide as claimed in any of claims 1 to 3 comprising a light chain having an amino acid sequence corresponding to amino acids 1649-2332 of the C terminal of full length Factor VIII.

5. A Factor VIII polypeptide as claimed in any of claims 1 to 3 comprising a light chain having an amino acid sequence corresponding to amino acids 1690-2332 of the C terminal full length Factor VIII.

6. A method for preparing a Factor VIII polypeptide comprising a heavy chain ha- ving an amino acid sequences corresponding to the amino acid sequence of the

N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the heavy chain is shorter than the A1- A2 domain of full length Factor VIII wherein a Factor VIII polypeptide comprising the full A1-A2 domain of full length Factor VIII is treated with a protease.

7. A pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid 5 sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein all or a part of the Faktor VIII has a heavy chain shorter than the A1-A2 domain of full length Factor VIII in admixture with a parenterally acceptable vehicle or excipient.

10 8. Use of a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than

15 the A1-A2 domain of full length Factor VIII for the preparation of a pharmaceutical preparation.

9. Use as claimed in Claim 7 wherein a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having 20 an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domain of full length Factor VIII 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.

25 10. 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 Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domain of full length Factor VIII in admixture with a pharmaceutically acceptable vehicle or excipient.

12. A method of preparing a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domain of full length Factor VIII with pharmaceutically acceptable vehicle and/or exhipient and forming a suitable dosis form of the pharmaceutical preparation.